Physiology and Behavior, Vol. 14, pp. 551--556. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Attempts to Influence Fighting and Threat Behaviors in Adult Isolated Female CFW Mice in Standard Opponent Aggression Tests Using Injected and Subcutaneously Implanted Androgens PAUL F. BRAIN AND CHARLES M. EVANS 3

Department o f Zoology, University College o f Swansea, Singleton Park, SA2 8PP South Wales, U.K.

(Received 26 September 1974) BRAIN, P. F. AND C. M. EANS. Attempts to influence fighting and threat behaviors m adult isolated female CFN mice in standard opponent aggression tests using in/ected and subcutaneously androgens. PHYSIOL. BEHAV. 14(5) 551-556, 1975. - Neither isolated or group-housed intact adult female CFW strata mice displayed overt fighting or threat behavior during standard opponent behavioral tests. Neither testosterone nor androstenedione administered as daily oil based IM inJections over 24 days markedly influenced overt fighting in adult ovariectomized females. However, testosterone administered m the form of SC 25 mg implants significantly increased overt fighting and threat behavior in ovariectomlzed females compared to sham-implanted mice over the same time interval. It was concluded that the aggressiveness of female mice of this strain could be significantly increased by androgen administration in adult hfe without previous neonatal hormonal manipulation, an effect in which the method of hormone administration appeared to be of critical importance. Some possible reasons for this result which is in conflict with most of the literature are suggested. Aggression

Testosterone

Androstenedlone

Fighting

T H E R E is a c o m m o n l y held view that female mice, of the strains routinely employed in laboratory studies, are relatively non-aggressive when compared with males [26,40]. However, under certain experimental conditions, females have been shown to evidence substantial levels of intra-specific fighting behavior. Fredericson [22] reported that food deprived females " f i g h t " over single pieces of food. Fighting has also been described in female mice in a number o f defined situations by other workers [ 23, 45, 46 ]. In contrast to the widely reported influence o f testosterone on the aggressiveness of male mice castrated as adults, androgenic hormones appeared in early studies to have little effect on this motivation in adult females [29,44] unless treatment had also been administered during early life [ 11,16]. This latter effect has been described as androgen

Ovariectomy

induced sexual differentiation of the neonatal hypothalamus.

Another way in which androgens can influence fighting behavior in male and female mice is by altering "attackability" (the ease with which a mouse is attacked by an aggressive fighter mouse). It has been demonstrated that females normally produce olfactory cues (pheromones) in their urine which inhibit attack by aggressive males [ 15,36], while males similarly release androgen-dependent olfactory cues which stimulate attack [32,38]. Further studies indicate that treatment with androgen either neonatally [28], or in adult life [37] will increase the "attackability" of female mice and that, such females can provoke a more violent reaction from male fighter mice than similarly treated or intact males [35].

1This work is partially supported by a M. R. C. project grant to PFB. 2Tnvml names used; Androstenedione: 4 androsten-3, 17 dlone; dihydrotestosterone. 5c~-androstan-17a-ol-3 one, testosterone. 4-androsten-17~ 1-3 one. 3Supported by a S. R. C. Studentship. Present address: Sub-department of Ethology, Department of Psychiatry, The Medical School, Birmingham. 551

552

BRAIN AND EVANS

A review of studies attempting to relate androgen levels to fighting behavior in mice reveals that a wide range of methodologies have been employed. For instance, the administration of the hormone may be by implantation or injection (in different sites) or vm oral application and may often involve widely variable doses of different androgens in different strains of mice. Behavmral testing methodologles may also vary greatly. Often no attempt is made to distinguish between the effects of androgens on the postulated internal motivation (aggressiveness) and peripheral hormone actions (attackability). Since differing results have been obtained and since variables tend to make it difficult to conclusively demonstrate an effect of androgens and aggressive motivation or attackability in female mice, it was felt that a further series of studies, using methodologies regularly employed in our laboratory, might yield some useful information relevant to this point. EXPERIMENT 1 The standard opponent test, (described in detail elsewhere [9]), has been found to be most useful for easy rating of aggressiveness in male mice. Scoring is on the basis of attack directed towards a non-aggressive opponent. Similar tests involving such opponents have been used by other workers [ 19, 31, 43] and it is generally agreed that the test has useful applications. The standard opponent test involves the exposure of experimental animals to young grouped male opponents. These opponents appear to be adequate stimuli, ehciting fighting behavior from the test mice, without initmting attacks themselves. In view of Edwards' [ 16] earlier reported findings that adult female mice vigorously attacked smaller male opponents and that in this respect, were indistinguishable from adult males, it was thought essentml to determine if intact adult female CFW strain mice, used in this series of experiments, would behave in a similar aggressive fashion towards smaller males. Method Antmals. Forty-eight female laboratory bred CFW strain mice which had been weaned at 1 8 - 2 2 days of age, then maintained in groups of six in opaque plastic cages measuring 30 × 12 × 11 cm, were used in this experiment. When 90 days of age, the mice were randomly allocated to two treatment categories each consisting of 24 mice. Mice belonging to the first category were housed individually while in the second category they were regrouped in 6's in cages of similar dimensions to those described above. Food and water were provided ad lib throughout the experiment. Procedure. After a period of three weeks of isolation/ grouping, the mice were given a series of three consecutive daffy standard opponent tests. Behavioral testing took place under dim red light during the initial part of the dark phase of the reversed lighting cycle (12 hr dark; 12 hr light) under which the animals were housed. Each individual aggression test took place in the home cage of the test animal as this has been reported to maximize the aggressive behavior of the resident animal while minimizing that of the intruder [12,251. The test involved removing the lid of the home cage and introducing the standard opponent. A p¢rspex lid was placed on top and the test animal and the standard opponent (a 3 0 - 4 0 day old male from a group of 6) observed

for a period of 7 min, after which the opponent was removed. After each test and on the day preceding the first test, the sawdust bedding of each experimental animal was replaced with fresh material in order to minimize the persistence of olfactory cues, which may influence the results of subsequent tests [ 25 ]. Measures of fighting behavior utilized in this study included: (1) whether or not overt fighting took place, (2) the latency to the first attack, (3) the accumulated attacking time, and (4) the number of attacks dtrected towards the standard opponent by the test animal. These measures are described in detail elsewhere [9]. Some indications of threat behavior were also noted. Threat has been defined by Moyer [34] as "behavior which attempts to communicate the intent to behave aggressively, which usually involves specms specific postures and gestures". Threat was recorded If the test animal approached the stimulus animal whilst evidencing plloerection and characteristically slitted it's eyes, rattled it's tail, postured or feinted towards it's opponent. Detailed descriptions of such rodent threat behavior are available in the literature [24,40]. R e s u l t s a n d Dtscusszon

Neither category of mice evidenced any overt fighting or threat behavior in this particular situation. It appears that isolation of this duration, which is effective in the case of the male [9], does not induce aggressiveness, as measured m a "standard opponent" test, in female mice of the CFW strain. This result differs from that reported by Edwards [16] and White e t al. [46] which initially suggested that the "bully" test (one involving the use of young male standard opponents) may not be suitable for work on intraspecific fighting in androgenized female mice. It therefore seemed reasonable to employ this test to evaluate the effects of endocrine manipulations, which may induce fighting behavior in females of this strain

EXPERIMENT 2 Since it appears that both testosterone (T) and androstenedlone (A) can induce fighting in isolated gonadectomized male mice of various strains [4, 22, 34] it was decided to investigate the achons of both these androgens on female mice. Unlike the majority of methodologies employed to study endocrines and aggresmon in female mice, it was decided to treat the animals in this study with androgens only in adulthood. While a daily dose of 120 #g T admimstered SC has been shown to enhance aggressiveness m castrate male C3 H mice, a higher dose ( 3 0 0 - 6 0 0 gg/day) has been found to have an opposite, inhibiting effect in Swiss Albino male mice [3]. In contrast with thss study, Luttge (30) and Luttge and Hall [31] found that a dose level of about 500 #g T/day was necessary to induce fighting behavior in 35 percent of castrate CD-1 and 60 percent of castrate Swiss Webster mice used in their studies and that progressively, lower doses induced correspondingly lower levels of this behavior. In view of these previous studies, T and A were administered at the relatively low dose of 50 #g/day. A further category of mice injected with a higher, perhaps more pharmacological, dose of 500 #g T/day was also included in this experiment for comparison with the Luttge and Hall [31] study.

ANDROGENS AND AGGRESSION

553 TABLE 1

THE INFLUENCE OF IM INJECTION OF TWO ANDROGENS ON AGONISTIC BEHAVIOR EVIDENCED BY ADULT OVARIECTOMIZED FEMALE Cb-W~MICE IN A SERIES OF THREE STANDARD OPPONENT TESTS

Treatment Category

Number of Observations per Category

Number of Mice Evidencing Threat in at least 2/3 Tests

Number of Mice Evidencing Overt Fighting in at least 1/3 Tests

M e a nLatency of the First Attack (see)

MeanNumber of Attacks

Mean Accumulated Attacking Time (sec)

Control injection (oil vehicle)

30

3

1

405.1

0.27

0.40

50 ~g Androstenedione

30

6

1

397.1

0.57

1.17

50 #g Testosterone

30

10"

2

356.2

2.20

4.57

500 ~g Testosterone

33

7

1

418.8

0.03

0.03

*Differs from control injection, p