The glucocorticosterone response to novelty as a psychological stressor

Physiology & Behavior, Vol. 23, pp. 649-652. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.

The Glucocorticosterone Response to N o v e l t y as a Psychological Stressor I H. P E T E R P F I S T E R

Department of Psychology, The University of Newcastle, Newcastle, N S W 2308, Australia R e c e i v e d 25 M a y 1979 PFISTER, H. P. The glucocorticosterone response to novelty as a psychological stressor. PHYSIOL. BEHAV. 23(4) 649-652, 1979.--The 1 l-hydroxycorticosterone response in non-pregnant and pregnant rats to novelty as a psychological stressor was examined. Past studies have mostly emphasized short duration of exposure to the novel environment. The present study aimed at investigating the elevation and habituation of the 11-hydroxycorticosterone response to repeated exposures (30 min daily) to the novel environment. It was found that on Day 1 of exposure in non-pregnant and pregnant rats the i l-hydroxycorticosterone elevation is approximately four fold, and then gradually habituating over days. No significant elevation above control levels were obtained after 5 days of exposure to the novel environment in nulliparous animals. Novelty is a powerful but simple tool in psychological stress research. Novelty

Psychological stress

11-Hydroxycorticosterone

P S Y C H O L O G I C A L stress is known to activate the pituitary-adrenocortical system [4, 5, 16, 17]. A wide variety of psychological stressors have been identified, such as fear motivated situations [3, 16, 17], conditioned anxiety [3,22], handling [6, 9, 14] and novelty [1, 3, 11, 12, 21] are well documented. If a naive laboratory rat is introduced into a novel environment, the ensuing elevation of glucocorticosterone (11-OHCS) has been regarded as a function of the novelty of that environment [21]. The rapid rise of 11-OHCS as a consequence of exposure to a novel environment has been documented [l, 3, ll, 12, 21]. As the rat reaches some degree of familiarity with a novel environment a corresponding reduction in emotional responsiveness and reduction in 11-OHCS has been observed [21]. However, it has been reported recently [10,12] that no such reductions were found in mice exposed to 10 daily 100 sec sessions, and in rats after 26 daily 15 min sessions in an empty chamber. This reported [i2] inability of the pituitary-adrenocortical system to habituate to a novel stimulus is unusual and calls for further systematic investigation. The present study examines the question of habituation of the 11-OHCS response in rats to a novel environment.

Rat

11-OHCS level was predicted. Since it has been reported that rats habituate to novelty after four hours of exposure [21] it was expected that initial l l-OHCS levels would be very high, decreasing gradually with repeated exposure until animals had fully habituated to the novel environment. In addition, handled rats were removed from their boxes held over the test apparatus and then returned to their respective boxes. This procedure controls for the handling component of the rats exposed to the novel test apparatus. A further control group which was not disturbed was also included. METHOD

Animals One-hundred and eighty nulliparous female Wistar rats 90-100 days old at the start of the experiment were used. Three weeks prior to testing all animals were housed singly in boxes (32×45x 17 cm) in a fully air conditioned holding room at 22 -- I°C. Light was on daily from 1100 hr-2300 hr. Food and water were provided ad lib.

Glucocorticoid Assay EXPERIMENT 1 This experiment was designed to examine further the question of habituation of the pituitary-adrenocortical system as indexed by the 11-OHCS response. As in some other experiments [12] animals were given repeated exposures to the same environment. The degree to which the novel environment (test apparatus) differed from the home cage was maximal [11] and consequently a substantial increase in the

The stress response measured was the plasma level of free 11-OHCS, the predominant glucocorticoid secreted in the rat [15,20]. At times indicated in the procedure animals were sacrificed by decapitation. The blood was collected in heparinized tubes and centrifuged to obtain cell-free plasma which was then frozen. Corticosterone levels in plasma were obtained subsequently by the tluorimetric method of Mattingly [18] which is specific for free plasma I1-OHCS.

1A critical reading of an earlier version of the manuscript by Professor M. G. King is gratefully acknowledged.

C o p y r i g h t © 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/100649-04500.90/0

650

PFi S i t;iR NOV

i

6

o

_

<

CON

o

HAN

o

Z " 3(]

[;!!iiiiii! O to i

DAY I

DAY 3

DAY @

DAY 7

DAY 9

FIG. 1. Mean values of ! I-OHCS (in/xg/100 ml of Plasma) for Novelty, Handled and Control groups for Days 1.3/5, 7 and 9.

Apparatus The novel apparatus consisted of a clear Plexiglas box with external dimensions of 2 3 x 3 5 x 3 0 cm. The grid floor was of stainless steel bars with a diameter of 0.5 cm and spaced 1.2 cm apart. The novel apparatus was placed inside the rat holding room described above.

Procedure The first 60 rats, of a total of 180, were randomly allocated to five groups of 12 rats each. These rats served as controls (CON) for plasma 11-OHCS base levels and were therefore not further disturbed. On Day 1 of the experiment, at 0900 hr, i.e., at the trough of the circadian rhythm in relation to 11-OHCS levels [21], animals of the first group (CON 1) were removed in their boxes from the holding room and taken to the preparation room. Blood plasma was collected from these animals within 60 sec of removal from the holding room. On Day 3 of the experiment animals of another group (CON 3) were similarly removed for plasma collection. On Day 5, Day 7 and Day 9, rats of groups CON 5, CON 7 and CON 9, respectively, were removed and their plasma was also collected. Similarly, the second batch of 60 rats were randomly allocated to five groups of 12 rats each. Animals of these five groups were subjected daily to one only 30 min exposure of the novel environment (NOV). Starting at 0900 hr a rat was picked up by the base of the tail and gently placed in the test apparatus described earlier. At 0930 hr, the rat was picked up again by the tail and returned to its holding box. Animals of the NOV 1 group received this treatment on 1 day only. Rats in group NOV 3 received the treatment for three successive days. Similarly, 5 days, or 7 days or 9 days of treatment were given to rats in groups NOV 5, NOV 7 and NOV 9, respectively. On the final day of their treatment, at 0930 hr, the rat was returned to its holding box and was then taken to the preparation room for plasma collection. Finally, the remaining 60 rats were randomly allocated to

the five handling groups (HAN) of 12 animals each, At 0900 hr, a rat was handled by picking it up by the base of the tail. holding it for 5 sec over the novel apparatus and then immediately returning it to its holding box. This treatment was given to all H A N animals daily for 1 day (HAN 1), or for 3 days (HAN 3), or for 5 days (HAN 5), or for 7 days ( H A N 7), or for 9 days (HAN 9). On the days indicated, at 0930 hr animals were removed from the holding room in their boxes and taken to the preparation room for plasma collection. The above procedure ensured common treatment o f all animals used in addition to which the rats Of the NOV groups were exposed to the novel apparatus, the animals of the H A N groups were handled while the rats of the CON groups remained undisturbed. RESULTS

AND

DISCUSSION

A two-way A N O V A R revealed a significant difference between treatments, F(2,165) =44.10, p <0.001, a significant habituation, F(4,165)= 13.11, p<0.001 and a Significant interaction effect, F(8,165)= 10.74, p<0.001. A series of post hoc Scheffe [131 comparisons of individual cell means showed that the mean 11-OHCS levels ofail CON and H A N groups did not differ significantly from each other (see Fig. 1). On the other hand, the mean 11-OHCS levels of the NOV groups for Day 1 and 3 were significantly higher (p<0.001) than those of the corresponding CON and HAN groups. On Day 5, the mean 11-OHCS level of group NOV 5 was significantly higher (p<0.05) than that of the HAN 5 group, but did not differ significantly from the CON 5 group. Thus the treatment effect obtained may be attributed to the NOV groups. The NOV 7 and NOV 9 groups, while having higher steroid levels than the CON 7 and CON 9 o r HAB 7 and HAB 9 groups, did not differ significantly from these groups. With respect to habituation, a series of Scheffe post hoc comparisons revealed no differences between-individual cell means of either the CON or H A N groups. Hence, the significant habituation effect was contained within the NOV groups (p<0.001). From the data outlined above (see Fig. 1)

NOVELTY--A PSYCHOLOGICAL STRESSOR

651

TABLE 1 MEAN VALUES OF 11-OHCS(IN p.G/100ML OF PLASMA)FOR NOVELTY, HANDLED AND CONTROL GROUPS FOR TRIMESTERS 1, 2 AND 3 AND DAYS 1, 3 AND 5 NOV

T1 T2 T3

1

3

61.03 53.73 71.63

38.37 38.02 50.93

HAN 5

37.37 35.40 58.29

1

3

13.70 13.43 24.60

8.77 8.48 20.94

it is evident that novelty is a powerful psychological stressor resulting in massive increases of the l l - O H C S output, Animals repeatedly exposed to the same novel test apparatus for 30 min daily appear to habituate to the novel apparatus after five days. EXPERIMENT 2 Experiment 1, confirmed that novelty is a powerful psychological stressor for rats [1, 3, 1 l, 12, 21]. This procedure is easily controlled, requires minimal equipment and is simple to administer. In the area of prenatal psychological stress research for instance, psychological stressors usually require sophisticated equipment and elaborate conditioning processes, which are costly and time consuming. Hence, a simple technique to administer psychological stress would be useful. Novelty as established in Experiment 1 would meet this criterion and would therefore be a useful tool for work in the prenatal and other psychological stress paradigms. However, I 1-OHCS levels are, apart from circadian fluctuations, subject to an increase during pregnancy. While it is generally accepted that l l - O H C S levels are elevated in the last few days of pregnancy [7, 8, 19], the steroid response to novelty stress in pregnant rats is not well established. This experiment was designed to evaluate novelty stress during pregnancy. Novelty stress, as established in Experiment 1, resulted in a significant elevation of 11-OHCS levels above the control groups for 5 days of testing. In this experiment, I or 3 or 5 days of novelty stress were given to pregnant animals in Term 1 or Term 2 or Term 3 of pregnancy. The l l - O H C S response to novelty stress was therefore evaluated in three distinct phases of pregnancy, taking into account possible changes in the steroid base level due to pregnancy. METHOD Animals Two hundred and seventy nulliparous female Wistar rats 75-85 days old were used. In addition, 30 experienced male Wistar rats approximately 150 days old drawn from the University rat breeding colony were available. Three weeks prior to mating, all rats were housed singly in boxes ( 3 2 x 4 5 x l 7 cm) in a fully air conditioned room at 22 -+ I°C. A 12:12 light/dark cycle was instituted. F o o d and water was provided ad lib. Procedure In the middle of the dark period, females were introduced into the boxes of the males for mating using the technique described by Barnett [2]. Successful copulation was observed on 249 out of 270 occasions and fertilization was as-

CON 5

7.95 9.74 17.47

1

3

13.82 12.78 15.58 12.00 20.95 20.28

5

11.03 12.13 18.88

sumed. Eighty-one pregnant females were randomly allocated for treatment in Term 1 (T1, Days 1-7 of pregnancy). Similarly, 81 pregnant females were randomly allocated for treatment each in Term 2 (T2, Days 8--14 of pregnancy) and Term 3 (T3, Days 15-21 of pregnancy). The 81 T1 pregnant females were randomly allocated to nine groups of 9 rats each. Rats were either allocated to the novelty stress group (T1 NOV), the handled group (T1 H A N ) or the control group (TI CON). Animals were either stressed, handled or served as controls for 1 day, or 3 days of 5 days. Animals o f T 2 and T3 were similarly allocated to nine groups as described above. This procedure resulted in a 3 (T1,T2,T3)x3 ( N O V , H A N , C O N ) x 3 (Day 1,Day 3,Day 5) factorial design. Otherwise, the procedure described was identical to that described in Experiment 1, with the one variation, that after animals were sacrificed pregnancy was confirmed by physical or histological examination. RESULTS AND DISCUSSION A three-way A N O V A R revealed three significant main effects, a treatment effect, F(2,297)=223.93, p<0.001, a trimester effect, F(2,297)=23.90, p<0.001, and a habituation effect over days, F(2,297)=14.20, p<0.001. A significant treatment by habituation interaction effect was also obtained, F(4,297)=5.05, p<0.001. In Table 1, no significant difference is apparent between any of the CON and H A N groups; a series of Scheffe post hoc comparisons confirmed this. On the other hand, the novelty groups differ significantly from both the CON and H A N groups (p<0.001). Hence, the main treatment effect observed in the A N O V A R is due to the significantly higher 11-OHCS levels obtained for the NOV groups. This effect is consistent for all three trimesters. An examination of the trimester effect revealed by Scheffe post hoc comparisons that no significant differences are obtained between trimester l, 2 and 3 with respect to the CON and H A N groups. However it is worth mentioning that the elevations in l l-OHCS levels of the CON and H A N groups in the third trimester approached significance (p<0.10). The Scheffe post hoc comparisons for NOV groups for trimester l, 2 and 3 revealed 3 significant differences. The T3 NOV 1 group has a significantly (p<0.01) higher steroid level than the corresponding T2 NOV 1 group. A similar result was obtained for the T3 NOV 5 comparison with T1 NOV 5. A highly significant difference was obtained for the T3 NOV 5 comparison with T2 NOV 5 (/9<0.001). With respect to habituation of novelty as measured over days, the Scheffe post hoc comparison revealed that the T1 NOV 1 group had a significantly higher l l - O H C S level (p<0.001) than the T1 NOV 3 or the T1 NOV 5 group. Similarly, the T2 NOV 1 group had a higher 11-OHCS level than

652

PFISTER

the T2 NOV 3 (0<0.05) or the T2 NOV 5 (p<0,01) group. Finally, the T3 NOV 1 group had a higher I I-OHCS level than the T3 NOV 3 (0<0.05) or the T3 NOV 5 (p<0.01) group. No other significant effect with respect to habituation was observed. Thus, the main habituation effect is due to the habituation component present in the NOV groups as described above. The significant interaction effect described earlier can now be attributed to the effect novelty treatment had on these animals over days. In summary, the rise in I 1-OHCS levels for the NOV groups above the CON and HAN groups is due to the novel environment. There is no appreciable difference in 11-OHCS levels of all three treatment groups between trimesters 1 and 2. However trimester 3 (see Table 1) shows significantly elevated steroid levels for animals of the NOV groups. The failure to obtain similar results for the CON and HAN groups is mainly due to the conservatism of the Scheffe post hoc comparison as compared with other tests. Habituation over days of testing was mainly due to the NOV groups which is also reflected in the interaction effect obtained. G E N E R A L DISCUSSION The results presented in both experiments clearly show that novelty is a powerful psychological stressor, resulting in a four fold increase in l l - O H C S levels after exposure of 30 min to the novel environment. Rats which were control handled did not show any appreciable difference in steroid levels from the control animals. This finding conforms with reports by others [1, 11, 12, 21]. It has been reported [21] that rats habituate to a novel environment after 4 hr of continued exposure. The results from Experiment 1 suggest that rats habituate to a novel environment after 5 days of repeated (30 min daily) exposures.

Furthermore both experiments demonstrate that habituation is a gradual process. As the rats reach some degree of familiarity with the novel apparatus a corresponding reduction in l l - O H C S levels is observed. This finding is in contrast with reports [11,121 maintaining that no habituation of the steroid response to a novel environment takes place. Novelty was equally stressful in nulliparous and pregnant rats. Elevated steroid levels were observed for those animals tested in the third trimester of pregnancy. This finding is in support of similar reports [7, 8, 19]. The results obtained in the second experiment demonstrate that novelty is a usefid, simple and effective technique for employment in psychological stress research, and is effective throughout pregnancy. Hennessey, Levin and Levine [12] argued that stimulus intensity may be the more effectively manipulated by varying the degree of difference between familiar and unfamiliar environments, rather than by varying the length of time that the animal is exposed to a standard novel environment, assuming that no habituation takes place. However, since it has been repeatedly shown that rats habituate to novel environments, it might be more appropriate to control stimulus intensity by the length of duration of exposure to the novel environment. Furthermore, it might be shown that a combination of both would result in the optimal control of the 11-OHCS response to novelty. However, this will have to be substantiated by further systematic research. Overall, the data suggests that novelty is a powerful psychological stressor in nulliparous and pregnant rats. The steroid response observed is massive on Day 1 and gradually habituates until after 5 days (30 min daily exposures) no significant elevation above the control levels is evident. Reports that the l l - O H C S response to novelty stress is not subject to habituation [121, are not supported and call for replication in the light of the present data.

REFERENCES

1. Ader, R. and S. B. Friedman. Plasma corticosterone response to environmental stimulation: Effects of duration of stimulation and the 24-hour adrenocortical rhythm. Neuroendocrinology 3: 378-386, 1968. 2. Barnett, S. A. The Rat: A Study in Behavior. Canberra: ANU Press, 1976. 3. Bassett, J. R., K. D. Cairncross and M. G. King. Parameters of novelty, shock predictability and response contingency in corticosterone release in the rat. Physiol. Behav. 10: 901-907, 1973. 4. Brain, P. F. Mammalian behavior and the adrenal cortex--A review. Behav. Biol. 7: 453--477, 1972. 5. DiGiusto, E. L., K. D. Cairncross and M. G. King. Hormonal influences in fear-motivated responses. Psychol. Bull. 75: 432444, 1971. 6. Dunn, J., L. Scheving and P. Millet. Circadian variation in stress-evoked increases in plasma corticosterone. Am. J. Physiol. 223: 402-406, 1972. 7. Dupouy, J. P., H. Coffigny and S. Marge. Maternal and foetal corticosterone levels during late pregnancy in rats. J. Endocr. 65: 347-352, 1975. 8. Eguchi, Y., K. Arishima, Y. Morikawa and Y. Hasimoto. Rise of plasma corticosterone concentrations in rats immediately before and after birth and in fetal rats after the ligation of maternal uterine blood vessels or the umbilical cord. Endocrinology 100: 1443--1447, 1977. 9. Grota, L. J. and R. Ader. Adrenocortical function in pregnant rats: Handling and the 24-hour rhythm. Physiol. Behav. 5: 739741, 1970. 10. Hennessy, M. B. and S. Levine. Effects of various habituation procedures on pituitary-adrenal responsiveness in the mouse. Physiol. Behav. 18: 799-802, 1977.

11. Hennessy, M. B. and S. Levine. Sensitive pituitary-adrenal responsiveness to varying intensities of psychological stimulation. Physiol. Behav. 21: 295--297, 1978. 12. Hennessy, J. W., R. Levin and S. Levine, Influence of experiential factors and gonadal hormones on pituitary-adrenal response of the mouse to novelty and electric shock. J. comp. physiol. Psychol. 91: 770--777, 1977. 13. Keppel, G. Design and Analysis: A Researcher's Handbook. New Jersey: Prentice Hall, 1973. 14. Knigge, K. M., C. H. Penrod and J. Schindler. In vitro and in vivo adrenal corticosterone secretion following stress. Am. J. Physiol. 196: 579--582, 1959. 15. LaPlanta, C. C., C. J. Geroud and J. Stachenko. Lack of appreciable 17-hydroxylase activity in the normal and regenerated rat adrenal tissues. Endocrinology 75: 825-827, 1964. 16. Mason, J. M. A review of psychoendocrine research on the pituitary adrenal cortical system. Psychosom. Med. 30: 576607, 1968. 17. Mason, J. M. A re-evaluation of the concept of non-specificity in stress theory. J. psychiat. Res. g: 323--333, 1971. 18. Mattingley, D. A. A simple fluorimetric method for estimation of free ll-hydroxycorticosteroids in human plasma. J. olin. Pathol. 15: 374--379, 1962. 19. Ota, K., T. Ota and A. Yokoyama. Plasma corticosterone concentrations and pituitary prolactin content in late pregnancy and their within-day fluctuations in the rat. J. Endocr. 61: 21-28, 1974. 20. Peron, F. G. The isolation and identification of some adrenocorticosteroids released by the rat adrenal tissue incubated in vitro. Endocrino!ogy 66: 458--469, 1960. 21. Pfister, H. P. and M. G. King. Adaptation of the glucocorticosterone response to novelty. Physiol. Behm'. 17: 43-46, 1976. 22. Thompson, W. R. Influence of prenatal maternal anxiety on emotionality in young rats. Science 125: 698-699, 1957.