Pineal influence on intermale aggression in adult house mice

Physiology & Behavior, Vol. 15, pp. 213--216. Pergamon Press and Brain Research Publ., 1975. Printed in the U.S.A.

Pineal Influence on Intermale Aggression in Adult House Mice 1 T. D. MCKINNEY

Division o f Allied Health and Life Sciences, University o f Texas at San Antonio San Antonio, TX 78285 AND M. K. VAUGHAN AND R. J. REITER 2

Department o f Anatomy, University o f Texas Health Sciences Center, San Antonio T X 78285

(Received 12 September 1974) MCKINNEY, T. D., M. K. VAUGHAN AND R. J. REITER. Pineal influence on intermale aggression in adult house mice. PHYSIOL. BEHAV. 15(2) 213-216, 1975. - Previously isolated, adult male mice of a wild-derived strain were sham-operated or pinealectomized (pinx) and 14 days later were paired for 15 rain, then grouped three per cage for 7 days. During 15 min pairings, mean latency to initiation of f'v~htingwas increased twofold and duration of fighting was reduced 35 to 41 percent in pairs comprised of one or both pinx animals. Although pinealectomy failed to influence which pair member initiated fighting, sham males were ranked as dominant in 75 percent of sham-pinx pairs. Pinealectomy reduced adrenal gland weights among isolated animals and increased in general the effects of social subordination in grouped males. Compared to sham subordinates, pinx subordinates experienced adrenal enlargraent and twofold greater wounding and had lighter thymus glands and final body weights. Additionally, however, pinealectomy prevented atrophy of seminal vesicles among subordinates. Thus, present results suggest a role of the pineal gland in influencing intermale aggression and linking social stimuli with testis dysfunction in adult house mice. Fighting

Pineal gland

Seminal vesicles

Social rank

EFFORTS to evaluate a possible link between behavior and pineal gland function have emphasized effects of gland ablation and/or exogenous hormone administration on locomotor activity. Although altered running activity [ 14,15] and exploratory behavior [21] have been observed in rats following pinealectomy, comparatively little evidence points to a behavioral influence of the pineal gland [13]. However, the adrenal cortex has been proposed as a site of a pineal action [17], and gonad-inhibiting capabilities of the pineal gland are well documented [16]. Intermale aggression also stimulates activity of the pituitary-adrenocortical axis [ 1 ], promotes testis dysfunction [ 11 ] and reduces circulating levels of gonadotropins [3,4], particularly among social subordinates. Thus, present experiments were designed to assess effects of pinealectomy on intermale aggressive behavior and on socially-induced changes in weights of adrenal glands and accessory sex organs.

METHOD

Animals and Surgery Laboratory-raised male descendants of a wild stock of house mice (Mus musculus) were weaned at 21 days of age and caged singly - a procedure known to induce aggressiveness in house mice [ 2 3 ] - in clear plastic cages (N = 105; this figure reflects survivors only). Our breeding colony was developed from animals trapped originally in eastern Pennsylvania. Subsequently, breeding pairs were established nonselectively, and wild-trapped males were introduced infrequently to reduce inbreeding. Animals in the present experiment were provided with excess food and water at all times in a room maintained at 25°C with 14 hr light and l 0 hr dark per 24 hr period. At 45 days of age, mice were anesthetized with methoxyflurane (Metofane) and either pinealectomized (pinx; 8) or sham-operated (sham) and

! This research supported in part by research grants HD MH 08545 to T. D. McKinney and HD-06523 to R. J. Reiter. 2R. J. Reiter is a recipient of USPHS Career Development Award, HD-42398. 213

214

MCKINNEY, VAUGHAN AND REITER

returned to their home cages for 14 days following which they were paired for 15 rain in a neutral cage. Sham surgery consisted of drilling through the skull without removing the resultant disc of bone.

Procedure Each pinx male was paired once with a sham male, and each animal also was paired once with an individual which had received similar surgical treatment. Each animal therefore was paired twice, but once with an individual which had received similar surgical treatment. The sequence of pairing was randomized, thus obviating interaction between prior surgical treatment and experience during pairing. During pairing, latency to first attack, total time spent fighting and total number of fights were recorded [5], incorporating blind grading on the part of the observer. An encounter was scored as a fight only if the attacker was observed to bite the opponent. Orientation of the attacker away from the attacked opponent for a m i n i m u m of 5 sec was taken as the endpoint of persistent fighting, and recorded duration of a given fight excluded this 5 sec period. Immediately after completion of all pairings, males were housed either one or three per cage. Animals in any given cage were of like surgical treatment. On Day 7 following caging singly or in groups, mice were killed by cervical dislocation, weighed, eviscerated and the remaining carcass fixed in neutral buffered Formalin prior to weighing seminal vesicles, thymus, adrenal glands, spleen and preputial glands. Social rank at termination of the experiment was assessed on the basis of wounding: dominant males were unwounded, while subordinates were wounded primarily on the rump and/or tail. Degree of wounding on Day 7 of grouping was rated on a scale of 0 (none) to 4 (most severe). Wounding of each animal was assessed following evisceration and without knowledge of that animal's treatment identity. This experiment also was repeated, excluding the 15 min behavioral observations. Statistically, body and organ weights and wounding in the former and latter experiments failed to differ between experiments, and results from both experiments were combined for final analyses of organ, body and wounding data (n shown in Table 3 represents combination of a comparable number of animals from each of the two experiments). Another group of males, housed singly through 60 days of age and treated surgically as described above (n = 24), were paired for 10 min in a neutral cage with intact stimulus males. The latter

had been grouped 5 per cage from 21 through 40 to 45 days of age and thus were nonaggressive. Analyses of behavioral data incorporated Student's t-test or adjusted t-test where nonhomogeneity of variance was indicated, while mean body and organ weights were compared using analysis of variance. RESULTS During 15 rain pairings, pinealectomy of one or both pair members reduced aggressiveness compared to that seen in sham pairs (sham-sham) (Table 1). Mean latency to initiation of fighting was more than doubled in pairs comprised of either one pinx and one sham (pinx-sham) or both pinx (pinx-pinx) animals (p<0.05 in each case). Moreover, total amount of time spent fighting was reduced 35 to 40 percent among pinx-sham and pinx-pinx pairs (p<0.05 in each case), although the total number of fights was independent of pair composition. Pinx and sham males initiated first attacks during pairings with equal frequency, but sham males became dominant in 75 percent of pairs (adjusted X2 = 3.76, 1 dr; p<0.05). All indices of aggressiveness were independent of prior surgical treatment of animals initiating first attacks or obtaining dominance. In contrast to the above findings, pinealectomy failed to influence aggressiveness toward stimulus males (Table 2). Pinealectomy also was without effect on body or organ weights among singlycaged animals, except that adrenal glands were 24 percent lighter (p<0.05) than control (isolate) values (Table 3). In general, prolonged intermale aggression led to heavier spleens and lower body and thymus weights among both pinx and sham subordinate males (Table 3; p<0.05 or better in each case). However, pinealectomy was associated with a nearly twofold increase in severity of wounding (p<0.01) among subordinates, and a differential influence of chronic defeat on organ weight was apparent between pinx and sham animals (Table 3). Subordination increased absolute adrenal gland weights by 30 percent (p<0.01) in pinx males, but was without significant influence on adrenal weights in sham animals. Moreover, mean final body weight of pinx subordinates was 11 percent below that of sham subordinates (p<0.01). Thymuses from pinx subordinates also were 70 percent lighter than those from sham subordinates (p<0.001). Importantly, pineal ablation prevented any significant effect of prolonged subordination on seminal vesicle weights, while subordination among sham males induced a 25 percent loss (p<0.001) in mean

TABLE 1 EFFECTS OF PINEALECTOMY ON AGGRESSIVE BEHAVIOR DURING 15 MINUTE ENCOUNTERS IN WHICH PAIR MEMBERS BOTH WERE SHAM-OPERATED, PINEALECTOMIZED, OR ONE ANIMAL SHAM-OPERATED AND THE OTHER PINEALECTOMIZED. DATA ARE PRESENTED AS MEAN +-SE.

Pair Composition

Number of Pairs

Latency to Attack (sec)

Total Fights

Duration of Fighting

Sham-sham

9

40.8 -+ 11.7

12.5 -+ 2.3

137.9 -+ 30.7

Pinx-sham

17

93.2 -+ 17.2

11.1 -+ 1.0

89.1 +- 10.2

Pinx-pinx

9

109.1 -+ 31.0

11.5 -+ 2.1

80.5 -+ 22.1

PINEAL AND AGGRESSION

215 TABLE 2

ATTACK OF STIMULUS (GROUP-RAISED) MALES BY SHAM-OPERATED AND PINEALECTOMIZED ADULT MALE HOUSE MICE. DATA ARE PRESENTED AS MEAN ±SE.

Number of Pairs

Latency to Attack (see)

Sham

11

110.5 ± 25.4

8.6 ± 1.0

48.1 ± 9.7

Pinx

13

71.1 ± 24.2

10.4 ± 1.2

51.6 ± 8.6

Treatment

Total Fights

Fighting Duration (see)

TABLE 3

EFFECTS O F P I N E A L E C T O M Y O N M E A N (±SE) B O D Y A N D O R G A N WEIGHTS A N D W O U N D I N G A M O N G A D U L T H O U S E MICE G R O U P E D 3/CAGE (ALL A N I M A L S IN E A C H G R O U P W E R E O F SIMILAR SURGICAL T R E A T M E N T ) F O R SEVEN DAYS.

Organs (mg wet tissue) Treatment

n

Social Experience

Body (g)

Seminal Vesicles

Adrenals

Thymus

Preputials

Spleen

Wounding

Sham

21 18 22

Isolate Dominant Subordinate

29.9 ± 0.7 30.2 ± 0.6 28.0 ± 0.5

159.6 ± 6.4 151.4 ± 7.8 119.2 ± 8.1

6.1 ± 0.3 5.8 ± 0.3 6.8 ± 0.5

26.2 -+ 1.5 22.5 ± 1.5 13.9 ± 1.2

123.2 ± 4.9 138.6 ± 10.7 110.8 ± 6.7

70.0 ± 3.1 112.9 ± 8.1 151.5 ± 18.0

1.8 ± 0.2

Pinx

16 13 15

Isolate Dominant Subordinate

28.1 ± 0.5 28.9 -+ 0.7 24.8 ± 1.0

142.6 ± 7.4 158.5 ± 11.2 131.5 ± 8.3

4.6 ± 0.3 4.6 ± 0.2 6.0 ± 0.4

26.0 ± 2.1 19.9 ± 1.8 6.8 -+ 0.9

120.4 ± 14.0 143.0 ± 10.5 95.8 ± 11.0

68.4 ± 7.7 85.3 ± 10.4 123.5 _+ 16.5

3.1 ± 0.2

weight o f s e m i n a l vesicles. Significant ( p < 0 . 0 0 1 ) s p l e n o megally o c c u r r e d in s h a m d o m i n a n t s , b u t all o t h e r o r g a n weights for s h a m a n d p i n x d o m i n a n t s were statistically e q u a l t o t h e i r respective c o n t r o l s ( T a b l e 3).

DISCUSSION Intermale aggression in house mice requires integrity of the olfactory system [ 19], and various olfactory cues are of probable importance in eliciting fighting and in developing and maintaining social position [2]. Conversely, visual stimuli appear to be of littleimportance in intermale aggression per se, but it would probably be presumptuous to negate this sensory modality entirely [22]. Both visual and olfactory modalities are important in regulating gonadal function by either direct or indirect influence on the pineal gland [17], but previous studies have considered relationships between exteroceptive stimuli and pineal gland function exclusive of the social environment in which an animal fives. P r e s e n t d a t a e x t e n d earlier w o r k b y finking social stimuli, pineal g l a n d a n d g o n a d f u n c t i o n a n d b y d e m o n strating a role of t h e pineal g l a n d in m o d u l a t i n g i n t e r m a l e aggression a n d t h e e f f e c t s of f i g h t i n g a n d d e f e a t o n accessory sex organs. During a b r i e f p e r i o d f o l l o w i n g pairing o f previously isolated males, aggression was r e d u c e d w h e n o n e or b o t h

pair m e m b e r s were pinx. In c o n t r a s t , t h e level o f aggression was increased in c h r o n i c a l l y m a i n t a i n e d g r o u p s in w h i c h all males were p i n x , as e v i d e n c e d b y d i f f e r e n t i a l w o u n d i n g , b o d y a n d organ weights b e t w e e n p i n x a n d s h a m s u b o r d i nates. While these results are in a p p a r e n t c o n f l i c t , t h e y n o n e t h e l e s s i n d i c a t e a role o f t h e pineal gland in i n f l u e n c i n g d e v e l o p m e n t a n d m a i n t e n a n c e o f social h i e r a r c h y , at least within the confines of laboratory study. Although no quantitative d a t a o n aggression were p r o v i d e d , V a u g h a n a n d R e i t e r [ 2 4 ] have also n o t e d increased aggressiveness w i t h i n p i n x g r o u p s o f a l b i n o mice. Specifically, p i n e a l e c t o m y a p p e a r s to i n f l u e n c e aggressiveness i n d i r e c t l y , since p i n x a n d s h a m males in t h e p r e s e n t s t u d y failed t o d i f f e r in aggressiveness t o w a r d s t i m u l u s males a n d since p i n x a n d s h a m a n i m a l s i n i t i a t e d first a t t a c k s w i t h e q u a l f r e q u e n c y f o l l o w i n g pairing. Moreover, n o d i f f e r e n c e in l a t e n c y t o first a t t a c k e x i s t e d w h e n a t t a c k s were i n i t i a t e d by p i n x as c o m p a r e d t o s h a m males. A l t h o u g h speculative, pinealect o m y m a y d i r e c t l y o r i n d i r e c t l y alter p e r c e p t i o n or expression o f s e n s o r y cues, i n c l u d i n g a c t i v i t y p a t t e r n s [ 15,21 ], p e r t i n e n t t o eliciting aggression a n d d e v e l o p i n g a n d m a i n t a i n i n g p o s i t i o n in a social h i e r a r c h y . This i n t e r p r e t a t i o n appears w a r r a n t e d since, a l t h o u g h aggressiveness o f p i n x males a p p e a r e d u n a l t e r e d , s h a m males were d o m i n a n t in a significant m a j o r i t y o f pairs. However, o n e m u s t n o t o v e r l o o k t h e possibility t h a t aggressiveness o b s e r v e d d u r i n g

216

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brief encounters following isolation may not accurately reflect aggressiveness during chronic interaction between dominant and subordinate males. Although considerable evidence points to a possible relationship between pineal and adrenocortical function [ 17], results in general are equivocal in terms of the effects of pinealectomy on the zonae fasciculata and reticularis. For example, in rats adrenal hypertrophy may [7,26] or may not [9,20] follow pinealectomy, and Kinson et al. [9] found only a transitory rise in plasma corticosterone levels following surgical removal of the pineal gland. Assuming that weight of adrenal glands reflects functional history of the zonae fasciculata and reticularis, previous studies with group-housed adult male house mice [25] also indicate increased adrenocortical activity following pinealectomy. In direct contrast is the finding of pinealectomy-induced adrenal atrophy among singly-caged animals in the present study. Our data further indicate that conditions of housing of experimental animals may influence response of adrenal glands to pinealectomy, at least with respect to house mice. Although adrenal atrophy occurred among singly-caged males, no differences in mean adrenal gland weights existed between sham isolates, sham subordinates and pinx subordinates. Thus, effects of social interaction among grouped pinx males may confound efforts to elucidate pinealadrenocortical interrelationships.

Dysfunction of the hypophysio-gonadal axis and atrophy of seminal vesicles in subordinate male house mice are well-documented phenomena [3, 10, 11, 12], although behavioral-endocrine mechanisms promoting testis dysfunction remain unclear. Adrenocorticotropin and adrenal androgens inhibit reproductive organ growth of females, but have little or no effect in males, at least as evidenced by morphological criteria [6]. Moreover, Bronson [3 ] recently demonstrated a lack of correlation between plasma corticosterone and gonadotropin levels in dominant and subordinate males. However, the present study suggests a role of the pineal gland in linking psychosocial stimuli with testis dysfunction in that pinealectomy effectively prevented regression of seminal vesicles among subordinates. Seminal vesicle weights from both dominant and subordinate animals remained equal to those from singly-caged controls even though social stress was increased in pinx groups. Thus, social subordination in house mice may potentiate antigonadal properties of the pineal gland, though mechanisms through which this is achieved are unknown. Other factors capable of potentiating pineal gland function also have been described, and it has been postulated that these, including anosmia, malnutrition and neonatal androgenization, may act by increasing sensitivity of the brain to pineal inhibitory substances [ 16,18 ].

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15. Reiss, M., R. H. Davis, M. B. Sideman and E. S. Plichta. Pineal gland and spontaneous activity of rats. J. Endocr. 28: 127-128, 1963. 16. Reiter, R. J. The role of the pineal in reproduction. In: Reproductive Biology, edited by H. Balin and S. Glasser. Amsterdam: Excerpta Medica, 1972, pp. 71-114. 17. Reiter, R. J. and F. Fraschini. Endocrine aspects of the mammalian pineal gland: a review. Neuroendocrinology 5: 219-255, 1969. 18. Reiter, R. J. and S. Sorrentino, Jr. Factors influential in determining the gonad-inhibiting activity of the pineal gland. In: The Pineal Gland, edited by G. E. Wolstenholme and J. Knoght. London: Churchill, 1971, pp. 329-344. 19. Ropartz, P. The relation between olfactory discrimination and aggressive behavior in mice. Anim. Behav. 16: 97-100, 1968. 20. Roth, W. D. Comments on J. Ariens Kappers' review and observations on pineal activity. Am. Zool. 4: 53-57, 1964. 21. Samson, P. H. and L. Bigelow. Pineal influence on exploratory behavior of the female rat. Physiol. Behav. 7: 713-715, 1971. 22. Scott, J. P. A~onistic behavior in mice and rats. Am. Zool. 6: 683-701, 1966. 23. Valzelli, L. Aggressive behavior induced by isolation. In: Aggressive Behavior, edited by S. Garattini and E. B. Sigg. Amsterdam: Excerpts Medica Foundation, 1969, pp. 70-76. 24. Vaughan, M. K. and R. J. Reiter. Transient hypertrophy of the ventral prostate and coagulating glands and accelerated thymic involution following pinealectomy in the mouse. Tex. Rep. Biol. Med. 29: 579-586, 1971. 25. Vaughan, M. K., G. M. Vaughan, R. J. Reiter and B. Benson. Effect on Melantonin and other pineal indoles on adrenal enlargment produced in male and female mice by pinealectomy, unilateral adrenalectomy, castration, and cold stress. Neuroendocrinology 10: 139-154, 1972. 26. Wurtman, R. J., M. D. Altschule and U. Holmgren. Effects of pinealectomy and of a bovine pineal extract in rats. Am. J. Physiol. 197: 108-110, 1959.