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Chronic physiological effects of fighting in mice
Gl?.Nl%RAE
AND
COMPARkTIVE
Chronic
ENDOCRINOLOGY
4, 9-14
Physiological
(1964)
Effects
of Fighting
in Mice1
F. H. BRONSON The
Jackson
Laboratory,
Bur
Harbo-r,
Maine
AND
B. E. ELEFTHERIOU? Depadment
of Biological
Sciences,
Purdue
Received
April
University,
West
Lafayette,
Indiuna
16, 1963
Mice of the C57BL/lOJ strain were exposed to t,rained fighters or empty cages at. ;he rate of 0, 1, 2, 4, or 8 times per day for 7 days. Each exposure lasted 1 minute. Mice exposed to fighters 2, 4, or 8 times per day or to an empty cage 8 times per day durin g the experiment. averaged losses in body weight Adrenal weight increased progressively as the number of exposures per day to fighters or empty cages increased, but the effects were much greater in ‘mice exposed to fighters. PIasma corticosterone levels, as measured 46 hours after the last exposure, were significantly higher in mice exposed to fighters than in those exposed to empty cages. Adrenal corticosterone levels, if adjusted for the changes in adrenal weight, showed no effects of the treatments. Seminal vesicle weights decreased as the number of exposures to fighters increased.
The impact of variations in population density on adrena physiology of rodents has been reviewed by Christian (1960). The direct cause of the adrenal changes observed under crowded conditions is probably in the nature of a psychological stressor associated with t,he social environment (Christian, 1957). Several workers have concluded that aggressive behavior may play a role as great or greater than crowding per se in stimulating adrenal response (e.g., Clarke, 1953; Barnett, 1958; Bronson and Eleftheriou, 1963). However, none of these laboratory st.udies, dealing either with cage density or aggression as t,he primary variable, has examined adrenal
responses when animals are allowed to contact members of the same sex only for short periods each day, a situation known to be normal for many rodents under field conditions. The present experiment evaluates adrenal and some other physiological responses when mice are exposed to trained fighting mice for a varying number of ‘Iminute periods each day. MATERISLS
An-D
METXODS
Adult male mice of the C57BIq’laT strain were isolated following weaning at 21-28 days of age. iit 75-85 days of age, 120 mice were exposed a varying number of times each day to eit.her (1) trained fighters of the same strain in the fighters’ home cages, or (2) handling similar to that. received by fighter-exposed animals, including exposure to an empty (clean) cage. Animals were exposed to these two types of treatments at the rate of 0, 1, 2, 4, or 8 times per day for 7 days. The experimental design, therefore, consisted of 10 treatments with 12 mice per treatment; however, since two treatments-no exposures to
‘Supported by PHS grant M-4481 from the National Institutes of Health, Public Health Service, Special thanks are due Dr. M. X. Zarrow of Purdue University for partial financial assistance and la.boratory facilities. ’ Present address : Department of Zoology, Kansas State University, Manhattan. 9
10
BROXSOK
AND
fighters and no exposures to empty cages-are identical, the 24 animals involved actually constituted a double control. Within these 24 mice, odd-numbered animals served as the controls for fighting a.nd even-numbered animals as controls for empty cage exposures. Each exposure to a fighter or an empty cage lasted 1 minute and, when more than one exposure per day was; required, a minimum of 5 minutes between exposures was maintained. All animals remained isolated in their home cages when not being exposed. A battery of 25 trained fighters (trained as suggested by Scomtt, 1946) was used in this investigation. Where possible, each animal scheduled for the fighter-exposure treatments was exposed to as many of the 25 fighters as its schedule would allow. Body weights were obtained on days 1 and 6 of the experiment by using a shadowgraph balance, and body lengths were obtained on day 6 from unanesthetized animals. Four to six hours after the last, exposure each animal was killed by decapitation, blood was collected, and the adrenals and seminal vesicles were weighed. Samples of blood and adrenals were pooled separately from 6 animals of each treatment and frozen for later biochemical analysis. It has been found in our laboratory that keeping plasma frozen up to 6 months does not alter corticosterone activity. Adrenal and plasma corticosterone levels were determined according to the method of Peron and Dorfman (1959) and Moncloa et al. (1959). Essentially, the procedure involved the following: adrenals were homogenized with 2.0 ml of 33% ethanol and made up to a volume of 5.0 ml with double-distilled water (original volume). Aliquots of 0.5, 0.7, 1.0, and 1.2 ml were taken and made up to 2 ml with 13% ethanol in a 12-ml ground glass-stoppered centrifuge tube. For plasma deterrminations, aliquots taken were: 0.2, 0.4, 0.6, and 0.8, ml. These were made up to a volume of 2.0 ml with 13% ethanol. Each aliquot was washed with 5 ml of ligroin, centrifuged, and the ligroin removed by aspiration. Samples were extracted with 5’ ml of dichloromethane and centrifuged. The aqueous phase was removed by aspiration, and the dichloromethane phase was washed with 1.0 ml of ice-cold 0.1 N NaOH and centrifuged. The NaOH phase was removed and 2.0 ml of 30N H&O, were added by transferring 4.0 ml of extract to 2.0 ml of H,SO,. The tubes were shaken vigorously and centrifuged. The H&O, phase was removed and the samples read in a Turner fluorimeter after 45 minutes. All extraction and washing steps were carried out by shaking vigorously by hand for exactly 1 minute. Cent,rifugation in
ELEFTHERIOU all cases was for 5 minutes at 2000 rpm. EmaPsions formed with NaOH could only be broken partially so that care was taken to remove 2.@ml aliquots of the clear dichloromethane extract and to transfer these to clean tubes prior to the addition of sulfuric acid. A standard curve was run with eorticosterone values of 0.02-0.1 pg per milliliter. A blank containing only 13% ethanol was also run. FIuorescence values for tissue and plasma were extrapolated to zero-tissue and plasma values, i.e., where the line intercepted the ordinate. These values were considered as true tissue blanks. Since the mice used in this study were SO-90 days old at the time of autopsy, the fluorescence method was presumably measuring only corticosterone. No interference was detected from other adrenal steroids such as cortisol. RESULTS
The treatments resulted in definite alterations of weight gain (Fig. 1). While control animals showed an average weight
FIG. 1. Relationship between mean proportion change of body weight (weight on day 6 minus weight on day 1 divided by weight on day 1) and various numbers of exposures per day to fighters or empty cages for one week. Twelve mice per average.
gain of about 25% of their body weight between days 1 and 6 of the experiment, weight gain decreased as the number of exposures to an empty cage or fighters increased. Mice exposed to 2, 4, or 8 fighters per day or exposed to empty cages 8 times per day lost weight during the experiment. Analysis of variance showed significance for empty cage vs. fighter-exposure treat-
EFFECTS
OF
$1
FIGHTIMG
ments (p < 0.05), and for the number of exposures per day (p < O.Ol), but, revealed a nonsignificant interaction, thus supporting the obvious trends in Fig. 1. Adrenal weights also changed drastically due to t,he various treatments (Table 1). _.- ..O,.. . ..___.___
:
TABLE
‘w-”
1
Enof, Cape-iia;ma -0..- . .. .. . . . . . . . . . ..______” ____o
MEAN PAIRED ADRENAL WEIGHTS~ 0~ MICE EXPOSED TO TRAINED FIGHTERS OR EMPTY CAGES FOR I WEEK Exposrdto Fighters Empty
cages
0 2.8 2.6
Number of exposuresper day 2 1 4 8 3.1 2.8
3.2 2.6
0 Paired adrenal weight (mg)/body X 100; 12 mice per mean.
3.8 3.0
4.0 3.1
length
(mm)
Because of the significant effects of the treatments on body weight, adrenal weights were adjusted to body length. Adrenal weight (mg)/body length (mm) X 1OQ is a conservative type of adjust.ment since 91% of tShe 120 mice used in this experiment had body lengths between 91 and 96 mm (range: 89-100 mm). Analysis of variance of the adrenal weights showed significance for (1) fighter vs. empty cage treatments (p < 0.001) ; (2) number of exposures per day (11< 0.001) ; and (3) t,he interact’ion (p < 0.01). It is obvious from Table 1 t,hat as the number of exposures per day to fighting or empty cages increased, adrenal weight increased also. It is also obvious that, of the two types of treatments, exposure to fighters had a much greater effect on adrenal weight. Plasma corticosterone levels, as measured 4-6 hours aft,er the final exposure to a Eghter or an empt,y cage, showed a distinct trend for higher levels in those mice that had been exposed to fighters (Fig, 2). This trend was tested by variance analysis and was significant (p < 0.01) ; however, there was no significance due to the number of exposures per day or to the interaction of these two variables. Adrenal corticost,erone levels (Fig. 2), if adjusted for the increased adrenal weight caused by the treatments, showed no significant differences in any m3pect.
0
iia
I
d 2 EXPOSURES/DAY
B
FIG. 2. Cortieosterone levels in the plasma (ag/lOO ml plasma) and adrenals (pgj100 mg adrenal tissue) after 0, 1, 2, 4, or 8 exposures pe: day for 1 week to fighters or empty cages. Two samples of plasma or adrenals {each sample pooled from 6 mice) per arerage.
In addition t.o the effects on body weight and the adrenal, definite effects of the t.reatments on seminal vesicle weights were found (Table 21. Analysis of variance revealed significant t,erms for figking vs. TABLE
2
MEAN SEMIXAL VESICLE WEIGHTGO ok bacx EXPOSED TO TRAINED FIGHTERS OR EMPTY @AGES FOR I WEEK Exposed to Fighters Empty
cage
0
1
2.4 2.6
2.3 2.6
2
4
5
2.2 2 4
2.1 2 .3
2.0 4.2
lengt,h
(mm)
0 Seminal vesicle weight (mg)/bodp X 100; 12 mice per mean.
empty cage (p < 0.01) and for number of exposures per day (p = 0.01)) but. a nonsignificant interaction, Because of the variat,ion among the conk01 samples, it would appear that, only many repeated exposures to empty cages had any ef?fect, while any amount of exposure to fighters caused some degree of decrease in seminal vesicle weight. DISCUSSIOX
The objectives of this study were t,wofold: (1) to investigate some possible
12
BRONSON
AND
physiological effects of exposure to very small amounts of aggression; and (2) to do so by utilizing a design that the authors feel has considerable merit for the laboratory study of the interrelationships between rodent population density and physiology. With respect to the first objective of the study, the data reveal that exposure to small amounts of aggression was consistently followed by relatively large physiological changes. Effects of a chronic nature were found in adrenal, body, and seminal vesicle weights. As little as 2 l-minute exposures to trained fighters per day for 7 days resulted in a 19% increase in adrenal weight over untreated controls and a 14% increase over empty cage controls. Eight exposures per day resulted in a 48% increase in adrenal weight over untreated controls and a 29% increase over empty cage controls. Body weight was affected as the number of exposures to fighters increased. Mice exposed to 2, 4, or 8 fighters per day averaged weight losses for the period as did mice exposed 8 times per day to an empty cage. Analyses of seminal vesicle weights showed a significant decline in weight as number of exposures to fighters increased, but probably only excessive exposure to a strange cage (8 times per day) had any real effect on the weight of these glands. Plasma corticosterone levels, as measured 4-6 hours after the final exposures to fighters or empty cages, averaged 20.2 pg/lOO ml plasma in fighter-exposed mice and 12.1 pg in mice exposed to empt,y cages. The unexposed control mice averaged 17.0 pg of corticosterone/lO9 ml plasma. Since mice exposed several times per day to empty cages showed effects on body and adrenal weight, the reason that plasma corticosterone levels in these mice averaged below those of unexposed controls is not readily apparent unless this is a reflection of the time relationships involved in length of treatments and time of autopsy. The failure of increased numbers of exposure to fighters to be reflected in plasma levels might be attributable to the same reasons. Plasma corticosterone levels were meas-
ELEFTHERIOU
ured between 2 and 4 P.M. and are similar in magnitude to those described for mice by Halberg et al. (1959). Adrenal corticosterone levels, when adjusted to micrograms/l00 mg of adrenal tissue, were not affected by the treatments. Both adrenal weight and amount of corticosterone in the adrenals increased fohowing fighter exposure, but, as expected, the relative ratio between the two remained essentially the same (O’Donnell & Preedy, 1961). Barnett (1958) reported lipid depletion in the adrenal cortex after fighting in rats, and Bronson and Eleftheriou (1963) reported depletion of adrenal ascorbic acid in two speciesof rodents exposed to trained fighters. Despite the problems associated with correlations of adrenal weight, ascorbic acid content, and secretory rates (Vogt,, 1957; Sayers, 1961; Gray et al., 1961; Schapiro et al., 1962), it would seem that there could be little doubt that social domination by fighting has large effects on adrenal morphology and secretion. AS shown by the present study, fighting can produce large adrenal changes even when occurring for only very short periods. An important point here is that. exposure to a trained fighter did not exclusively involve attacks on the subject mice, nor were the subjects wounded to any great degree. A random sample of 20 cumulative attack times obtained during the experiment revealed an average of 20 seconds and a range of O-43 seconds of fighting during l-minute exposures. Wounds were rare in mice exposed t.o one fight per day and common in those exposed to 8 fights per day. However, wounds were almost exclusively restricted to the tail and no severe wounds were found which parallel those that are often found when mice of this strain are isolated at weaning and then grouped as adults. Southwick (1959) attributed increases in adrenal weight among crowded groups primarily to the wounded mice in those groups, but Christian (1959) found no correlation between wounding and adrenal weight in his caged populations. Barnett (1958) observed that actual
EFFECTS
OF
FIGHTIKG
13.
fighting was not always necessary to cause ulations of highly aggressive rodents since all social contacts were made aggressive in adrenal hypertrophy in crowded rats. In addition, Mason (1959) reported an in- nature by training some animals. The same design, however, could be used to sludy the crease in circulat.ing corticoid in a monkey relationships between density and physithat was watching two other monkeys fight. The physiological effects found in ology for less aggressive species of rodents (see Terman, 1959) by using only naive t.he present study were, therefore, probanimals or giving some mice a different ably more a result of psychological factors type of training procedure (e.g., avoidan~ than wounding per se. of other mice). The large changes in adrenal and body weight, as well as the fact t.hat the altered REFERENCES corticosterone levels were found 4-6 hours B.~RNETT, S. A. (1958). Physiological effects of after exposure to fighters, would tend to “social stress” in wild rats. I. The adrenal corargue that the psychological responses were tex. J. Psychosow Res. 3, l-11. sustained over a much longer span of time BROKSOK, F. H., AND E,LEFTHERIOIJ, S. E. (19631. than just during the short exposures. HowAdrenal responses to crowning in Peromyscus ever, the degree to which short-t,erm and C57BL/lOJ mice. Physiol. Zool. 36, 161.. 166. “alarm” responses contributed to the CHRISTIAK, J. J. (1957). A review of the endoaltered plasma corticosterone levels cannot crine responses in rats and mice to increasing be evaluated by the present experiment. population size including delayed effect.s on With respect to the second objective of offspring. Nazjal ilIed. Res. Inst. Lect. Rel! the study, i.e., examination of the general Series No. 57-2, pp. 443462. design for possible use in laboratory studies CB[RISTI.4N, J. J. (1959). Lack Of COm?&iOR brof the relationships between population tween adrenal weight and injury from fighting density and rodent physiology, the authors in grouped male albino’ mice, PWC. Isoc. Expti. feel t,hat this design has considerable value Bid. Med. 1101, 1666168. for such investigations. The traditional CHRIS~IAX, J. J. (1960). Endocrine adaptive models used in such studies are: (1) varymechanisms and the physiologic regulation of populat’ion growth. Na,val Med. Res. Inst. Lect. ing the number of animals per cage ; and Rev. f&es No. 60-2, pp. 49-150. (2) sampling freely growing caged populaCLARKE, J. R. (1953). The effect of fighting on tions. The advantage of the present model the adrenals, thymus and spleen of the vole is that all animals are always socially iso(Microtus agrestis). J. Endoc~inof. 9, 113-226. lated except, for a variable number of very GRAY, 6. H., GI~EER'BwAY, J. M., AND EIOLNESS, short periods each day, a situation that N. J. (1961). I?2 “Hormones in Blood” (G. H. comes much closer to duplicating field conGray and A. L. Bacharach, eds.), pp. 446-,514. dit,ions for the majority of rodents (at least Scademic Press, New York. with respect to members of the same sex). HALBERG, F., PETERSON, R. E., nsn SILVER, 2,. a. The effect of increasing density can there(19598). Phase relations of 24-hour periodicities in blood corticosterone, mitoses in cortical adfore be examined under conditions approxirenal parenchyma, and total body act.ivity. mating field conditions by increasing the Endocrinology 64, 222-230. number of social contacts per day. An inM.~soN, J. W. (1959). Psychological influences on teresting conclusion of the present study t,he pituitary-a.drena,l cortical system. Recent is that effects were found on adrenal and Progr. Hormone Res. 15, 343-389. seminal vesicle weights which parallel those MONCLOA, F., PERON, F. G., AND DORFMAN, R. 1. found in studies where the number of mice (1959). The fiuorimetric determination of cortiper cage was varied (cf. Christian, 1960) ; costerone in rat adrenal tissue and plasma: however, in the present study a design was Effect of administering ACTH subcutaneousiy. used which avoids criticisms of unnaturally Endoc&ology 65, 717-724. high densities (Negus et al., 1961). WEGUS, K. C., GOULD, E., ASD CBIPMAK, It. K. The applicability of the results from the (1961). Ecology of the rice rat, Qryzornys poEztstris (Harlan), on Breton Island, Gulf of present study is probably restricted to pop-
14
BRONSON
AND
Mexico, with a critique of the social stress theory. Tulane Studies Zool. 8, 95-123. PERON, F. G., AND DORFMAN, R. I. (1959). A method for the evaluation of adrenocorticotropic hormone suppressing action of corticoids. Endocrinology 64, 431-436’. O’DONNELL, V. J., AND PREEDY, J. R. K. (1961). In “Hormones in Blood” (C. H. Gray and A. L. Bacharach, eds.), pp. 303354. Academic Press, New York. SAYERS, G. (1961). In “Hormones in Blood” (C. H. Gray and A. L. Bacharach, eds.), pp. l-10. Academic Press, New York. SCHAPIRO, S., GELLER, E., AND EIDUSON, S. (1962). Corticoid response to stress in the steroidinhibited rat. Proc. Sot. Exptl. Biol. Med. 109,
935-937.
ELEFTHERIOU
Scour, J. P. (19%). Incomplete adjustment caused by frustration of untrained fighting mice. J. Comp. Psych. 39, 37%390. SOUTHWICK, C. H., AND BLAND, V. P. (1959). Effect of population density on adrenal glands and reproductive organs of CFW mice. Am. 1. Physiol. 197, 111-114. TERMAN, C. R. (1962). Spatial and homing consequences of the introduction of aliens into semi-natural populations of prairie deermice. Ecology 43, 216-223. VOGT, M. (1957). In “Hormones in Blood” (G. E. W. Wolstenholme and E. C. P. Millar, eds.), pp. 193-199. Ciba Foundation Colloquia on Endocrinology. Little, Brown, Boston, Massachusetts.
AND
COMPARkTIVE
Chronic
ENDOCRINOLOGY
4, 9-14
Physiological
(1964)
Effects
of Fighting
in Mice1
F. H. BRONSON The
Jackson
Laboratory,
Bur
Harbo-r,
Maine
AND
B. E. ELEFTHERIOU? Depadment
of Biological
Sciences,
Purdue
Received
April
University,
West
Lafayette,
Indiuna
16, 1963
Mice of the C57BL/lOJ strain were exposed to t,rained fighters or empty cages at. ;he rate of 0, 1, 2, 4, or 8 times per day for 7 days. Each exposure lasted 1 minute. Mice exposed to fighters 2, 4, or 8 times per day or to an empty cage 8 times per day durin g the experiment. averaged losses in body weight Adrenal weight increased progressively as the number of exposures per day to fighters or empty cages increased, but the effects were much greater in ‘mice exposed to fighters. PIasma corticosterone levels, as measured 46 hours after the last exposure, were significantly higher in mice exposed to fighters than in those exposed to empty cages. Adrenal corticosterone levels, if adjusted for the changes in adrenal weight, showed no effects of the treatments. Seminal vesicle weights decreased as the number of exposures to fighters increased.
The impact of variations in population density on adrena physiology of rodents has been reviewed by Christian (1960). The direct cause of the adrenal changes observed under crowded conditions is probably in the nature of a psychological stressor associated with t,he social environment (Christian, 1957). Several workers have concluded that aggressive behavior may play a role as great or greater than crowding per se in stimulating adrenal response (e.g., Clarke, 1953; Barnett, 1958; Bronson and Eleftheriou, 1963). However, none of these laboratory st.udies, dealing either with cage density or aggression as t,he primary variable, has examined adrenal
responses when animals are allowed to contact members of the same sex only for short periods each day, a situation known to be normal for many rodents under field conditions. The present experiment evaluates adrenal and some other physiological responses when mice are exposed to trained fighting mice for a varying number of ‘Iminute periods each day. MATERISLS
An-D
METXODS
Adult male mice of the C57BIq’laT strain were isolated following weaning at 21-28 days of age. iit 75-85 days of age, 120 mice were exposed a varying number of times each day to eit.her (1) trained fighters of the same strain in the fighters’ home cages, or (2) handling similar to that. received by fighter-exposed animals, including exposure to an empty (clean) cage. Animals were exposed to these two types of treatments at the rate of 0, 1, 2, 4, or 8 times per day for 7 days. The experimental design, therefore, consisted of 10 treatments with 12 mice per treatment; however, since two treatments-no exposures to
‘Supported by PHS grant M-4481 from the National Institutes of Health, Public Health Service, Special thanks are due Dr. M. X. Zarrow of Purdue University for partial financial assistance and la.boratory facilities. ’ Present address : Department of Zoology, Kansas State University, Manhattan. 9
10
BROXSOK
AND
fighters and no exposures to empty cages-are identical, the 24 animals involved actually constituted a double control. Within these 24 mice, odd-numbered animals served as the controls for fighting a.nd even-numbered animals as controls for empty cage exposures. Each exposure to a fighter or an empty cage lasted 1 minute and, when more than one exposure per day was; required, a minimum of 5 minutes between exposures was maintained. All animals remained isolated in their home cages when not being exposed. A battery of 25 trained fighters (trained as suggested by Scomtt, 1946) was used in this investigation. Where possible, each animal scheduled for the fighter-exposure treatments was exposed to as many of the 25 fighters as its schedule would allow. Body weights were obtained on days 1 and 6 of the experiment by using a shadowgraph balance, and body lengths were obtained on day 6 from unanesthetized animals. Four to six hours after the last, exposure each animal was killed by decapitation, blood was collected, and the adrenals and seminal vesicles were weighed. Samples of blood and adrenals were pooled separately from 6 animals of each treatment and frozen for later biochemical analysis. It has been found in our laboratory that keeping plasma frozen up to 6 months does not alter corticosterone activity. Adrenal and plasma corticosterone levels were determined according to the method of Peron and Dorfman (1959) and Moncloa et al. (1959). Essentially, the procedure involved the following: adrenals were homogenized with 2.0 ml of 33% ethanol and made up to a volume of 5.0 ml with double-distilled water (original volume). Aliquots of 0.5, 0.7, 1.0, and 1.2 ml were taken and made up to 2 ml with 13% ethanol in a 12-ml ground glass-stoppered centrifuge tube. For plasma deterrminations, aliquots taken were: 0.2, 0.4, 0.6, and 0.8, ml. These were made up to a volume of 2.0 ml with 13% ethanol. Each aliquot was washed with 5 ml of ligroin, centrifuged, and the ligroin removed by aspiration. Samples were extracted with 5’ ml of dichloromethane and centrifuged. The aqueous phase was removed by aspiration, and the dichloromethane phase was washed with 1.0 ml of ice-cold 0.1 N NaOH and centrifuged. The NaOH phase was removed and 2.0 ml of 30N H&O, were added by transferring 4.0 ml of extract to 2.0 ml of H,SO,. The tubes were shaken vigorously and centrifuged. The H&O, phase was removed and the samples read in a Turner fluorimeter after 45 minutes. All extraction and washing steps were carried out by shaking vigorously by hand for exactly 1 minute. Cent,rifugation in
ELEFTHERIOU all cases was for 5 minutes at 2000 rpm. EmaPsions formed with NaOH could only be broken partially so that care was taken to remove 2.@ml aliquots of the clear dichloromethane extract and to transfer these to clean tubes prior to the addition of sulfuric acid. A standard curve was run with eorticosterone values of 0.02-0.1 pg per milliliter. A blank containing only 13% ethanol was also run. FIuorescence values for tissue and plasma were extrapolated to zero-tissue and plasma values, i.e., where the line intercepted the ordinate. These values were considered as true tissue blanks. Since the mice used in this study were SO-90 days old at the time of autopsy, the fluorescence method was presumably measuring only corticosterone. No interference was detected from other adrenal steroids such as cortisol. RESULTS
The treatments resulted in definite alterations of weight gain (Fig. 1). While control animals showed an average weight
FIG. 1. Relationship between mean proportion change of body weight (weight on day 6 minus weight on day 1 divided by weight on day 1) and various numbers of exposures per day to fighters or empty cages for one week. Twelve mice per average.
gain of about 25% of their body weight between days 1 and 6 of the experiment, weight gain decreased as the number of exposures to an empty cage or fighters increased. Mice exposed to 2, 4, or 8 fighters per day or exposed to empty cages 8 times per day lost weight during the experiment. Analysis of variance showed significance for empty cage vs. fighter-exposure treat-
EFFECTS
OF
$1
FIGHTIMG
ments (p < 0.05), and for the number of exposures per day (p < O.Ol), but, revealed a nonsignificant interaction, thus supporting the obvious trends in Fig. 1. Adrenal weights also changed drastically due to t,he various treatments (Table 1). _.- ..O,.. . ..___.___
:
TABLE
‘w-”
1
Enof, Cape-iia;ma -0..- . .. .. . . . . . . . . . ..______” ____o
MEAN PAIRED ADRENAL WEIGHTS~ 0~ MICE EXPOSED TO TRAINED FIGHTERS OR EMPTY CAGES FOR I WEEK Exposrdto Fighters Empty
cages
0 2.8 2.6
Number of exposuresper day 2 1 4 8 3.1 2.8
3.2 2.6
0 Paired adrenal weight (mg)/body X 100; 12 mice per mean.
3.8 3.0
4.0 3.1
length
(mm)
Because of the significant effects of the treatments on body weight, adrenal weights were adjusted to body length. Adrenal weight (mg)/body length (mm) X 1OQ is a conservative type of adjust.ment since 91% of tShe 120 mice used in this experiment had body lengths between 91 and 96 mm (range: 89-100 mm). Analysis of variance of the adrenal weights showed significance for (1) fighter vs. empty cage treatments (p < 0.001) ; (2) number of exposures per day (11< 0.001) ; and (3) t,he interact’ion (p < 0.01). It is obvious from Table 1 t,hat as the number of exposures per day to fighting or empty cages increased, adrenal weight increased also. It is also obvious that, of the two types of treatments, exposure to fighters had a much greater effect on adrenal weight. Plasma corticosterone levels, as measured 4-6 hours aft,er the final exposure to a Eghter or an empt,y cage, showed a distinct trend for higher levels in those mice that had been exposed to fighters (Fig, 2). This trend was tested by variance analysis and was significant (p < 0.01) ; however, there was no significance due to the number of exposures per day or to the interaction of these two variables. Adrenal corticost,erone levels (Fig. 2), if adjusted for the increased adrenal weight caused by the treatments, showed no significant differences in any m3pect.
0
iia
I
d 2 EXPOSURES/DAY
B
FIG. 2. Cortieosterone levels in the plasma (ag/lOO ml plasma) and adrenals (pgj100 mg adrenal tissue) after 0, 1, 2, 4, or 8 exposures pe: day for 1 week to fighters or empty cages. Two samples of plasma or adrenals {each sample pooled from 6 mice) per arerage.
In addition t.o the effects on body weight and the adrenal, definite effects of the t.reatments on seminal vesicle weights were found (Table 21. Analysis of variance revealed significant t,erms for figking vs. TABLE
2
MEAN SEMIXAL VESICLE WEIGHTGO ok bacx EXPOSED TO TRAINED FIGHTERS OR EMPTY @AGES FOR I WEEK Exposed to Fighters Empty
cage
0
1
2.4 2.6
2.3 2.6
2
4
5
2.2 2 4
2.1 2 .3
2.0 4.2
lengt,h
(mm)
0 Seminal vesicle weight (mg)/bodp X 100; 12 mice per mean.
empty cage (p < 0.01) and for number of exposures per day (p = 0.01)) but. a nonsignificant interaction, Because of the variat,ion among the conk01 samples, it would appear that, only many repeated exposures to empty cages had any ef?fect, while any amount of exposure to fighters caused some degree of decrease in seminal vesicle weight. DISCUSSIOX
The objectives of this study were t,wofold: (1) to investigate some possible
12
BRONSON
AND
physiological effects of exposure to very small amounts of aggression; and (2) to do so by utilizing a design that the authors feel has considerable merit for the laboratory study of the interrelationships between rodent population density and physiology. With respect to the first objective of the study, the data reveal that exposure to small amounts of aggression was consistently followed by relatively large physiological changes. Effects of a chronic nature were found in adrenal, body, and seminal vesicle weights. As little as 2 l-minute exposures to trained fighters per day for 7 days resulted in a 19% increase in adrenal weight over untreated controls and a 14% increase over empty cage controls. Eight exposures per day resulted in a 48% increase in adrenal weight over untreated controls and a 29% increase over empty cage controls. Body weight was affected as the number of exposures to fighters increased. Mice exposed to 2, 4, or 8 fighters per day averaged weight losses for the period as did mice exposed 8 times per day to an empty cage. Analyses of seminal vesicle weights showed a significant decline in weight as number of exposures to fighters increased, but probably only excessive exposure to a strange cage (8 times per day) had any real effect on the weight of these glands. Plasma corticosterone levels, as measured 4-6 hours after the final exposures to fighters or empty cages, averaged 20.2 pg/lOO ml plasma in fighter-exposed mice and 12.1 pg in mice exposed to empt,y cages. The unexposed control mice averaged 17.0 pg of corticosterone/lO9 ml plasma. Since mice exposed several times per day to empty cages showed effects on body and adrenal weight, the reason that plasma corticosterone levels in these mice averaged below those of unexposed controls is not readily apparent unless this is a reflection of the time relationships involved in length of treatments and time of autopsy. The failure of increased numbers of exposure to fighters to be reflected in plasma levels might be attributable to the same reasons. Plasma corticosterone levels were meas-
ELEFTHERIOU
ured between 2 and 4 P.M. and are similar in magnitude to those described for mice by Halberg et al. (1959). Adrenal corticosterone levels, when adjusted to micrograms/l00 mg of adrenal tissue, were not affected by the treatments. Both adrenal weight and amount of corticosterone in the adrenals increased fohowing fighter exposure, but, as expected, the relative ratio between the two remained essentially the same (O’Donnell & Preedy, 1961). Barnett (1958) reported lipid depletion in the adrenal cortex after fighting in rats, and Bronson and Eleftheriou (1963) reported depletion of adrenal ascorbic acid in two speciesof rodents exposed to trained fighters. Despite the problems associated with correlations of adrenal weight, ascorbic acid content, and secretory rates (Vogt,, 1957; Sayers, 1961; Gray et al., 1961; Schapiro et al., 1962), it would seem that there could be little doubt that social domination by fighting has large effects on adrenal morphology and secretion. AS shown by the present study, fighting can produce large adrenal changes even when occurring for only very short periods. An important point here is that. exposure to a trained fighter did not exclusively involve attacks on the subject mice, nor were the subjects wounded to any great degree. A random sample of 20 cumulative attack times obtained during the experiment revealed an average of 20 seconds and a range of O-43 seconds of fighting during l-minute exposures. Wounds were rare in mice exposed t.o one fight per day and common in those exposed to 8 fights per day. However, wounds were almost exclusively restricted to the tail and no severe wounds were found which parallel those that are often found when mice of this strain are isolated at weaning and then grouped as adults. Southwick (1959) attributed increases in adrenal weight among crowded groups primarily to the wounded mice in those groups, but Christian (1959) found no correlation between wounding and adrenal weight in his caged populations. Barnett (1958) observed that actual
EFFECTS
OF
FIGHTIKG
13.
fighting was not always necessary to cause ulations of highly aggressive rodents since all social contacts were made aggressive in adrenal hypertrophy in crowded rats. In addition, Mason (1959) reported an in- nature by training some animals. The same design, however, could be used to sludy the crease in circulat.ing corticoid in a monkey relationships between density and physithat was watching two other monkeys fight. The physiological effects found in ology for less aggressive species of rodents (see Terman, 1959) by using only naive t.he present study were, therefore, probanimals or giving some mice a different ably more a result of psychological factors type of training procedure (e.g., avoidan~ than wounding per se. of other mice). The large changes in adrenal and body weight, as well as the fact t.hat the altered REFERENCES corticosterone levels were found 4-6 hours B.~RNETT, S. A. (1958). Physiological effects of after exposure to fighters, would tend to “social stress” in wild rats. I. The adrenal corargue that the psychological responses were tex. J. Psychosow Res. 3, l-11. sustained over a much longer span of time BROKSOK, F. H., AND E,LEFTHERIOIJ, S. E. (19631. than just during the short exposures. HowAdrenal responses to crowning in Peromyscus ever, the degree to which short-t,erm and C57BL/lOJ mice. Physiol. Zool. 36, 161.. 166. “alarm” responses contributed to the CHRISTIAK, J. J. (1957). A review of the endoaltered plasma corticosterone levels cannot crine responses in rats and mice to increasing be evaluated by the present experiment. population size including delayed effect.s on With respect to the second objective of offspring. Nazjal ilIed. Res. Inst. Lect. Rel! the study, i.e., examination of the general Series No. 57-2, pp. 443462. design for possible use in laboratory studies CB[RISTI.4N, J. J. (1959). Lack Of COm?&iOR brof the relationships between population tween adrenal weight and injury from fighting density and rodent physiology, the authors in grouped male albino’ mice, PWC. Isoc. Expti. feel t,hat this design has considerable value Bid. Med. 1101, 1666168. for such investigations. The traditional CHRIS~IAX, J. J. (1960). Endocrine adaptive models used in such studies are: (1) varymechanisms and the physiologic regulation of populat’ion growth. Na,val Med. Res. Inst. Lect. ing the number of animals per cage ; and Rev. f&es No. 60-2, pp. 49-150. (2) sampling freely growing caged populaCLARKE, J. R. (1953). The effect of fighting on tions. The advantage of the present model the adrenals, thymus and spleen of the vole is that all animals are always socially iso(Microtus agrestis). J. Endoc~inof. 9, 113-226. lated except, for a variable number of very GRAY, 6. H., GI~EER'BwAY, J. M., AND EIOLNESS, short periods each day, a situation that N. J. (1961). I?2 “Hormones in Blood” (G. H. comes much closer to duplicating field conGray and A. L. Bacharach, eds.), pp. 446-,514. dit,ions for the majority of rodents (at least Scademic Press, New York. with respect to members of the same sex). HALBERG, F., PETERSON, R. E., nsn SILVER, 2,. a. The effect of increasing density can there(19598). Phase relations of 24-hour periodicities in blood corticosterone, mitoses in cortical adfore be examined under conditions approxirenal parenchyma, and total body act.ivity. mating field conditions by increasing the Endocrinology 64, 222-230. number of social contacts per day. An inM.~soN, J. W. (1959). Psychological influences on teresting conclusion of the present study t,he pituitary-a.drena,l cortical system. Recent is that effects were found on adrenal and Progr. Hormone Res. 15, 343-389. seminal vesicle weights which parallel those MONCLOA, F., PERON, F. G., AND DORFMAN, R. 1. found in studies where the number of mice (1959). The fiuorimetric determination of cortiper cage was varied (cf. Christian, 1960) ; costerone in rat adrenal tissue and plasma: however, in the present study a design was Effect of administering ACTH subcutaneousiy. used which avoids criticisms of unnaturally Endoc&ology 65, 717-724. high densities (Negus et al., 1961). WEGUS, K. C., GOULD, E., ASD CBIPMAK, It. K. The applicability of the results from the (1961). Ecology of the rice rat, Qryzornys poEztstris (Harlan), on Breton Island, Gulf of present study is probably restricted to pop-
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Mexico, with a critique of the social stress theory. Tulane Studies Zool. 8, 95-123. PERON, F. G., AND DORFMAN, R. I. (1959). A method for the evaluation of adrenocorticotropic hormone suppressing action of corticoids. Endocrinology 64, 431-436’. O’DONNELL, V. J., AND PREEDY, J. R. K. (1961). In “Hormones in Blood” (C. H. Gray and A. L. Bacharach, eds.), pp. 303354. Academic Press, New York. SAYERS, G. (1961). In “Hormones in Blood” (C. H. Gray and A. L. Bacharach, eds.), pp. l-10. Academic Press, New York. SCHAPIRO, S., GELLER, E., AND EIDUSON, S. (1962). Corticoid response to stress in the steroidinhibited rat. Proc. Sot. Exptl. Biol. Med. 109,
935-937.
ELEFTHERIOU
Scour, J. P. (19%). Incomplete adjustment caused by frustration of untrained fighting mice. J. Comp. Psych. 39, 37%390. SOUTHWICK, C. H., AND BLAND, V. P. (1959). Effect of population density on adrenal glands and reproductive organs of CFW mice. Am. 1. Physiol. 197, 111-114. TERMAN, C. R. (1962). Spatial and homing consequences of the introduction of aliens into semi-natural populations of prairie deermice. Ecology 43, 216-223. VOGT, M. (1957). In “Hormones in Blood” (G. E. W. Wolstenholme and E. C. P. Millar, eds.), pp. 193-199. Ciba Foundation Colloquia on Endocrinology. Little, Brown, Boston, Massachusetts.