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Individual housing does not influence the adaptation of the pituitary-adrenal axis and other physiological variables to chronic stress in adult male rats
Physiology & Behavior, Vol. 45, pp. 477-481. ©Pergamon Press pie, 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Individual Housing Does not Influence the Adaptation of the Pituitary-Adrenal Axis and Other Physiological Variables to Chronic Stress in Adult Male Rats MERCE
GIRALT
AND ANTONIO
ARMARIO
1
D e p a r t a m e n t o de Biologfa Celular y Fisiologia, F a c u l t a d de Ciencias Universidad A u t 6 n o m a de Barcelona, Bellaterra, Barcelona, Spain R e c e i v e d 28 M a y 1987 GIRALT, M. AND A. ARMARIO. Individual housing does not influence the adaptation of the pituitary-adrenal axis and other physiological variables to chronic stress in adult male rats. PHYSIOL BEHAV 45(3) 477-481, 1989.-Although the influence of housing conditions on the physiological response to stress has been extensively studied for several years, no attempts have been made to investigate the effect of this variable on the capacity for adaptation to chronic stress. To this end, adult male rats were housed either individually or in groups of four per cage and subjected to 2 hr of daily immobilization stress for 14 days. Housing did not influence any of the physiological variables measured either in unstressed or in stressed rats except the corticosterone response to stress which was higher in individually housed rats. Of the behavioral measures, individual housing significantly decreased defecation rate in the novel environment. Other behavioral measures were not influenced by housing. Chronic stress significantly reduced ambulation but no significant interaction between housing and chronic stress was observed. Taken together, these data indicate that a short period of individual housing did not affect the physiological and behavioral consequences of repeated exposure to chronic stress. Housing conditions Individual housing Food intake Body weight
Adaptation
Chronic stress
S O C I A L interaction can play an important role in the maintenance of normal behavior in some animals. Thus, the effect of individual housing (sometimes inappropriately termed isolation) on behavioral and physiological reactivity to novel stimuli has been extensively studied in rodents [see (9, 10, 28) for review]. It has been established in rats that individual housing from weaning to adulthood usually induces hyperactivity in novel environments (7, 11, 13, 17, 23, 27) and normal or altered physiological reactivity to aversive stimuli [see (30)]. Shorter periods of individual housing are often required in some experimental designs. For this reason, attention has been paid to the influence of maintaining adult animals individually for a short period o f time (usually a few weeks) on behavioral and physiological reactivity to aversive stimuli. No consistent effect of housing conditions has been reported (4, 8, 12, 14, 21, 22, 24, 25). More specifically it has been usually found that individually housed rats show normal or reduced pituitary-adrenal (PA) response to acute stress (4, 8, 12, 21). However, to our knowledge possible effects of individual housing on behavioral and physiological response to chronic stress have not been investigated.
Immobilization
Pituitary-adrenal axis
Previously, we considered the possibility (16) that some discrepancies between experiments investigating the effects of chronic shock on the defecation rate in a novel environment could be due to the different housing conditions used in the studies (16,20), the individually-housed rats being more susceptible to chronic stress than the group-housed rats. To test this hypothesis, we compared the response to chronic stress in individually and group-housed rats. Both behavioral (food intake, exploratory activity) and physiological (body weight changes, pituitary-adrenal activity) variables were assessed. METHOD Male Sprague-Dawley rats approximately 50 days old upon their arrival at the laboratory were used. They were housed four per cage ( 4 8 x 2 3 x 1 4 cm) in a controlled environment (lights on from 07.00 to 19.00 hr temperature 22°C) for 7 days before the experiment. Then they were weighed (average body weight was 275 g) and randomly assigned to four groups as follows: 15 rats to individual housing without stress, 36 rats to group housing (4 rats per cage) without stress, 15 rats to indi-
I Requests for reprints should be addressed to Antonio Armario
477
478
100
GIRALT AND A RM A R I O
FOOD INTAKE
~t
BODY WEIGHT CHANGES
80 6o
FIG. 1. Effect of housing condition on food intake during chronic immobilization. Means and SEM (n=9) of the percentage of food ingested by each group with respect to the corresponding unstressed group are indicated. The means of the food intake of unstressed rats were 26.0 and 26.1 g/rat/day for individually and group-housed rats, respectively, ~ p<0.001 vs. their respective unstressed group. Open circles indicate group-housed and closed circles individually-housed rats.
I.~" ~
_.,,~*
-20
3 vidual housing with stress, and 36 to group housing with stress. Stress consisted of exposure to 2 hr of immobilization daily in the prone position by attaching the animals' fore and rearlimbs to metal mounts fixed to a board. Head motion was restricted by metal loops fixed over the neck area. Stress exposure began at 10 a.m. Food in pellets and tap water were available ad lib. Food intake was determined by measuring the difference between the amount of food put into the cages and that which remained at the designated times. In group-housed rats, body weight was controlled in one randomly chosen rat per cage and food intake, expressed as g/rat, was controlled in all cages. For individually-housed rats, body weight and food intake were controlled in 9 cages for each stress condition. Food intake and body weight changes were measured in the morning of the 3rd, 8th, 1 lth and 14th days. On day 13 the rats were tested for 4 rain in a holeboard apparatus (15). The test was carried out in the early morning before the stress exposure period. The platform of the holeboard was divided into 16 areas of approximately the same size in order to allow measurement of locomotor activity. The number of boluses produced (defecation), areas crossed, rears and head-dips were manually recorded. The time spent head-dipping was measured using a stopwatch. A dip was considered to have taken place when the introduction of the head into the holes was at least to the level of the eyes. After each test the holeboard was cleaned. On day 15, several rats from each group (n=7) were killed without stress. Other rats (n=8) were subjected to acute immobilization for 30 min and then killed. The remainder of the group-housed rats were used for other experimental purposes. Trunk blood was collected in plastic tubes maintained at 4°C to prevent A C T H degradation. The adrenals were weighed. ACTH and corticosterone were analyzed by radioimmunoassay as previously described (2). All samples were processed in the same assay to avoid interassay variations. The statistical significance of the results were evaluated by the Student's t-test or by ANOVA with housing conditions and chronic stress as main factors. Food intake and body weight gain were analyzed by a 2 x 2 x 4 ANOVA with repeated measures on the third factor. To achieve homogeneity of variances, ACTH and defecation data were subjected to log and log (x+l) transformations, respectively.
6
9
12 DAYS 15
FIG. 2. Effect of housing conditions on body weight changes during chronic immobilization. Means and SEM (n=9) are represented. Squares indicate nonchronically stressed rats and circles chronically immobilized rats. Open symbols indicate group-housed and closed symbols individually-housed rats. *p<0.001 vs. their respective unstressed group.
RESULTS Three-way ANOVA revealed that chronic immobilization stress (p<0.001), but not housing conditions, had a significant effect on food intake. The effect of time as well as the interactions of time by housing and time by stress were significant (p<0.001 in all cases). No differences in food intake were observed between individually and group-housed rats in unstressed conditions and food intake remained essentially constant over all the experimental period. Figure 1 shows the changes in food intake during the stress period expressed as a percentage of the corresponding unstressed group. ANOVA revealed that chronic stress (p<0.001), but not housing conditions had a significant effect on body weight gain (Fig. 2). The interaction of time by stress was significant (p<0.001) while time by housing was not. Two-way ANOVA revealed a significant effect of chronic stress on relative adrenal weight (p<0.001). Similar results were obtained with regard to basal serum corticosterone (p<0.001). However, no effect of housing on the two variables was observed (Table 1). Corticosterone response to acute immobilization was significantly greater in individually-housed than in group-housed rats (p<0.05), but the hormone levels were not influenced by previous chronic immobilization (Fig. 3). ANOVA showed a significant effect of chronic stress (p<0.001) but not of housing on the ACTH response to acute immobilization. Thus, the two experimental groups showed the same decrease in the A C T H response to acute immobilization after previous chronic exposure to the same stressor (Fig. 3). The behavioral response to the novel environment (holeboard) in the two housing conditions is depicted in Table 2. Two-way ANOYA revealed a significant effect of housing conditions (p
I N D I V I D U A L H O U S I N G A N D A D A P T A T I O N TO STRESS
479
DIU
TABLE 1 THE EFFECT OF HOUSING CONDITIONS AND CHRONIC IMMOBILIZATIONSTRESS ON ADRENAL WEIGHT AND BASAL SERUM CORTICOSTERONELEVELS Adrenal Weight mg/100 g b.wt.
Corticosterone ~tg/dl
Individual no stress (n--7)
13.1 _+0.8
2.9_+0.3
Individual stress (n=6)
20.4 _+1.3
7.5 -+2.5
Group no stress (n=7)
15.9+_ 1. l
2.9_+0.3
Group stress (n=7)
19.7_+1.7
8.9_+2.4
Treatment
Mean_+SEM are represented. The number of animals per group is in parentheses. ANOVA revealed a significant effect of chronic immobilization but not of housing conditions on the two variables.
Ills Dau los
I
"~
ao
influenced by housing. The only significant effect o f chronic stress was on ambulation ( p < 0 . 0 5 ) . The interaction of housing conditions by chronic stress was always nonsignificant. DISCUSSION In unstressed conditions, individual housing did not influence either food intake or body weight gain. Likewise, neither adrenal or thymus weights nor basal corticosterone levels were affected by housing. These data are in agreement with several reports [e.g., (25)], although conflicting results have often been reported [see (10) for review]. The reasons for the discrepancies between experiments are at present unclear. Nevertheless, the higher basal corticosterone levels often reported in grouphoused rats (8,14) may have been due to a stress on rats resulting from the removal o f a cage-mate. The A C T H response to acute stress, considered a good index of emotional reactivity (5), was equal in the two housing conditions. These results are consistent with a previous report from our laboratory in which adrenocortical response to 20-min exposure to a novel environment was assessed. It was found that adult rats housed individually for 20 days showed the same corticosterone response to the novel environment as grouphoused rats (4). In accordance with previous reports (3, 18, 26), the chronically immobilized rats showed reduced food intake and loss of body weight. These effects were more marked in the initial period of exposure to chronic stress. Subsequently, the animals showed signs of partial adaptation characterized by a less marked decrease of food intake and a recovery of body weight. Single housing did not modify either of the two variables. Similarly, increases in adrenal weight were the same in the two housing conditions. These data suggest that housing conditions did not influence adaptation. This hypothesis was confirmed by measurement o f the P A response to 30 min immobilization. This response was significantly lower in the chronically immobilized rats, but the reduction was similar in the two housing conditions. Despite a significant decrease in the A C T H response to immobilization in the chronically stressed rats, their corticosterone response was not modified by previous chronic
FIG. 3. Effect of housing conditions and chronic immobilization on the pituitary adrenal response to 30 min immobilization. Means and SEM (n=7-8) are represented. Groups are indicated as follows: individual housing not subjected to any stress (IU), group housing not subjected to any stress (GU), individual housing subjected to chronic immobilization (IS) and group housing subjected to chronic immobilization (GS). The ANOVA revealed a significant effect of housing conditions (p<0.05) but not of chronic stress on the corticosterone response to acute immobilization. A significant effect of chronic stress (/7<0.001) but not of housing conditions on the ACTH response was found.
stress. This apparently surprising effect has been previously observed in our laboratory (3), and was most likely due to the increased adrenocortical responsiveness to circulating A C T H developed in chronically immobilized rats (3). Unexpectedly the corticosterone response to immobilization was slightly greater in individually-housed than in group-housed rats. Since A C T H response to stress was similar in the two experimental groups, it appears that individual housing might enhance adrenocortical responsiveness to A C T H , without a concomittant increase in adrenal mass. In the present experiment, individually housed rats showed a lower defecation rate but normal activity (as measured by ambulation, rearing and head-dipping) in a novel environment. Chronic immobilization stress reduced ambulation only, but no interaction with housing was found. The significance o f the reduced defecation rate of individually-housed rats is uncertain because neither exploratory activity nor the A C T H response to stress were found altered in them. Therefore it appears that housing did not alter emotional reactivity. The influence of individual housing on emotional reactivity in adult rats is controversial. Inconsistent results have been reported regarding the
480
GIRALT AND ARMARIO TABLE 2 EFFECT OF HOUSING CONDITIONS AND CHRONIC IMMOBILIZATION STRESS ON BEHAVIORAL PERFORMANCES IN THE HOLE BOARD
Treatment
Defecation (number)
Ambulation (number)
Rearing (number)
Head-Dips (number)
Head-Dipping (sec)
Individual no stress
0.1_+0.1
57.1+_5.4
10.0+2.8
7.7_+1.3
15.8_+5.9
Individual stress
1.7+_0.8
49.7+_7.1
11.6+_2.0
5.9+_0.9
7.5_+1.5
Group no stress
2.6_+0.9
78,0+_3.5
20.1_+3.4
7,3_+0.9
10.9_+1.2
Group stress
2.4+-1.0
48.1+8.9
11.6+_1.8
7,6_+1.1
11.1_+1.6
Means+SEM (n=7) are represented. The results of the ANOVA of the variables are indicated in the text.
differences between individually- a n d g r o u p - h o u s e d rats in l o c o m o t o r a n d / o r exploratory activities (19, 24, 25). T h e discrepancies between experiments apparently cannot be explained by the d u r a t i o n of the individual housing. It is n o t e w o r t h y t h a t other models o f chronic stress, using milder stressors t h a n that used in the present experiment as evidenced by the lesser increase in adrenal weight (6), were able to reduce all measures of exploratory activity in rats (16). These
models of chronic stress were characterized by some degree of unpredictability. Therefore, it seems that factors other than the intensity o f stress, as assessed by the P A activity, might be responsible for the reduction o f exploratory activity observed with o t h e r chronic stress models. In conclusion, the present data suggest that individual housing did n o t exert any m a j o r effect o n behavioral a n d physiological responses to chronic stress in adult male rats.
REFERENCES 1. Ader, R.; Plaut, S. M. Effect of prenatal maternal handling and differential housing on offspring emotionality, plasma corticosterone levels, and susceptibility to gastric erosions. Psychosom. Med. 30:277-286; 1968. 2. Armario, A.; Castellanos, J. M.; Balasch, J. Effect of crowding on emotional reactivity in adult male rats. Neuroendocrinology 39: 330-333; 1984. 3. Armario, A.; Hidalgo, J.; Giralt, M. Evidence that the pituitaryadrenal axis does not cross-adapt to stressors: comparison to other physiological variables. Neuroendocrinology 47:263-267; 1988, 4. Armario, A.; Luna, G.; Balasch, J. The effect of conspecifics on corticoadrenal response of rats to a novel environment. Behav. Neural Biol. 37:332-337; 1983. 5. Armario, A.; Montero, J. L.; Balasch, J. Sensitivity of corticosterone and some metabolic variables to graded levels of low intensity stresses in adult male rats. Physiol. Behav. 37:559-561; 1986. 6. Armario, A.; Restrepo, C.; Castellanos, J. M.; Balasch, J. Dissociation between adrenocorticotropin and corticosterone responses to restraint after previous chronic exposure to stress. Life Sci. 36: 2085-2092; 1985. 7. Baenninger, L. P. Comparison of behavioural development in socially isolated and grouped rats. Anita. Behav. 15:312-323; 1967. 8. Barrett, A. M.; Stockham, M. A. The effect of prior housing conditions and simple experimental procedures upon the corticosterone level in the plasma of rats. J. Endocrinol. 26:97-105; 1963. 9. Brain, P. F. What does individual housing mean to a mouse? Life Sci. 16:187-200; 1975. 10. Brain, P. F.; Benton, D. The interpretation of physiological correlates of differential housing in laboratory rats. Life Sci. 24:99-116; 1979.
11. Einon, D. F.; Morgan, M. J.; Sahakian, B. J. The development of intersession habituation and emergence in socially reared and isolated rats. Dev. Psychobiol. 8:553-559; 1975. 12. Fagin, F. D.; Shinsako, J.; Dallman, M. F. Effects of housing and chronic cannulation on plasma ACTH and corticosterone in the rat. Am. J. Physiol. 240:RI66-R172; 1983. 13. File, S. E. Exploration, distraction, and habituation in rats reared in isolation. Dev. Psychobiol. 11:73-81; 1978. 14. File, S. E.; Peet, L. A. The sensitivity of the rat corticosterone response to environmental manipulations and to chronic chlordiazepoxide treatment. Physiol. Behav. 25:753-758; 1980. 15. File, S. E.; Wardill, A. G. Validity of head-dipping as a measure of exploration in a modified holeboard. Psychopharmacologia 44: 53-59; 1975. 16. Garcia-M~irquez, C.; Armario, A. Chronic stress depresses exploratory activity and behavioral performance in the forced swimming test without altering ACTH response to a novel acute stressor. Physiol. Behav. 40:33-38; 1987. 17. Gentsch, C.; Lichtsteiner, M.; Feer, H. Locomotor activity, defecation score and corticosterone levels during an openfield exposure: A comparison among individually and group-housed rats and genetically selected rat lines. Physiol. Behav. 27:183-186; 1981. 18. Giralt, M.; Garcia-M~rquez, C.; Armario, A. Previous chronic ACTH administration does not protect against the effects of acute or chronic stress in male rats. Physiol. Behav. 40:165-170; 1987. 19. Hahn, W. H. Some effects of group size on behavior and physiology of the rat. J. Psychosom. Res. 8:455-465; 1965. 20. Levine, S.; Madden, J., IV; Conner, R. L.; Moskal, J. R.; Anderson, C. Physiological and behavioral effects of prior aversive stimulation (preshock) in the rat. Physiol. Behav. 10:467-471; 1973.
INDIVIDUAL HOUSING AND ADAPTATION TO STRESS
21. Lovely, R. H.; Pagano, R. R. Shuttle-box-avoidance performance and basal corticosterone levels as a function of duration of individual housing in rats. J. Comp. Physiol. Psychol. 81:331-335; 1972. 22. Par6, W. P.; Valdsar, E. The effects of housing and preshock on activity-stress ulcer. Physiol. Psychol. 13:33-36; 1985. 23. Sahakian, B. J.; Robbins, T. W.; Iversen, S. D. The effect of isolation rearing on exploration in the rat. Anim. Learn. Behav. 5: 193-198; 1977. 24. Segal, D. S.; Knapp, S.; Kuczenski, R. T.; Mandell, A. J. The effects of environmental isolation on behavior and regional brain tyrosine hydroxylase and tryptophan hydroxylase activities. Behav. Biol. 8:47-53; 1973.
481 25. Stern, J. A.; Winokur, G.; Einsenstein, A.; Taylor, R.; Sly, M. The effect of group vs. individual housing on behaviour and physiological responses to stress in the albino rats. J. Psychosom. Res. 4:185-190; 1960. 26. Stone, E. A.; Platt, J. E. Brain adrenergic receptors and resistance to stress. Brain Res. 237:405-414; 1982. 27. Syme, L. A. Social isolation at weaning: some effects on two measures of activity. Anim. Learn. Behav. 1:161-163; 1973. 28. Valzelli, L. The isolation syndrome in mice. Psychopharmacologia 31:305-320; 1973.
0031-9384/89 $3.00 + .00
Individual Housing Does not Influence the Adaptation of the Pituitary-Adrenal Axis and Other Physiological Variables to Chronic Stress in Adult Male Rats MERCE
GIRALT
AND ANTONIO
ARMARIO
1
D e p a r t a m e n t o de Biologfa Celular y Fisiologia, F a c u l t a d de Ciencias Universidad A u t 6 n o m a de Barcelona, Bellaterra, Barcelona, Spain R e c e i v e d 28 M a y 1987 GIRALT, M. AND A. ARMARIO. Individual housing does not influence the adaptation of the pituitary-adrenal axis and other physiological variables to chronic stress in adult male rats. PHYSIOL BEHAV 45(3) 477-481, 1989.-Although the influence of housing conditions on the physiological response to stress has been extensively studied for several years, no attempts have been made to investigate the effect of this variable on the capacity for adaptation to chronic stress. To this end, adult male rats were housed either individually or in groups of four per cage and subjected to 2 hr of daily immobilization stress for 14 days. Housing did not influence any of the physiological variables measured either in unstressed or in stressed rats except the corticosterone response to stress which was higher in individually housed rats. Of the behavioral measures, individual housing significantly decreased defecation rate in the novel environment. Other behavioral measures were not influenced by housing. Chronic stress significantly reduced ambulation but no significant interaction between housing and chronic stress was observed. Taken together, these data indicate that a short period of individual housing did not affect the physiological and behavioral consequences of repeated exposure to chronic stress. Housing conditions Individual housing Food intake Body weight
Adaptation
Chronic stress
S O C I A L interaction can play an important role in the maintenance of normal behavior in some animals. Thus, the effect of individual housing (sometimes inappropriately termed isolation) on behavioral and physiological reactivity to novel stimuli has been extensively studied in rodents [see (9, 10, 28) for review]. It has been established in rats that individual housing from weaning to adulthood usually induces hyperactivity in novel environments (7, 11, 13, 17, 23, 27) and normal or altered physiological reactivity to aversive stimuli [see (30)]. Shorter periods of individual housing are often required in some experimental designs. For this reason, attention has been paid to the influence of maintaining adult animals individually for a short period o f time (usually a few weeks) on behavioral and physiological reactivity to aversive stimuli. No consistent effect of housing conditions has been reported (4, 8, 12, 14, 21, 22, 24, 25). More specifically it has been usually found that individually housed rats show normal or reduced pituitary-adrenal (PA) response to acute stress (4, 8, 12, 21). However, to our knowledge possible effects of individual housing on behavioral and physiological response to chronic stress have not been investigated.
Immobilization
Pituitary-adrenal axis
Previously, we considered the possibility (16) that some discrepancies between experiments investigating the effects of chronic shock on the defecation rate in a novel environment could be due to the different housing conditions used in the studies (16,20), the individually-housed rats being more susceptible to chronic stress than the group-housed rats. To test this hypothesis, we compared the response to chronic stress in individually and group-housed rats. Both behavioral (food intake, exploratory activity) and physiological (body weight changes, pituitary-adrenal activity) variables were assessed. METHOD Male Sprague-Dawley rats approximately 50 days old upon their arrival at the laboratory were used. They were housed four per cage ( 4 8 x 2 3 x 1 4 cm) in a controlled environment (lights on from 07.00 to 19.00 hr temperature 22°C) for 7 days before the experiment. Then they were weighed (average body weight was 275 g) and randomly assigned to four groups as follows: 15 rats to individual housing without stress, 36 rats to group housing (4 rats per cage) without stress, 15 rats to indi-
I Requests for reprints should be addressed to Antonio Armario
477
478
100
GIRALT AND A RM A R I O
FOOD INTAKE
~t
BODY WEIGHT CHANGES
80 6o
FIG. 1. Effect of housing condition on food intake during chronic immobilization. Means and SEM (n=9) of the percentage of food ingested by each group with respect to the corresponding unstressed group are indicated. The means of the food intake of unstressed rats were 26.0 and 26.1 g/rat/day for individually and group-housed rats, respectively, ~ p<0.001 vs. their respective unstressed group. Open circles indicate group-housed and closed circles individually-housed rats.
I.~" ~
_.,,~*
-20
3 vidual housing with stress, and 36 to group housing with stress. Stress consisted of exposure to 2 hr of immobilization daily in the prone position by attaching the animals' fore and rearlimbs to metal mounts fixed to a board. Head motion was restricted by metal loops fixed over the neck area. Stress exposure began at 10 a.m. Food in pellets and tap water were available ad lib. Food intake was determined by measuring the difference between the amount of food put into the cages and that which remained at the designated times. In group-housed rats, body weight was controlled in one randomly chosen rat per cage and food intake, expressed as g/rat, was controlled in all cages. For individually-housed rats, body weight and food intake were controlled in 9 cages for each stress condition. Food intake and body weight changes were measured in the morning of the 3rd, 8th, 1 lth and 14th days. On day 13 the rats were tested for 4 rain in a holeboard apparatus (15). The test was carried out in the early morning before the stress exposure period. The platform of the holeboard was divided into 16 areas of approximately the same size in order to allow measurement of locomotor activity. The number of boluses produced (defecation), areas crossed, rears and head-dips were manually recorded. The time spent head-dipping was measured using a stopwatch. A dip was considered to have taken place when the introduction of the head into the holes was at least to the level of the eyes. After each test the holeboard was cleaned. On day 15, several rats from each group (n=7) were killed without stress. Other rats (n=8) were subjected to acute immobilization for 30 min and then killed. The remainder of the group-housed rats were used for other experimental purposes. Trunk blood was collected in plastic tubes maintained at 4°C to prevent A C T H degradation. The adrenals were weighed. ACTH and corticosterone were analyzed by radioimmunoassay as previously described (2). All samples were processed in the same assay to avoid interassay variations. The statistical significance of the results were evaluated by the Student's t-test or by ANOVA with housing conditions and chronic stress as main factors. Food intake and body weight gain were analyzed by a 2 x 2 x 4 ANOVA with repeated measures on the third factor. To achieve homogeneity of variances, ACTH and defecation data were subjected to log and log (x+l) transformations, respectively.
6
9
12 DAYS 15
FIG. 2. Effect of housing conditions on body weight changes during chronic immobilization. Means and SEM (n=9) are represented. Squares indicate nonchronically stressed rats and circles chronically immobilized rats. Open symbols indicate group-housed and closed symbols individually-housed rats. *p<0.001 vs. their respective unstressed group.
RESULTS Three-way ANOVA revealed that chronic immobilization stress (p<0.001), but not housing conditions, had a significant effect on food intake. The effect of time as well as the interactions of time by housing and time by stress were significant (p<0.001 in all cases). No differences in food intake were observed between individually and group-housed rats in unstressed conditions and food intake remained essentially constant over all the experimental period. Figure 1 shows the changes in food intake during the stress period expressed as a percentage of the corresponding unstressed group. ANOVA revealed that chronic stress (p<0.001), but not housing conditions had a significant effect on body weight gain (Fig. 2). The interaction of time by stress was significant (p<0.001) while time by housing was not. Two-way ANOVA revealed a significant effect of chronic stress on relative adrenal weight (p<0.001). Similar results were obtained with regard to basal serum corticosterone (p<0.001). However, no effect of housing on the two variables was observed (Table 1). Corticosterone response to acute immobilization was significantly greater in individually-housed than in group-housed rats (p<0.05), but the hormone levels were not influenced by previous chronic immobilization (Fig. 3). ANOVA showed a significant effect of chronic stress (p<0.001) but not of housing on the ACTH response to acute immobilization. Thus, the two experimental groups showed the same decrease in the A C T H response to acute immobilization after previous chronic exposure to the same stressor (Fig. 3). The behavioral response to the novel environment (holeboard) in the two housing conditions is depicted in Table 2. Two-way ANOYA revealed a significant effect of housing conditions (p
I N D I V I D U A L H O U S I N G A N D A D A P T A T I O N TO STRESS
479
DIU
TABLE 1 THE EFFECT OF HOUSING CONDITIONS AND CHRONIC IMMOBILIZATIONSTRESS ON ADRENAL WEIGHT AND BASAL SERUM CORTICOSTERONELEVELS Adrenal Weight mg/100 g b.wt.
Corticosterone ~tg/dl
Individual no stress (n--7)
13.1 _+0.8
2.9_+0.3
Individual stress (n=6)
20.4 _+1.3
7.5 -+2.5
Group no stress (n=7)
15.9+_ 1. l
2.9_+0.3
Group stress (n=7)
19.7_+1.7
8.9_+2.4
Treatment
Mean_+SEM are represented. The number of animals per group is in parentheses. ANOVA revealed a significant effect of chronic immobilization but not of housing conditions on the two variables.
Ills Dau los
I
"~
ao
influenced by housing. The only significant effect o f chronic stress was on ambulation ( p < 0 . 0 5 ) . The interaction of housing conditions by chronic stress was always nonsignificant. DISCUSSION In unstressed conditions, individual housing did not influence either food intake or body weight gain. Likewise, neither adrenal or thymus weights nor basal corticosterone levels were affected by housing. These data are in agreement with several reports [e.g., (25)], although conflicting results have often been reported [see (10) for review]. The reasons for the discrepancies between experiments are at present unclear. Nevertheless, the higher basal corticosterone levels often reported in grouphoused rats (8,14) may have been due to a stress on rats resulting from the removal o f a cage-mate. The A C T H response to acute stress, considered a good index of emotional reactivity (5), was equal in the two housing conditions. These results are consistent with a previous report from our laboratory in which adrenocortical response to 20-min exposure to a novel environment was assessed. It was found that adult rats housed individually for 20 days showed the same corticosterone response to the novel environment as grouphoused rats (4). In accordance with previous reports (3, 18, 26), the chronically immobilized rats showed reduced food intake and loss of body weight. These effects were more marked in the initial period of exposure to chronic stress. Subsequently, the animals showed signs of partial adaptation characterized by a less marked decrease of food intake and a recovery of body weight. Single housing did not modify either of the two variables. Similarly, increases in adrenal weight were the same in the two housing conditions. These data suggest that housing conditions did not influence adaptation. This hypothesis was confirmed by measurement o f the P A response to 30 min immobilization. This response was significantly lower in the chronically immobilized rats, but the reduction was similar in the two housing conditions. Despite a significant decrease in the A C T H response to immobilization in the chronically stressed rats, their corticosterone response was not modified by previous chronic
FIG. 3. Effect of housing conditions and chronic immobilization on the pituitary adrenal response to 30 min immobilization. Means and SEM (n=7-8) are represented. Groups are indicated as follows: individual housing not subjected to any stress (IU), group housing not subjected to any stress (GU), individual housing subjected to chronic immobilization (IS) and group housing subjected to chronic immobilization (GS). The ANOVA revealed a significant effect of housing conditions (p<0.05) but not of chronic stress on the corticosterone response to acute immobilization. A significant effect of chronic stress (/7<0.001) but not of housing conditions on the ACTH response was found.
stress. This apparently surprising effect has been previously observed in our laboratory (3), and was most likely due to the increased adrenocortical responsiveness to circulating A C T H developed in chronically immobilized rats (3). Unexpectedly the corticosterone response to immobilization was slightly greater in individually-housed than in group-housed rats. Since A C T H response to stress was similar in the two experimental groups, it appears that individual housing might enhance adrenocortical responsiveness to A C T H , without a concomittant increase in adrenal mass. In the present experiment, individually housed rats showed a lower defecation rate but normal activity (as measured by ambulation, rearing and head-dipping) in a novel environment. Chronic immobilization stress reduced ambulation only, but no interaction with housing was found. The significance o f the reduced defecation rate of individually-housed rats is uncertain because neither exploratory activity nor the A C T H response to stress were found altered in them. Therefore it appears that housing did not alter emotional reactivity. The influence of individual housing on emotional reactivity in adult rats is controversial. Inconsistent results have been reported regarding the
480
GIRALT AND ARMARIO TABLE 2 EFFECT OF HOUSING CONDITIONS AND CHRONIC IMMOBILIZATION STRESS ON BEHAVIORAL PERFORMANCES IN THE HOLE BOARD
Treatment
Defecation (number)
Ambulation (number)
Rearing (number)
Head-Dips (number)
Head-Dipping (sec)
Individual no stress
0.1_+0.1
57.1+_5.4
10.0+2.8
7.7_+1.3
15.8_+5.9
Individual stress
1.7+_0.8
49.7+_7.1
11.6+_2.0
5.9+_0.9
7.5_+1.5
Group no stress
2.6_+0.9
78,0+_3.5
20.1_+3.4
7,3_+0.9
10.9_+1.2
Group stress
2.4+-1.0
48.1+8.9
11.6+_1.8
7,6_+1.1
11.1_+1.6
Means+SEM (n=7) are represented. The results of the ANOVA of the variables are indicated in the text.
differences between individually- a n d g r o u p - h o u s e d rats in l o c o m o t o r a n d / o r exploratory activities (19, 24, 25). T h e discrepancies between experiments apparently cannot be explained by the d u r a t i o n of the individual housing. It is n o t e w o r t h y t h a t other models o f chronic stress, using milder stressors t h a n that used in the present experiment as evidenced by the lesser increase in adrenal weight (6), were able to reduce all measures of exploratory activity in rats (16). These
models of chronic stress were characterized by some degree of unpredictability. Therefore, it seems that factors other than the intensity o f stress, as assessed by the P A activity, might be responsible for the reduction o f exploratory activity observed with o t h e r chronic stress models. In conclusion, the present data suggest that individual housing did n o t exert any m a j o r effect o n behavioral a n d physiological responses to chronic stress in adult male rats.
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INDIVIDUAL HOUSING AND ADAPTATION TO STRESS
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