Effects of housing on corticosterone rhythm and stress responses in female mice

Physiology & Behavior, Vol. 27, pp. 1-5. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.

Effects of Housing on Corticosterone Rhythm and Stress Responses in Female Mice D. J. N I C H O L S

A N D P. F. D. C H E V I N S

Department of Biological Sciences, University of Keele, Keele Staffordshire, ST5 5BG, England R e c e i v e d 16 S e p t e m b e r 1980 N I C H O L S , D. J. A N D P. F. D. C H E V I N S . Effects o f housing on corticosterone rhythm and stress responses in female

mice. PHYSIOL. BEHAV. 27(1) 1-5, 1981.--In a longitudinal study of grouping of virgin female mice, basal plasma corticosterone levels were significantly higher at 1 hour, 5 hours and 9 hours before lights off when the animals were grouped than when individually housed. There were no differences 3 hours, 7 hours and 11 hours after lights off. A week of daily vaginal smearing at the expected circadian peak elevated plasma corticosterone at that time of day but not at other times. A circadian rhythm of basal plasma corticosterone with highest values 1 hour before lights out was demonstrated, but there were no apparent differences in the rhythm phase in differentially housed mice. Group housed females, blood sampled and ether stressed, but not mice only ether stressed, had significantly higher plasma corticosterone levels than individually housed mice. There was no circadian rhythm in stress levels. Corticosterone

Circadian rhythm

T H E circadian rhythm of pituitary-adrenal function is considered endogenous and is usually entrained to the light-dark regime [ 19]. However many factors can alter both the steroid response at different times of the day and the phase of the rhythm. Thus food and water deprivation can alter the phasing of plasma corticosterone [17, 23, 24, 44] as can acute and chronic stressors [6,29]. Likewise reproductive condition can alter the adrenal rhythm. Male and female rats have different phasing of corticotrophin-releasing factor (CRF) [22] and ovariectomy advances the peak of CRF. Also it has been shown that pregnant rats have a longer peak of corticosterone [18], lactating rats have no rhythm [13] and infertile mice and rats may have no rhythm (or desynchronised free-running rhythms) [29,31]. Since reproductive condition can alter rhythmic pituitary adrenal function and social factors including housing conditions strongly influence reproductive function in female mice [43] we have examined the plasma corticosterone in individually and group housed females. With the development of a method for repeated blood sampling it has been possible to carry out a longitudinal study of mice subjected to different housing conditions. We have shown elsewhere [28] that basal plasma corticosterone levels o f animals at proestrus are elevated well above those at other stages of the cycle. Knowledge of the estrous cycle is thus important in studies o f plasma corticosterone, and as daily handling or disturbance can alter adrenal function [21, 25, 27, 29, 31, 36] we have also examined the effect of daily vaginal smearing. Apart from the circadian rhythm of basal adrenocortical secretion, pituitary adrenal responsiveness to stress can vary throughout the day. It has been shown with various stressors that a higher corticosterone response occurs at the circadian trough than at the peak [1, 2, 11, 12, 20, 39-41]. In contrast, others have shown the same response at peak and trough [32,45]. Brain and co-workers [4, 5, 15, 16] have argued that

Stress

Housing

Isolation

individually housed females may be more stress-responsive than group-housed females. It is not known whether this difference is apparent throughout the day. METHOD

Sixty virgin TO females housed in 4s at weaning were randomly assigned to 12 groups of 5 animals at the age of 86 days and rehoused in single cages. Twelve days later blood sampling commenced at l 1 a.m., then at 4 hourly intervals for 48 hours. Ten animals were blood sampled under unstressed conditions (i.e. within 3 rain of disturbance) at each time point. Each animal was sampled twice, the second sample being 12 or 36 hours after the first (see Table 1). A vaginal smear was taken after each blood sample then daily (between 9 a.m.-12 a.m.) for 6 days. At the end of this time the blood sampling procedure was repeated starting at l 1 a.m. with the same mice being sampled at the same times as previously sampled. A third blood sample (for stress levels) was taken from each mouse 15 min after the second. All the animals were then rehoused into 6 groups of 10 animals in large cages ensuring that blood samples could be taken from the groups of 10 mice at the same times of the day as before without disturbing their cage for 12 hours prior to blood sampling. Seventeen days after grouping all the mice were blood sampled at the same times as used previously. They were smeared after each blood sample and then daily between 9 a.m. and 12 a.m. for 6 days. Resampling took place, including a third blood sample 15 min after the second. To summarise, a total of 10 blood samples have been taken from each mouse. Two blood samples from unstressed mice, 12 hours (or 36 hr) apart were taken from animals whilst in four experimental conditions; individually housed (I), individually housed and smeared (Is), group housed (G) and group housed and smeared (Gs). In addition two stress samples were taken, one at the end of individual housing and

C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s

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NICH()I~S AND CHt;VINS 'FABLE 1

T I M E T A B L E S H O W I N G S E Q U E N C E O F B L O O D SAMPLES, V A G I N A L SMEARING AND H O U S I N G C H A N G E S FOR O N I Ol, I'HI, I W E I \'~ G R O U P S O F MICE IN E X P E R I M E N T 1 Blood sample no.

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The sequence shown above was conducted for 12 separate groups of animals, at 6 times of day. After sample 5 the mice in each pair of groups being sampled at the same time of day were put into a single large cage.

,4, Group of lO, sme~red ~-7~ rou0 o, ,o, .~d.-~ Individually tlou,s~_____l~-~l ana s m e a r ea-~--~j..-~ Individually h o u s e d ~ ,

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FIG. 1. Plasma corticosterone levels throughout the day in female mice subjected to different conditions of housing and to vaginal smearing.

one at the end of group housing. Different groups of animals were sampled to give values at 4 hourly intervals throughout 48 hours. In a second experiment 40 virgin TO females aged 120 days, housed in 4s from weaning were randomly assigned to four experimental groups, two groups of 10 per cage and two groups of 10 individually housed. All animals were undisturbed for 18 or 19 days then blood sampled at 11 a.m. One group of 10 and 10 individually housed mice were sampled within 3 min of disturbance (i.e. unstressed) then resampled 15 min later (stressed sample). The other two groups of mice were etherised only (until unconscious) then blood sampled 15 rain later. Individually housed animals were kept in opaque plastic cages 30 x 13 x 11 cm and grouped animals in similar cages sized 42 x 26 x 11 cm. Cages had wire tops with ad lib supply of food and water. The temperature was controlled at (18-23°C) and reversed lighting used, with 14 hour light, 10

hours dim red light (lights on at 10 p.m.). All experimental animals were housed in a quiet, "male-smell free" room with air extraction and were undisturbed except for weekly bedding changes until used in experiments. Blood sampling was accomplished by retro-orbital puncture [33] under rapid ether anesthesia. Less than 400/zl of blood was taken from any animal. Cages were quietly removed from the animal house to an adjacent room and blood sampling was completed within 3 minutes of entering the room. The animal room was not entered more than 5 times per sampling session. Blood was centrifuged within 45 minutes of sampling and plasma frozen and stored at -20°C until assayed. Individual plasma samples were assayed by RIA using anti-corticosterone-thyroglobulinantisera (MilesYeda) [28]. Recovery of corticosterone added to plasma is 94%+_4.8%, n=34. The limit of detection is less than 5 ng/ml and interassay variation averages -+9.1%. Vaginal smears were taken, stained, and staged as described previously [28].

CORTICOSTERONE RHYTHM AND STRESS

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FIG. 2. Basal and stress corticosterone levels throughout the day in female mice subjected to different housing conditions.

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n RESULTS Mean basal plasma corticosterone levels for the first experiment are shown in Fig. 1. Friedman ANOVA revealed significant differences between the four conditions I, Is, G and Gs at 3 a.m., 7 a.m. and 11 a.m. (O<0.01; p<0.01; p<0.001) but not at 3 p.m., 7 p.m. or 11 p.m. Further analysis using Wilcoxon test revealed significant differences between individually housed (I) and group housed (G) at 3 a.m., 7 a.m. and 11 a.m. (p<0.01; p<0.01; p<0.005) and between individually housed and smeared and group housed and smeared at 3 a.m., 7 a.m. and 11 a.m. (O<0.025; p<0.005; p <0.025). Smearing had a significant effect only at 7 a.m. in group housed mice and at 11 a.m. in individually housed mice (.o<0.025; p<0.05). However at both these times unsmeared animals had lower values than the equivalent, smeared. Stressed and unstressed mean plasma corticosterone values for individually housed and smeared and group housed and smeared are shown in Fig. 2. Kruskal-Wallis ANOVA of stress values throughout the day reveal no significant difference in either grouped (p<0.1) or individually housed (p<0.3) implying an absence of circadian rhythm in stress levels. However Wilcoxon signed rank test for differences between matched pairs revealed a highly significant difference between individually and group housed animals (p <0.00001). Table 2 shows that, as expected, when housed individually the mice showed predominantly short estrous cycles of 6 days or less, but when group-housed, cycles lengthened. This difference was highly significant whether judged according to cycle length (O<0.001: Chi square) or mean estrus frequency (p <0.001: Wilcoxon). Mean plasma corticosterone values for experiment 2 are shown in Fig. 3. There are no significant differences between individually and group housed unstressed or ether stress values. However in agreement with experiment 1 group-housed mice have a significantly higher stress level after blood sampiing and ether than individually housed mice (o<0.05).

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I G I G I G Unstressed Blood s~npled Ether and ether stress stress FIG. 3. Plasma corticosterone levels in female mice subjected to different housing conditions and kinds of stress.

TABLE 2 PROPORTION OF MICE SHOWINGSHORTAND LONG ESTRUS CYCLES, AND MEAN PROESTRUS/ESTRUSFREQUENCYIN INDIVIDUALAND GROUPHOUSING Individual Housing

Group Housing

Number of mice showing ~<6 day cycles

54

20

Number of mice showing long or abnormal cycles

6

40

1.87 +_ 0.08

1.03 ± 0.06

Mean number of proestrus or estrus smears in 9 days (±S.E.M.)

Long cycles are defined by 5 or more consecutive diestrus smears; abnormal cycles by 4 or more consecutive estrus smears.

4

NICH(!I.S ,~NI) ~I-qEVIN,'~ DISCUSSION

A major finding of the present study is that the raised basal plasma concentrations of corticosterone found by some authors [5,37] in group housed mice as compared with individually-housed, but not found by others [7,16] is present through the latter part of the animal's day, and especially just before lights off but is not apparent during the hours of darkness. In previous, unpublished studies, the present authors found such a difference at the circadian peak only after the elevated levels of proestrus were excluded from all the data. In this experiment, removal of proestrus data from the analysis does not affect the results as only a relatively small proportion of the individually housed animals (16%) were in proestrus. The significance of a difference in plasma corticosterone in differentially housed female mice at only some times of the day is unclear. It is possible that the higher basal levels at and before the circadian peak are related to increased social interaction in a group as postulated by Christian [8]. Hyperactivity of a single individual in a group cage could disturb the rest of the group, so giving very variable results from different cages. Alternatively the difference may not be a direct behavioural effect on pituitary-adrenal function but be mediated by the reproductive system. The argument that the difference found in basal levels may reflect methodological factors (e.g. [4, 15, 16]) such as previous disturbance, blood sampling, or housing conditions seems unlikely since elevation of values for grouped animals occurred at only three out of six times of the day. Further, we have shown that plasma corticosterone is unaffected by blood sampling 12 hours (Nichols and Chevins, unpublished data) or one week previously or by a change of housing and cage mates. One week of daily vaginal smearing at the circadian peak of adrenal activity did elevate plasma corticosterone slightly but only at and around this time of day. This is in agreement with others [21,36] who found that the pituitary-adrenal system in mice is sensitized by daily "training" rather than habituated to it. The presence of a circadian rhythm of basal plasma corticosterone in mice with highest values just before lights off [19, 37-40] is confirmed here. However neither housing nor smearing had an effect on the rhythm phase. Paris and Ramaley [29] found a change in phase of plasma corticostetone rhythms in mice as a result of hi-daily stress (but not

vaginal smearing) and noted altered ferlilil3. Ramaley !3t lbund that a large percentage of rats that had no apparenv corticosterone rhythm, also had irregular wlgmal smear c3 ties. It is well known that grouping femalc mice leads tt spontaneous pseudopregmmcy 134,421 and anoestrus [43i. Both long and abnormal cycles were apparent in our grouped mice, but the evidence presented here indicates lhal this altered reproductivc state does not affecl the phase of the pituitary-adrenal rhythm of at least this strain of mice The finding that there is no circadian rhythm of strcs~ levels and therefore, that the corticosteronc response to a stressor is greater at the trough than the peak is m agreement with others (in r a t s [ l , 2, I1, 12, 30], and mice [20, 39-41]). However, contrary to other reports [4, 5, 15, 16]. stress levels were higher in grouped animals than individually housed ones, using blood sampling and ether as stressors. This result was consistent throughout the day and was found in mice previously unused or disturbed. However in animals subjected only to ether stress there was no difference in plasma corticosterone levels, in agreement with Brain and Nowell [51. It is known that pituitary-adrenal response to stressors can be dependent on the type of stressor and its intensity [1,9, 14, 25]. Blood sampling and ether may constitute a more intense or different type of stress than ether alone. Activation of the pituitary-adrenal system can operate through different pathways [10] and it has been shown thai ether can stimulate the system at the median eminence, without higher brain centres [26]. Since higher brain centres would be involved in "'social stress" [3,8] the relevance of ether stress responsiveness to social behaviour is questionable. A problem raised by Ader eta/. [1] and as yet unresolved, is that of which parameter of pituitary-adrenal function should be used as a correlate of behavioural stress. We would add that the concept of measurement of basal plasma corticosterone levels is equally problematical, being open to interference from reproductive factors in females, and social interactions in groups of both sexes. In this paper we have examined basal levels, circadian rhythm phase and stress levels (at a fixed time point after stress was applied) in relation to housing density of female mice. Whilst we have found some differences between individually and group housed mice, there is no evidence of the permanently elevated basal levels associated with the chronic stress of Selyes GAS [351 and implied in Christian's hypothesis of regulation of population size by social behaviour [81.

REFERENCES 1. Ader, R., S. B. Friedman and L. J. Grota. "Emotionality'" and adrenal cortical function: effects of strain, test and the 24 hour corticosterone rhythm. Anita. Behav. 15: 37-44, 1967. 2. Allen, C. F., J. P. Allen and M. A. Greer. Absence of nyctohemeral variation in stress-induced ACTH secretion in the rat. Aviat. space environ. Med. 46: 296-299, 1975. 3. Bajusz, E. The pituitary-adrenocortical system, its regulation and adaptive functions. In: Physiology and Pathology of Adaptation Mechanisms, edited by E. Bajusz. Oxford: Pergamon, 1969, p. 89. 4. Brain, P. F. What does individual housing mean to a mouse. Lift" Sci. 16: 187-200, 1975. 5. Brain, P. F. and N. W. Nowell. Isolation versus grouping effects on adrenal and gonadal function in albino mice. II. Female. Gen. comp. Endocr. 16: 155-159, 1971.

6. Brodish, A. Hormonal and behavioural influence on the circadian rhythmicity of the hypothalamic pituitary-adrenal system. In: Biological Rhythms in Neuroendocrine Activity, edited by M. Kawakami. Tokyo: Igaku Shoin, 1974, p. 253. 7. Champlin, A. K. Changes in the oestrous cycle and adrenal glands of the mouse as a result of differences in the number of females housed in a cage. Ph.D. Thesis, University of Rochester, 1979. 8. Christian, J. J. Population density and reproductive efficiency. Biol. Reprod. 1: 248-294, 1971. 9. Dallman, M. F. and M. T. Jones. Corticosteroid feedback control of ACTH secretion: effect of stress induced corticosterone secretion on subsequent stress responses in the rat. Endocrinology 92: 1367-1375, 1973. 10. Dallman, M. F. and F. E. Yates. Anatomical and functional mapping of central neural input and feedback pathways of the adrenocortical system. Mere. Soc, Endocr. 17: 39-72, 1968.

CORTICOSTERONE

RHYTHM AND STRESS

11. Dunn, J., L. Scheving and P. Millet. Circadian variation in stress evoked increases in plasma corticosterone. Am. J. Physiol. 223: 402-406, 1972. 12. Engeland, W. C., J. Shinsako, C. M. Winget, J. VernikosDanellis and M. F. Dallman. Circadian patterns of stress induced ACTH secretion are modified by corticosterone responses. Endocrinology 100: 138--147, 1977. 13. Endroczi, E. Circadian rhythm in the pituitary-adrenal function: limbic clock and its influence by nervous and humoral factors. In: Biological Rhythms in Neuroendocrine Activity, edited by M. Kawakami. Tokyo: Igaku Shoin, 1974, p. 281. 14. Friedman, S. B., R. Ader, L. J. Grota and T. Larson. Plasma corticosterone response to parameter of electric shock in the rat. Psychosom. Med. 29: 323-328, 1967. 15. Goldsmith, J. F., P. F. Brain and D. Benton. A re-investigation of "isolation' as a possible stressor in the male Tuck 'TO' strain albino mice. J. Endocr. 71: 92p--93p, 1976. 16. Goldsmith, J. F., P. F. Brain and D. Benton. Interpretation of differences in plasma corticosterone titres in the circulation of individually and group-housed male and female mice. J. Endocr. 72: 63p--64p, 1977. 17. Gray, G. D., A. M. Bergfors, R. Levin and S. Levine. Comparison of the effects of restricted morning or evening water intake on adrenocortical activity in female rats. Neuroendocrinology 25: 236-247, 1978. 18. Grota, L. J. and R. Ader. Adrenocortical function in pregnant rats: handling and the 24 hour rhythm. Physiol. Behav. 5: 739741, 1970. 19. Halberg, F., C. P. Barnum, R. H. Silber and J. J. Bittner. 24 hour rhythms at several levels of integration in mice on different lighting regimes. Proe. Sac. exp. Biol. Med. 97: 897-900, 1958. 20. Haus, E. and F. Halberg. Stage of adrenal cycle determining different corticosterone responses of C mice to unspecified stimulation and ACTH. Acta. Endocr. 35: 219, 1960. 21. Hennesey, M. B. and S. Levine. Effects of various habituation procedures on pituitary-adrenal responsiveness in the mouse. Physiol. Behav. 18: 799-802, 1977. 22. Hiroshige, R. Circadian rhythm of corticotropin-releasing activity in the rat hypothalamus: An attempt at physiological validation. In: Biological Rhythms in Neuroendocrine Activity, edited by M. Kawakami. Tokyo: Igaku Shoin, 1974, p. 267. 23. Johnson, J. J. and S. Levine. Influence of water deprivation on adrenocortical rhythms. Neuroendocrinology 11: 268-273, 1973. 24. Krieger, D. T. Rhythms in CRF, ACTH and corticosteroids. In: Endocrine Rhythms, Comprehensive Endocrinology, 1, edited by D. T. Krieger. New York: Raven Press, 1979, p. 123. 25. Levine, S. and D. M. Treiman. Determinants of individuals differences in the steroid response to stress. In: Physiology and Pathology of Adaptation Mechanisms, edited by E. Bajusz. Oxford: Pergamon, 1969, p. 171. 26. Matsuda, K., C. Duyck, J. W. Kendall and M. A. Greer. Pathways by which traumatic stress and ether induce increased ACTH released in the rat. Endocrinology 74: 981-985, 1964.

5

27. Nelson, M. L., A. M. Cullin and J. C. Hoffman. Circadian rhythms of serum estradiol and corticosterone and related organ weight changes in the prepubertal rat. Biol. Reprod. 18: 125132, 1978. 28. Nichols, D. J. and P. F. D. Chevins. Plasma corticosterone fluctuations during the oestrous cycle of the house mouse. Experientia 37: 319-320, 1981. 29. Paris, A. L. and J, A. P,amaley. Adrenal-gonadal relations and fertility: effects of repeated stress upon the adrenal rhythm. Neuroendocrinology 15: 126--136, 1974. 30. Pfister, H. P. and M. G. King. Adaptation of the glucocorticosteroid response to novelty. Physiol. Behav. 17: 43-46, 1976. 31. Ramaley, J. A. Differences in serum corticosterone patterns in individual rats: relationships to ovulatory cycles. J. Endocr. 66: 421-426, 1975. 32. Retiene, K., F. Schultz and J. Mareo. Circadian rhythmicity of pituitary adrenal function under resting and stress conditions in rats. Rass. Neural. Veg. 21: 217-221, 1967. 33. Riley, V. Adaptation of orbital bleeding technique to rapid serial blood studies. Proc. Sac. exp. Biol. Med. 104: 751-754, 1960. 34. Ryan, K. D. and N. B. Schwartz. Grouped female mice: demonstration of pseudo-pregnancy. Biol. Reprod. 17: 578-584, 1977. 35. Selye, H. The Physiology and Pathology of Exposure to Stress. Montreal: Acta. Inc., 1950. 36. Smolensley, H. M., F. Halberg, J. Harter, B. Hsi and W. Nelson. Higher corticosterone values at a fixed single time point in serum from mice trained by prior handling. Chronobiology 1: 1-14, 1978. 37. Salem, J. H. Plasma corticosteroids in mice. Scand. d. chem. lab. Inv. 15: 1-36, 1966. 38. Spackman, D. and V. Riley. Increased corticosterone a factor in LDH-virus induced alterations of immunological responses in mice. Proc. Am. Ass. Cancer Res. 15: 143, 1974, 39. Ungar, F. and F. Halberg. In vitro exploration of a circadian rhythm in adrenocorticotropic activity of C mouse hypophysis. Experientia 19: 158-160, 1962. 40. Ungar, F. In vitro studies of adrenal-pituitary circadian rhythms in the mouse. Ann. N. Y. Acad. Sci. 117: 374-385, 1964. 41. Ungar, F. and F. Halberg. Circadian rhythm in the in vitro response of mouse adrenal to adrenocorticotropic hormone. Science 137: 1058-1060, 1962. 42. Van der Lee, S. and L. M. Boot. Spontaneous pseudopregnancy in mice. Acta physiol, pharmac, neerl. 4: 442, 1955. 43. Whitten, W, K. Pheromones and mammalian reproduction. Reprod. Physiol. 1: 155-177, 1966. 44. Wilkinson, C. W., J. Shinsako and M. F. Dallman. Daily rhythms in adrenal responsiveness to ACTH are determined primarily by the time of feeding in the rat. Endocrinology 104: 350-360, 1979. 45. Zimmermann, E. and V. Critchlow. Effects of diurnal variation in plasma corticosterone levels on adrenocortical response to stress. Proc. Sac. exp. Biol. Med. 125: 658-663, 1967.