Olfactory bulb removal: Influences on the aggressive behaviors of male mice

Physiobgy and Behhavior.Vol.7, pp. 889-892. Pergamon Press. 1971. Printed in Great Britain

Olfactory Bulb Removal Influences on the Aggressive Behaviors of Male Mice’ :

FRANK

A. ROWE8 AND DAVID A. EDWARDS

Department of Psychology, Emory University, Atlanta, Georgia 30322

(Received 2 July 1971) Rows, F. A. AND D. A. EDWARDS. OIfactory bulb removal: Influences on the aggressive behaviors of male mice. P~SIOL. BEHAV.7 (6) 889-892, 1971.-Biiteral removal of the olfactory bulbs of castrated male mice completely prevented the arousal of aggressiveness by exogenous administration of androgen. Unilateralally bulbectomized mice showed fighting comparable to that shown by sham-operated control mice. It was concluded that earlier demonstrations of the abolition of intermale aggressive behavior in mice following olfactory bulb removal could not be attributed to impairment in pituitary-got&al function. Although in thii experiment bulbectomy completely prevented the androgenic arousal of intermale aggression, bulbectomy did not affect the display of aggressive behavior in a competition for food situation. Mice

Aggression

Olfactory bulbs

Androgen

IT IS WELL established that intermale aggressive behavior in mice is, at least partially, under hormonal control. Castration of male mice prior to the onset of puberty results in the absence of spontaneous fighting; replacement therapy with adrogen results in a striking increase in aggressiveness in these castrates [23. Several years ago, Ropartz 191 reported that bilateral removal of the olfactory bulbs of highly aggressive male mice resulted in the total abolition of intermale aggressive behavior. Although Ropartz concluded that the olfactory bulbs constituted a part of some central neural mechanism involved in the control of aggressive behavior, the deRcits he observed could have been due to impaired gonadotrophic function affecting the androgenic secretory activity of the testes. The primary purpose of the present experiment was to determine the extent to which bilateral and unilateral removal of the olfactory bulbs would impair the arousal of aggressiveness in castrated male mice administered exogenous androgen. Second, provided that deticits in intermale aggression in bulbectomized mice could not be ameliorated by replacement therapy with androgen, to what extent would similar impairments be evident in forms of aggressive behavior less susceptible to hormonal influences? Edwards [21 demonstrated that many gonadectomixed male and female mice would vigorously attack a younger, and consequently much smaller, opponent even in the absence of replacement therapy with androgen, although they would not attack an adult opponent of the same size. Fred&son [4] demonstrated that food deprived female mice, normally not highly aggressive towards each other, would vigorously bite and wrestle each other in competition for a small piece of food. To what extent fighting

against a small opponent, and fighting in a competition for food situation be a&ted by either bilateral or unilateral removal of the olfactory bulbs? In brief, our experimental strategy involved the administration of exogenous androgen to castrated non-aggressive male mice which were either bilaterally bulbectomized, unilaterally bulbectomized sham-operated, or unoperated. These mice were tested for aggressive behavior against an adult opponent of the same size. Following the completion of these tests, mice were tested for aggressive behavior towards a younger and much smaller opponent. Finally, all mice were deprived of food, and tested in within group pairs, for fighting in a competition for food situation. MATERIALS AND PROCEDURE Male mice of the Swiss-Webster albino strain were commercially obtained (Marland Farms, N.J.) at about 25 days of age. At 30 days of age all mice were castrated under ether anesthesia and were caged in groups of 5 until they were 60 days of age. At this time, all experimental mice were isolated in indivdual stainless steel pans. On the 6th and 7th days of isolation, each mouse was tested for the display of aggressive behavior towards a non-experimental stimulus male. Stimulus males were drawn from a population of males rendered nonaggressive by castration at 30 days of age, and were of the same strain, age, and approximate weight as the experimental males. A single test for aggression consisted of placing the experimental mouse in a small (4 in. x 10 in. x 5 in. deep) stainless steel pan, identical in construction to the home cage of the

‘This work was supported by research grant MH 18791 from the National Institutes of Mental Health to DAR. FAR was supported by an NDRA predoctoral fellowship. Testosterone propionate was provided through the courtesy of Dr. Preston Perlman of the Schering Corporation, Bloomfield, New Jersey. *Present Address: Department of Psychology, Illinois Institute of Technology, Chicago, Illinois 60616. 889

890 experimental male. A stimulus male was then introduced into this cage. An attack was scored if the experimental male repeatedly lunged at, and bit, the stimulus male. Once an attack was scored, the stimulus male was removed from the cage. If no attack was observed within 10 min of starting the test was scored negative and was terminated. A few experimental males fought on one or both of these tests, and were eliminated from the rest of the study. The remaining mice were assigned to one of four groups. Some males were bilaterally bulbectomized; others were unilaterally bulbectomized; others were sham operated; and still others served as unoperated controls. All surgery was performed under ether anesthesia preceded by an intraperitoneal injection of atropine sulphate. Once anaesthetized, the mouse's head was secured in the stereotaxie instrument and an incision was made in the skin overlying the frontal bone. The bone was thinned with a dental burr, and then removed with forceps to expose the olfactory bulbs. The bulbs were then aspirated either unilaterally or bilaterally, Gelfoam was packed in the resulting space to control bleeding, and the wound was closed with sutures. An identical procedure was employed for mice in the sham-operated group with the exception that the olfactory bulbs were left intact. Two weeks following surgery, all mice were injected with corn oil daily for one week, and were tested for aggressive behavior on the 6th and 7th days of this week. On the subsequent 4 weeks, all experimental mice received daily injections of 25, 50, 100 and 500 ~g testosterone propionate (TP) respectively, and were tested on the 6th and 7th days of each week for fighting. As described above, all experimental mice were tested against adult stimulus males. On the two days following the last test for aggressive behavior against the adult stimulus castrates, all mice were tested in a similar fashion against male mice of between 25-30 days of age. These stimulus males were about one half to one-third smaller by weight than the experimentaI males. Following the second test for aggressive behavior against the small opponent, all experimental mice were deprived of food for 48 hr. Mice within each surgical group were then paired, on the basis of comparable weight, and placed together in a situation designed to produce fighting in competition for food. In this test, pairs of mice were placed in a neutral cage and a small pellet (1 g) of lab chow was introduced into the cage. This pellet was small enough so that it could be effectively controlled by only one member of the pair at a time. This test was continued for 10 min. During this test we noted which (if any) members of each pair initiated aggressive responses (lunging and biting) related to control of the food pellet, and whether or not fighting also occurred which was not apparently related to the control of the food pellet. All behavior tests were conducted during the dark phase of a reversed lighting cycle. All hormones were delivered in a volume of 0.05 ml corn oil, and were administered subcutaneously. At the conclusion of behavioral testing, all bilaterally and unilaterally bulbectomized mice were sacrificed with an overdose of sodium pentobarbitol. These mice were perfused intracardially with normal saline followed by 10 per cent formalin. Following perfusion mice were decapitated and the heads soaked in formalin for several days. Brains were then removed from the skull casing, and the extent of brain damage was determined by examination of the whole brains under a dissecting microscope, and later by microscopic examination of 30 ~ sagital sections stained with cresyl violet.

ROW[! AND EDWARDS RESUL'[S Since there were no apparent differences in the aggressive performances of the sham-operated and unoperated mice, data from these groups were combined into a sham-normal group for comparison with unilaterally and bilaterally bulbectomized groups. Following the administration of TP 88 percent of the sham-normals fought against an adult opponent at least once, and 83 per cent of the unilaterally bulbectomized males fought at least once. in contrast, none of the bilaterally bulbectomized males fought against an adult opponent. As shown in Fig. 1, as TP dose was increased each week the percent of sham-normals and unilaterally bulbectomized mice displaying aggression correspondingly increased. Once a mouse began to fight, it usually continued to fight on most subsequent tests. The close correspondence of the curves for the sham-normal group and the unilateral group indicates that mice in these groups were little different from each other in their response to exogenous androgen administration. Bilaterally bulbectomized mice, however, appeared totally unresponsive to androgen administration, failing to fight even under the highest androgen dose.

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FIG. 1. The per cent of the total number of castrate male mice in each group fighting at each dose level of androgen. Different groups of males were either bilaterally bulbectomized (BL), unilaterally bulbectomized (UL), or sham operated o r unoperated (S + N) prior to androgen administration. At a given dose level, data points reflect the percent of mice in each group which fought on at least one of the two tests at that dose level. The abscissa shows the dose levels of testosterone propionate employed.

Results similar to these were obtained when mice of each group were tested for aggressive behavior towards a much smaller opponent. Bilaterally bulbectomized mice failed to fight, while a relatively high percent of males in the s h a m normal group and the unilateral group fought (Table 1), In contrast to the results obtained when mice were tested against the same size or smaller opponent, in the competition for food tests 70 per cent of the bilaterally bulbec!omized mice showed at least one instance of initiating aggression in,

AGGRESSION

AND

OLFACTORY

TABLE

BULBS

1

FIQHIING BEHAVIOROP CASTRATEDSTALEMICE GIVEN REPLACEMENTT-Y WITH EXOOENOUS ANDROOEN. PRIORTO HORMONE TREATMENT MICE WBREEITHERUNILATERAUY (UL) OR BILATERALLY (BL) BULBECTOMIZED, OR WERE SHAI)(OPERATEDOR UNOPERATED (S + N). MICE WERE TESTEDFIRST AGAINST AN ADULT OPPONENT,SECONDAGAINST A YOUNGER AND SMALLEROPPONENT AND FINALLYIN A COMPETITION M)R Foot SIIIIAIION

Per cent Fighting Group

n

Adult Small vs. Opponent vs. Opponent

Food Competition*

8

88

85

15

UL

12

83

15

80

BL

12

S+N

0t

0t

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*Data is combined for two independent replications of this experiment. Because of an odd number of mice in operated groups

in each replication, not all mice could be paired for a food competition test. Percentages for this test are based upon n=lO, 10 and 8 for BL, UL, and S+ N groups respectively. tbrdicates significantly different from S + N group at beyond the 0.01 level by Chi-square analysis of frequencies employed in calculating percentages.

as best we could judge, direct competition for control of the food pellet. In this resepct, bilaterally bulbectomized males appeared little different from the sham-normal and unilaterally bulbectomized males (Table 1). In most cases, the initiator of an attack was the member of the pair which was not in control of the pellet; attack usually followed an unsuccessful attempt to pull the food pellet away from the other member of the pair. Occasionally, however, a mouse would defend his control over the pellet by attacking the other member of the pair when that member attempted to pull the pellet away. During the competition for food tests we noted many instances of fighting which, in our judgement, were nor related to the control of the food pellet. These instances were restricted to pairs of sham-normals and unilaterally bulbectomized pairs.

HISTOLOGY

Examination of whole brains and of cresyl violet stained saggital sections revealed a variable amount of olfactory bulb removal in both unilaterally and bilaterally bulbectomized males. In mice from the bilaterally operated group bulbectomies ranged in completeness from total removal of both olfactory bulbs and olfactory peduncles to removal of only the rostra1 third of both bulbs. In the case of one male in which tissue removal included both olfactory peduncles there was slight unilateral damage to the prepiriform cortex. Unilateral bulbectomies typically involved one complete bulb and a varying amount of its peduncle. In no instance was there complete unilateral removal of the olfactory peduncle. In two of the unilateral males the medial surface of the rostal pole of the remaining bulb was slightly damaged. Damage to the neocortex was present in five males (four

bilateral and one unilateral). In all instances it was restricted to the neocortex of the ventral surface of the frontal poles. In no instance was damage to subcortical structures observed. DISCUSSION

The present data clearly demonstrate that bilateral ablation of the olfactory bulbs prevents the androgenic arousal of aggressive behavior in male mice. This tinding is in accord with Ropartz’s [9] earlier demonstration that bulbectomy abolishes intermale aggressiveness in intact male mice, and indicates that the abolition of aggressiveness observed by Ropartz can probably not be ascribed to altered pituitarygonadal function. Research on the behavioral effects of anosmia in rodents has generally employed the technique of surgical removal of the olfactory bulbs [7, 8, 91. Where behavioral deficits following surgery are observed, however, it is not clear whether such deficits occur as a consequence of anosmia per se, or as the consequence of removal of neural tissue more directly involved in the mediation of the behavior in question. With respect to intermale aggression in mice, Ropartz [9] has argued that the olfactory bulbs are part of some larger neural system involved in the control of aggressive behavior, and that deficits in fighting observed following bulbectomy reflect disruption of this mechanism. In the present experiment, unilateral bulbectomy failed to affect intermale aggressive behavior. If the bulbs do constitute some part of a neural mechanism for aggression, it is apparent that the activity of a single bulb is sufficient for the support of vigorous fighting. On the other hand, since the unilaterally bulbcctomized males were presumably not entirely anosmic, our data is not inconsistent with the notion that anosmia is the condition crucial for the abolition of intermale fighting. Obviously, resolution of the anosmia vs. neural mechanism question depends on the development of techniques for rendering mice anosmic without removing the olfactory bulbs. Alberts [l] has recently reported that anosmia produced by an intranasal application of a zinc sulphate solution abolished territorial aggression in wild rats. The use of this technique in mice might help to clarify the nature of the olfactory control of aggressive behavior in mice, and work along this line has already been started in our laboratory. Our failure to arouse aggressiveness in castrated bulbectomized male mice is consistent with the somewhat novel notion that the olfactory bulbs constitute part of some androgen sensitive mechanism involved in the control of aggressive behavior in male mice. This hypothesis is probably not tenable since bulbectomized mice failed to light when tested against a much smaller opponent, a circumstance in which fighting has been shown to be somewhat independent of hormonal state [2]. Our present view is that bulbectomy results in a general deficit in most forms of social behavior in mice. Gandleman [5], for example has reported that maternal behavior in mice is totally abolished following bilateral removal of the olfactory bulbs. Bilateral removal of the olfactory bulbs totally abolishes copulatory behavior in male mice (Rowe and Edwards, unpublished observations). In addition, bilaterally bulbcctomized female mice appear incapable of displaying sexual receptivity in response to exogenously administered ovarian hormones (Thompson and Edwards, unpublished observations). It seems reasonable to presume that specific and separate neural mechanisms exist for the

892

ROWE AN[) El)WARDS

mediation of aggressive, sexual, and maternal behavior respectively in male and female mice. The finding that removal of the olfactory bulbs is followed by deficits in all of these behaviors suggests the view that olfaction may be a condition necessary for the occurrence of most social behaviors, but that the bulbs may not be involved in the specific mediation of any one of these behaviors. One further point is worthy of consideration. The term aggression has been used to describe a variety of behaviors occurring in a variety of contexts. These contexts include: intermale fighting among mice [2, 9]; shock induced sparring and biting in rats and mice [10, 11]; mouse killing behavior in rats [6]; and struggling among food deprived mice in a competition for food situation [3, 4]. It is almost certainly true that, despite the common label of aggression, these behaviors do not share a common neural mediating mech-

anism. A case in point is the example of fighting in competition over food among mice. Conditions necessary for the display of such fighting include a period of food deprivation and the presence of a small amount of food in the test situation. Unlike more spontaneous fighting among mice, fighting in competition for food appears to be relatively independent of the hormonal state of the competing mice, and sex differences in competitive fighting are not observed [3, 4], In addition, data from the present experiment demonstrates that competitive fighting among mice is not affected by removal of the olfactory bulbs, although more spontaneous intermale fighting is totally abolished by bulb removal. Thus, although stereotyped fighting may occur in a variety of contexts, this stereotypy is not sufficient grounds for presuming a neural mediating system common to all these contexts.

REFERENCES

1. Alberts, J. R. Olfactory control of territorial aggression in wild rats. Paper presented at the Eastern Psychological Association meeting, New York, 1971. 2. Edwards, D. A. Early androgen stimulation and aggressive behavior in male and female mice. Physiol. Behav. 4: 333-338, 1969. 3. Fredericson, E. The effects of food deprivation upon competitive and spontaneous combat in C57 black mice. J. Psychol. 29: 89-100, 1950. 4. Frederic.son, E. Aggressiveness in female mice. J. comp. physiol. Psychol. 45: 254--257, 1952. 5. Gandelman, R., M. X. Zarrow, V. H. Denenberg, and M. Myers. Olfactory bulb removal eliminates maternal behavior in the mouse. Science. 171: 210-211, 1971. 6. Karli, P., M. Vergnes and F. Didiergeorges. Rat-mouse interspecific aggressive behavior and its manipulation by brain ablation and by brain stimulation. In: Aggressive

7. 8. 9. 10.

11.

Behavior, edited by S. Garattini and E. B. Sigg. New York: John Wiley, 1969, pp. 47-55. Larsson, K. Failure of gonadal and gonadotrophic hormones to compensate for an impaired sexual function in anosmic male rats. Physiol. Behav, 4: 733-737, 1967. Murphy, M. R. and G. E, Schneider. Olfactory bulb removal climates mating behavior in male hamsters. Science 167: 302-303, 1970. Ropartz, P. The relation between olfactory stimulation and aggressive behavior in mice. Anita. Behav. 16: 97-100, 1968. Tedeschi, R. E., D. H. Tedeschi, A. Mucha, L. Cook, P. A. Mattis, and E. J. Fellows. Effects of various centrally acting drugs on fighting behavior of mice. J. Pharmac. exp Ther. 125: 28-34, 1959. Ulrich, R. Pain as a cause of aggression. Ant. ZooL 6: 643-662, 1966.