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Adrenocortical activity and avoidance learning as a function of time after avoidance training
Physiology and Behavior. Vol. 2, pp. 385-388. Pergamon Press Ltd., 1967 Printed in Great Britain
Adrenocortical Activity and Avoidance Learning as a Function of Time after Avoidance Training' S E Y M O U R L E V I N E ' A N D F. R O B E R T B R U S H
Stanford University School of Medicine, and University of Oregon Medical School (Received 8 M a r c h 1967) LEVINE, S. AND F. R. BRUSH. Adrenocortical activity and avoidance learning as a function of time after avoidance training. PHYSIOL.BEHAV.2 (4) 385-388, 1967.--Shuttlebox avoidance learning in rats is a U-shaped function of time after original avoidance training. The descending limb of the function is correlated with a decrease in plasma concentration of corticosterone. When high corticosteroid levels are maintained, either by exogenous ACTH administration or by hydrocortisone replacement, then high levels of performance are maintained. These findings support the hypothesis that avoidance learning decrements at intermediate intersession intervals are directly related to the functioning of the neuroendocrine system. Adrenocortical activity
Avoidance learning
ACTH
IN RECENTexperiments we showed that avoidance learning by rats in a shuttlebox is a U-shaped function of time after fear conditioning, the minimum of the function being at 4 hr, and that the descending limb of this function is closely associated with circulating levels of plasma corticosterone [2, 5]. The purpose of the present experiments is to determine (a) whether a similar association between avoidance learning and circulating corticosteroids could be demonstrated following avoidance training as had been shown to follow fear conditioning, and (b) whether by manipulating the steroid levels a direct relation between circulating corticosteroids and avoidance behavior could be shown to exist. Such a relationship was hypothesized in our previous report; specifically, if the poor performance at intermediate intervals after original training is related to the low level of plasma steroids and/or a reduced capacity of the adrenal cortex to respond to a second avoidance training session, then maintained steroid elevation, however accomplished, should mitigate the observed decrement in performance.
Corticosterone
Hydrocortisone
US was a scrambled a.c. shock of 0.25 mA delivered to the grid floor of the shuttlebox.
Procedure Following a 2 rain period of adaptation to the shuttlebox each rat received ten pretest trials during which only the CS was presented. On each trial the CS was terminated either by a shuttle response or after 45 sec, whichever occurred first. A one rain intertrial interval was used. This pretest procedure results in approximately 18 per cent of animals being rejected by one of the following criteria: (a) response latency less than 5 sec from CS onset on five or more of the ten pretest trials or (b) response latency less than 5 sec on three or more of the last five pretest trials. One min after the last pretest trial, original avoidance training began and continued until a learning criterion of three avoidance responses (not necessarily consecutive) had been met, or until a maximum of forty training trials had been given. A 5 sec CS-US interval was used in a delayedconditioning paradigm; the intertrial interval was constant at 1 rain. Of animals receiving training, 28 per cent failed to meet the learning criterion and were rejected. The shuttlebox data reported here are from animals that satisfied both pretest and original learning criteria. See Brush [3] for a discussion of the effect of these selection criteria. Two min after the final avoidance response the animals were removed to their home cages and assigned, in groups of ten, to one of the following intersession intervals: 0.08, 1, 4, 24, or 168 hr. In order to control for any possible effects due to differences in rate of original learning all groups were
METHOD
Subjects and Apparatus A total of 393 experimentally naive male hooded (LongEvans) rats were used for this research. They were approximately 90 days of age and were housed individually where food and water were continually available. The training apparatus was an automatic shuttlebox which has been described elsewhere [1, 6]. The CS was a 78 dB white noise (8 dB above background) and a concomitant increase in illumination in the compartment occupied by the rat. The
1This research was supported by Grants MH~)3337, HE~6336, and MH-07435 from the National Institute of Health, USPHS. Organon, Inc., West Orange, New Jersey, supplied the placebo gel used in these experiments. The Upjohn Co., Kalamazoo, Michigan, supplied the corticosterone and hydrocortisone acetate. 2Supported by Public Health Service research career development award (K3-MH-19, 936) from the National Institute of Mental Health. 385
386
LEVINE AND BRUSH
matched on the median number of trials required to reach criterion. Fifty animals received the sequence of pretest, original avoidance training, intersession interval, and forty trials of avoidance training in which the procedures were identical to those of original training. An additional fifty animals were treated identically to the first group except that after the appropriate intersession interval they were decapitated instead of being given avoidance training. The blood was collected in heparinized beakers, centrifuged, and the plasma extracted and frozen for later determination of corticosterone concentration by the method of Glick et al. [8]. Ten untreated rats served to determine the basal cortico" steroid level. Ninety additional animals (in three replications of thirty animals each) received the following treatment: pretest, original learning, injection, 1 hr intersession interval, and avoidance training. Thirty more animals received the same treatments but were decapitated for corticosteroid determination after the 1 hr interval. These 120 animals were injected subcutaneously with 0.2 cc either of ACTH (Organon Cortrophin Gel, 80 units/cc), gel placebo, or physiological saline. Thirty-three additional animals, in three groups of 11 each, were given the same procedures but were injected subcutaneously with 0.2 cc either of hydrocortisone acetate (10 mg/cc suspended in physiological saline), corticosterone (10 mg/cc suspension), or physiological saline. For thirty other animals the procedure was as follows: pretest, original learning, 168 hr intersession interval, injection (ACTH, placebo or saline as previously described), 1 hr post-injection interval (also spent in the home cage), and avoidance training. Thirty more animals received the same treatments but were decapitated for steroid determination 1 hr after injection. Effects of diurnal fluctuations in corticosterone secretion were eliminated by maintaining the rats under constant illumination for three weeks prior to and during the experiment. Also, steroid and behavioral measurements were obtained between 11:00 a.m. and 5:00 p.m., and the times within these limits were randomized among the experimental groups. In order to conduct the above injection experiments it was necessary first to determine the dose-time function of steroid elevation by ACTH. For this purpose sixty animals were injected subcutaneously with 0.2 cc Organon Cortrophin Gel (ACTH), half receiving a dose of 80 tmits/cc, the other half 40 units/cc. Ten animals within each dose group were decapitated 1, 2, or 4 hr after injection using the technique described above. Ten untreated animals served to determine basal corticosteroid levels. The results presented in Table 1
TABLE 1 CHANGES IN PLASMA CORTICOSTERONE(t~g/100 ml) FOLLOWING ACTH ADMINISTI~ATION Dose Units 8.0
Hours After Injection 1
59.04± 1.99" 28.89~z5.18
16.0
59.41 ± 2.92
Control
8.76 ± 1.42
*Mean ± SE.
2
33.10d: 6.09
8.52± 1.69 10.86± 1.40
indicate that a dose of 16 units (80 units/cc) would elevate and sustain high steroid levels for at least 1 hr following ACTH administration. However, after 4 hr steroid values were close to basal levels. Therefore, we used a 1 hr postinjection interval in the above experiments even though in earlier experiments the maximum decrement in performance occurred following an intersession interval of 4 hr. RESULTS
The behavioral data of these experiments arc summarized in Fig. I which plots the median total number of avoidance responses as a function of intersession interval. This U-shaped function replicates the data of earlier experiments [2, 4],
r.,,,)
40,
z o.. r~
.A
30. $
E ,qc In.. 0
P
20,
c~ Z ..,.I o
I0,
:IE
.08
1
4
24
168
INTERSESSION INTERVAL IN HOURS (10g scale) FIG. 1. Median total number of avoidance responses as a function of intersession interval. Isolated points at 1 hr are for animals that were injected with ACTH (A), placebo (P), or saline (S) immediately after original training and tested 1 hr later. The isolated points at 168 hr are for animals that were injected 168 hr after original learning and tested 1 hr after injection.
except that here the minimum of the function is at 1 hr, rather than at 4 hr. It should be noted, however, that in the cited experiments training routines and CS and US parameters were different from those used in this experiment. The data were subjected to a treatment (intersession interval) by levels (rate of original learning, each group being split at the median) analysis of variance which showed that the intersession interval effect is significant (F : 2.93; df-- 4, 40; p < 0.05), that the effect of original learning rate is not significant (F = !.42; d f = 1, 40; p > 0.05), and that the effects of intersession interval and rate of original learning interact (F = 2.99; d f : 4, 40; p < 0.05). This interaction is such that the minimum of the function is at 4 hr for fast learning animals, whereas it is at 1 hr for slow learning rats, an effect just opposite to that reported earlier by Kamin [9]. Figure 2 presents the median plasma concentration of corticosterone as a function of intersession interval. The corticosteroid level is initially elevated and decreases with time after original avoidance training, reaching basal level
ADRENOCORTICAL ACTIVITY AND LEARNING
387
within an hour. An analysis of variance applied to these data showed that the effect of intersession interval is highly significant (F = 25.45; df= 4, 45; p < 0.001), whereas neither the original learning rate nor the interaction effect is significant (F < 1.0, and F -- 2.56; d['= 4, 40; 0.05 < p < 0.10), respectively.
60
p < 0.001), an effect illustrated in Fig. 2. The corresponding behavioral effect, illustrated in Fig. 1, is the predicted facilitation of avoidance learning, but the effect is less reliable than the steroid effect. Mann-Whitney U tests showed that A C T H facilitates avoidance learning over placebo controls (U = 23, p < 0.025) but not in comparison to the saline control group (U = 33, p > 0.05). The failure to find a significant difference between the ACTH and saline groups is probably attributable to sampling error in the latter.
A
DISCUSSION • A
o
.A
40
20
..j. m o =E
.08
1
4
24
168
INTERSESSION INTERVAL IN HOURS (10g scale) FIG. 2. Median plasma corticosterone concentration as a function of intersession interval. The isolated points are analogous to those of Fig. 1. The horizontal dashed line represents basal level obtained from untreated control rats.
Also plotted in Fig. 2 are the steroid levels for the ! hr intersession interval groups that were injected with A C T H (A), placebo (P), or saline (S) immediately after completing original learning. Analyses of variance showed that the corticosterone concentration I hr following A C T H injection is significantly higher than that of the corresponding injected control groups (F - 28.12; df-- 2, 27; p < 0.001) and that the steroid levels of the injected control groups do not differ from each other or from that of the uninjected group (F < 1.0 in each case). The corresponding points in Figure 1 show that the behavioral effect of A C T H is the predicted facilitation of avoidance learning over that of placebo, saline, and uninjected controls. A Kruskal-Wallis analysis of variance by ranks was applied to the data of the injected groups because of heterogeneity of variance and skewness of the distributions. This analysis showed that the apparent effect of ACTH injection is significant (H =~ 10.00, df= 2, p < 0.01). Hydrocortisone replacement produces essentially the same effect as exogenous ACTH, whereas corticosterone and saline treatments do not affect avoidance behavior. The median total number of avoidance responses for the animals injected with hydrocortisone, corticosterone and saline are 26.0, 14.0, and 16.0, respectively. These data were also subjected to a Kruskal-Wallis analysis of variance by ranks and just fail to reach statistical significance (H = 5.24; df-: 2; 0.05 < p < 0.10). Finally, animals injected with ACTH 168 hr after completion of original avoidance training show a significant elevation of corticosteroid level over controls (F = 70.54; df= 2, 26;
Avoidance learning has once again been shown to be a U-shaped function of time after an initial training session. The function shown here following avoidance training is similar in form to the one previously shown to follow fear conditioning. We have also shown that the changes in steroid level following the initial avoidance training session are virtually identical to the changes observed after fear conditioning. Thus it is apparent that the relationship between avoidance learning and circulating corticosteroids after fear conditioning also occurs after avoidance training. The stability of this relation gives further support to the hypothesis that avoidance learning decrements at intermediate intersession intervals may be directly related to the functioning of the neuroendocrine mechanisms regulating steroidogenesis. Support for this hypothesis rests in the finding that when high corticosteroid levels are maintained, either by exogenous A C T H administration or by hydrocortisone replacement, then high levels of performance are maintained. It may also be noted that the steroid concentration in plasma 1 hr after injection of 16 units of A C T H is less in stressed animals (those previously given avoidance training) than in unstressed subjects. This finding suggests that the capacity of the adrenal cortex to respond to a second stress is reduced by the original avoidance training. However, the steroid response to A C T H in animals injected 168 hr after original training suggests that this reduction in adrenal capacity is temporary. Somewhat paradoxical is the failure of corticosterone replacement to effect a change in behavior similar to that produced by ACTH. However, there are at least two possible explanations of this paradox. First, with respect to the many known biochemical actions of steroids, hydrocortisone is known to be much more potent than corticosterone. Thus it is possible that the dose of corticosterone used in this experiment was insufficient to cause behavioral changes. A second possible explanation is that although corticosterone is the predominant glucocorticoid produced by the rat adrenal cortex it is not necessarily the corticosteroid which exerts CNS control [7]. Although these data seem to implicate the corticosteroids in controlling behavior these experiments do not preclude extra-adrenal behavioral effects of ACTH. One possible interpretation of these data is that hormones somehow affect the memory process. It could be argued, for example, that maintenance of high steroid levels prevents forgetting of previously learned responses over a 1 hr intersession interval. However, the facilitation of avoidance learning by ACTH in the 168 hr group refutes such an interpretation. Thus, it is unlikely that a steroid elevation produced by ACTH administration 168 hr after learning could preserve an already decayed memory trace. Rather, it is more likely that A C T H treatment or corticosteroid replacement reinstates motivational cues associated with the avoidance training situation thereby facilitating performance.
388
LEVINE A N D BRUSH
REFERENCES 1. Brush, F. R. The effects of intertrial interval on avoidance learning in the rat. J. comp. physiol. Psyehol. 55: 888-892, 1962. 2. Brush, F. R. Avoidance learning as a function of time after fear conditioning and unsignalled shock. Psychonom. Sci. 1: 405-406, 1964. 3. Brush, F. R. On the differences between animals that learn and do not learn to avoid electric shock. Psychonom. Sci. 5: 123-124, 1966. 4. Brush, F. R., J. S. Myer and M. E. Palmer. The effects of kind of prior training and intersession interval upon subsequent avoidance learning. ,jr. comp. physiol. Psychol. 56: 539-545, 1963. 5. Brush, F. R. and S. Levine. Andrenocortical activity and avoidance learning as a function of time after fear conditioning. Physiol. Behav. 1 : 309-311, 1966.
6. Brush, F. R. and P. R. Knaff. A device for detecting and controlling automatic programming of avoidance conditioning in a shuttlebox. Am. J. Psychol. 72: 275-278, 1959. 7. Davidson, J. M., L. E. Jones and S. Levine. Effects of hypothalamic implantation of steroids on plasma cortieosterone. Fedn Proc. 24: 191, 1965. 8. Glick, D., D. Von Redlich and S. Lcvine. Fluorometric determination of corticosterone and cortisol in 0.02-0.05 milliliters of plasma or submilligram samples of adrenal tissue. Endocrinology 74: 653-655, 1964. 9. Kamin, L. J. Retention of an incompletely learned avoidance response: some further analyses. J. comp. physiol. Psychol. 56: 713-718, 1963.
Adrenocortical Activity and Avoidance Learning as a Function of Time after Avoidance Training' S E Y M O U R L E V I N E ' A N D F. R O B E R T B R U S H
Stanford University School of Medicine, and University of Oregon Medical School (Received 8 M a r c h 1967) LEVINE, S. AND F. R. BRUSH. Adrenocortical activity and avoidance learning as a function of time after avoidance training. PHYSIOL.BEHAV.2 (4) 385-388, 1967.--Shuttlebox avoidance learning in rats is a U-shaped function of time after original avoidance training. The descending limb of the function is correlated with a decrease in plasma concentration of corticosterone. When high corticosteroid levels are maintained, either by exogenous ACTH administration or by hydrocortisone replacement, then high levels of performance are maintained. These findings support the hypothesis that avoidance learning decrements at intermediate intersession intervals are directly related to the functioning of the neuroendocrine system. Adrenocortical activity
Avoidance learning
ACTH
IN RECENTexperiments we showed that avoidance learning by rats in a shuttlebox is a U-shaped function of time after fear conditioning, the minimum of the function being at 4 hr, and that the descending limb of this function is closely associated with circulating levels of plasma corticosterone [2, 5]. The purpose of the present experiments is to determine (a) whether a similar association between avoidance learning and circulating corticosteroids could be demonstrated following avoidance training as had been shown to follow fear conditioning, and (b) whether by manipulating the steroid levels a direct relation between circulating corticosteroids and avoidance behavior could be shown to exist. Such a relationship was hypothesized in our previous report; specifically, if the poor performance at intermediate intervals after original training is related to the low level of plasma steroids and/or a reduced capacity of the adrenal cortex to respond to a second avoidance training session, then maintained steroid elevation, however accomplished, should mitigate the observed decrement in performance.
Corticosterone
Hydrocortisone
US was a scrambled a.c. shock of 0.25 mA delivered to the grid floor of the shuttlebox.
Procedure Following a 2 rain period of adaptation to the shuttlebox each rat received ten pretest trials during which only the CS was presented. On each trial the CS was terminated either by a shuttle response or after 45 sec, whichever occurred first. A one rain intertrial interval was used. This pretest procedure results in approximately 18 per cent of animals being rejected by one of the following criteria: (a) response latency less than 5 sec from CS onset on five or more of the ten pretest trials or (b) response latency less than 5 sec on three or more of the last five pretest trials. One min after the last pretest trial, original avoidance training began and continued until a learning criterion of three avoidance responses (not necessarily consecutive) had been met, or until a maximum of forty training trials had been given. A 5 sec CS-US interval was used in a delayedconditioning paradigm; the intertrial interval was constant at 1 rain. Of animals receiving training, 28 per cent failed to meet the learning criterion and were rejected. The shuttlebox data reported here are from animals that satisfied both pretest and original learning criteria. See Brush [3] for a discussion of the effect of these selection criteria. Two min after the final avoidance response the animals were removed to their home cages and assigned, in groups of ten, to one of the following intersession intervals: 0.08, 1, 4, 24, or 168 hr. In order to control for any possible effects due to differences in rate of original learning all groups were
METHOD
Subjects and Apparatus A total of 393 experimentally naive male hooded (LongEvans) rats were used for this research. They were approximately 90 days of age and were housed individually where food and water were continually available. The training apparatus was an automatic shuttlebox which has been described elsewhere [1, 6]. The CS was a 78 dB white noise (8 dB above background) and a concomitant increase in illumination in the compartment occupied by the rat. The
1This research was supported by Grants MH~)3337, HE~6336, and MH-07435 from the National Institute of Health, USPHS. Organon, Inc., West Orange, New Jersey, supplied the placebo gel used in these experiments. The Upjohn Co., Kalamazoo, Michigan, supplied the corticosterone and hydrocortisone acetate. 2Supported by Public Health Service research career development award (K3-MH-19, 936) from the National Institute of Mental Health. 385
386
LEVINE AND BRUSH
matched on the median number of trials required to reach criterion. Fifty animals received the sequence of pretest, original avoidance training, intersession interval, and forty trials of avoidance training in which the procedures were identical to those of original training. An additional fifty animals were treated identically to the first group except that after the appropriate intersession interval they were decapitated instead of being given avoidance training. The blood was collected in heparinized beakers, centrifuged, and the plasma extracted and frozen for later determination of corticosterone concentration by the method of Glick et al. [8]. Ten untreated rats served to determine the basal cortico" steroid level. Ninety additional animals (in three replications of thirty animals each) received the following treatment: pretest, original learning, injection, 1 hr intersession interval, and avoidance training. Thirty more animals received the same treatments but were decapitated for corticosteroid determination after the 1 hr interval. These 120 animals were injected subcutaneously with 0.2 cc either of ACTH (Organon Cortrophin Gel, 80 units/cc), gel placebo, or physiological saline. Thirty-three additional animals, in three groups of 11 each, were given the same procedures but were injected subcutaneously with 0.2 cc either of hydrocortisone acetate (10 mg/cc suspended in physiological saline), corticosterone (10 mg/cc suspension), or physiological saline. For thirty other animals the procedure was as follows: pretest, original learning, 168 hr intersession interval, injection (ACTH, placebo or saline as previously described), 1 hr post-injection interval (also spent in the home cage), and avoidance training. Thirty more animals received the same treatments but were decapitated for steroid determination 1 hr after injection. Effects of diurnal fluctuations in corticosterone secretion were eliminated by maintaining the rats under constant illumination for three weeks prior to and during the experiment. Also, steroid and behavioral measurements were obtained between 11:00 a.m. and 5:00 p.m., and the times within these limits were randomized among the experimental groups. In order to conduct the above injection experiments it was necessary first to determine the dose-time function of steroid elevation by ACTH. For this purpose sixty animals were injected subcutaneously with 0.2 cc Organon Cortrophin Gel (ACTH), half receiving a dose of 80 tmits/cc, the other half 40 units/cc. Ten animals within each dose group were decapitated 1, 2, or 4 hr after injection using the technique described above. Ten untreated animals served to determine basal corticosteroid levels. The results presented in Table 1
TABLE 1 CHANGES IN PLASMA CORTICOSTERONE(t~g/100 ml) FOLLOWING ACTH ADMINISTI~ATION Dose Units 8.0
Hours After Injection 1
59.04± 1.99" 28.89~z5.18
16.0
59.41 ± 2.92
Control
8.76 ± 1.42
*Mean ± SE.
2
33.10d: 6.09
8.52± 1.69 10.86± 1.40
indicate that a dose of 16 units (80 units/cc) would elevate and sustain high steroid levels for at least 1 hr following ACTH administration. However, after 4 hr steroid values were close to basal levels. Therefore, we used a 1 hr postinjection interval in the above experiments even though in earlier experiments the maximum decrement in performance occurred following an intersession interval of 4 hr. RESULTS
The behavioral data of these experiments arc summarized in Fig. I which plots the median total number of avoidance responses as a function of intersession interval. This U-shaped function replicates the data of earlier experiments [2, 4],
r.,,,)
40,
z o.. r~
.A
30. $
E ,qc In.. 0
P
20,
c~ Z ..,.I o
I0,
:IE
.08
1
4
24
168
INTERSESSION INTERVAL IN HOURS (10g scale) FIG. 1. Median total number of avoidance responses as a function of intersession interval. Isolated points at 1 hr are for animals that were injected with ACTH (A), placebo (P), or saline (S) immediately after original training and tested 1 hr later. The isolated points at 168 hr are for animals that were injected 168 hr after original learning and tested 1 hr after injection.
except that here the minimum of the function is at 1 hr, rather than at 4 hr. It should be noted, however, that in the cited experiments training routines and CS and US parameters were different from those used in this experiment. The data were subjected to a treatment (intersession interval) by levels (rate of original learning, each group being split at the median) analysis of variance which showed that the intersession interval effect is significant (F : 2.93; df-- 4, 40; p < 0.05), that the effect of original learning rate is not significant (F = !.42; d f = 1, 40; p > 0.05), and that the effects of intersession interval and rate of original learning interact (F = 2.99; d f : 4, 40; p < 0.05). This interaction is such that the minimum of the function is at 4 hr for fast learning animals, whereas it is at 1 hr for slow learning rats, an effect just opposite to that reported earlier by Kamin [9]. Figure 2 presents the median plasma concentration of corticosterone as a function of intersession interval. The corticosteroid level is initially elevated and decreases with time after original avoidance training, reaching basal level
ADRENOCORTICAL ACTIVITY AND LEARNING
387
within an hour. An analysis of variance applied to these data showed that the effect of intersession interval is highly significant (F = 25.45; df= 4, 45; p < 0.001), whereas neither the original learning rate nor the interaction effect is significant (F < 1.0, and F -- 2.56; d['= 4, 40; 0.05 < p < 0.10), respectively.
60
p < 0.001), an effect illustrated in Fig. 2. The corresponding behavioral effect, illustrated in Fig. 1, is the predicted facilitation of avoidance learning, but the effect is less reliable than the steroid effect. Mann-Whitney U tests showed that A C T H facilitates avoidance learning over placebo controls (U = 23, p < 0.025) but not in comparison to the saline control group (U = 33, p > 0.05). The failure to find a significant difference between the ACTH and saline groups is probably attributable to sampling error in the latter.
A
DISCUSSION • A
o
.A
40
20
..j. m o =E
.08
1
4
24
168
INTERSESSION INTERVAL IN HOURS (10g scale) FIG. 2. Median plasma corticosterone concentration as a function of intersession interval. The isolated points are analogous to those of Fig. 1. The horizontal dashed line represents basal level obtained from untreated control rats.
Also plotted in Fig. 2 are the steroid levels for the ! hr intersession interval groups that were injected with A C T H (A), placebo (P), or saline (S) immediately after completing original learning. Analyses of variance showed that the corticosterone concentration I hr following A C T H injection is significantly higher than that of the corresponding injected control groups (F - 28.12; df-- 2, 27; p < 0.001) and that the steroid levels of the injected control groups do not differ from each other or from that of the uninjected group (F < 1.0 in each case). The corresponding points in Figure 1 show that the behavioral effect of A C T H is the predicted facilitation of avoidance learning over that of placebo, saline, and uninjected controls. A Kruskal-Wallis analysis of variance by ranks was applied to the data of the injected groups because of heterogeneity of variance and skewness of the distributions. This analysis showed that the apparent effect of ACTH injection is significant (H =~ 10.00, df= 2, p < 0.01). Hydrocortisone replacement produces essentially the same effect as exogenous ACTH, whereas corticosterone and saline treatments do not affect avoidance behavior. The median total number of avoidance responses for the animals injected with hydrocortisone, corticosterone and saline are 26.0, 14.0, and 16.0, respectively. These data were also subjected to a Kruskal-Wallis analysis of variance by ranks and just fail to reach statistical significance (H = 5.24; df-: 2; 0.05 < p < 0.10). Finally, animals injected with ACTH 168 hr after completion of original avoidance training show a significant elevation of corticosteroid level over controls (F = 70.54; df= 2, 26;
Avoidance learning has once again been shown to be a U-shaped function of time after an initial training session. The function shown here following avoidance training is similar in form to the one previously shown to follow fear conditioning. We have also shown that the changes in steroid level following the initial avoidance training session are virtually identical to the changes observed after fear conditioning. Thus it is apparent that the relationship between avoidance learning and circulating corticosteroids after fear conditioning also occurs after avoidance training. The stability of this relation gives further support to the hypothesis that avoidance learning decrements at intermediate intersession intervals may be directly related to the functioning of the neuroendocrine mechanisms regulating steroidogenesis. Support for this hypothesis rests in the finding that when high corticosteroid levels are maintained, either by exogenous A C T H administration or by hydrocortisone replacement, then high levels of performance are maintained. It may also be noted that the steroid concentration in plasma 1 hr after injection of 16 units of A C T H is less in stressed animals (those previously given avoidance training) than in unstressed subjects. This finding suggests that the capacity of the adrenal cortex to respond to a second stress is reduced by the original avoidance training. However, the steroid response to A C T H in animals injected 168 hr after original training suggests that this reduction in adrenal capacity is temporary. Somewhat paradoxical is the failure of corticosterone replacement to effect a change in behavior similar to that produced by ACTH. However, there are at least two possible explanations of this paradox. First, with respect to the many known biochemical actions of steroids, hydrocortisone is known to be much more potent than corticosterone. Thus it is possible that the dose of corticosterone used in this experiment was insufficient to cause behavioral changes. A second possible explanation is that although corticosterone is the predominant glucocorticoid produced by the rat adrenal cortex it is not necessarily the corticosteroid which exerts CNS control [7]. Although these data seem to implicate the corticosteroids in controlling behavior these experiments do not preclude extra-adrenal behavioral effects of ACTH. One possible interpretation of these data is that hormones somehow affect the memory process. It could be argued, for example, that maintenance of high steroid levels prevents forgetting of previously learned responses over a 1 hr intersession interval. However, the facilitation of avoidance learning by ACTH in the 168 hr group refutes such an interpretation. Thus, it is unlikely that a steroid elevation produced by ACTH administration 168 hr after learning could preserve an already decayed memory trace. Rather, it is more likely that A C T H treatment or corticosteroid replacement reinstates motivational cues associated with the avoidance training situation thereby facilitating performance.
388
LEVINE A N D BRUSH
REFERENCES 1. Brush, F. R. The effects of intertrial interval on avoidance learning in the rat. J. comp. physiol. Psyehol. 55: 888-892, 1962. 2. Brush, F. R. Avoidance learning as a function of time after fear conditioning and unsignalled shock. Psychonom. Sci. 1: 405-406, 1964. 3. Brush, F. R. On the differences between animals that learn and do not learn to avoid electric shock. Psychonom. Sci. 5: 123-124, 1966. 4. Brush, F. R., J. S. Myer and M. E. Palmer. The effects of kind of prior training and intersession interval upon subsequent avoidance learning. ,jr. comp. physiol. Psychol. 56: 539-545, 1963. 5. Brush, F. R. and S. Levine. Andrenocortical activity and avoidance learning as a function of time after fear conditioning. Physiol. Behav. 1 : 309-311, 1966.
6. Brush, F. R. and P. R. Knaff. A device for detecting and controlling automatic programming of avoidance conditioning in a shuttlebox. Am. J. Psychol. 72: 275-278, 1959. 7. Davidson, J. M., L. E. Jones and S. Levine. Effects of hypothalamic implantation of steroids on plasma cortieosterone. Fedn Proc. 24: 191, 1965. 8. Glick, D., D. Von Redlich and S. Lcvine. Fluorometric determination of corticosterone and cortisol in 0.02-0.05 milliliters of plasma or submilligram samples of adrenal tissue. Endocrinology 74: 653-655, 1964. 9. Kamin, L. J. Retention of an incompletely learned avoidance response: some further analyses. J. comp. physiol. Psychol. 56: 713-718, 1963.