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Learned aversion to the odors of male mice: Effects on agonistic behavior
Physiology & Behavior, Vol. 27, pp. 1%25. PergamonPress and BrainResearch Publ., 1981. Primedin the U.S.A.
Learned Aversion to the Odors of Male Mice: Effects on Agonistic Behavior T H O M A S F. S A W Y E R
Psychology Department, North Central College N a p e r v i l l e , I L 60566 R e c e i v e d 24 D e c e m b e r 1980 SAWYER, T. F. Learned aversion to the odors of male mice: Effects on agonistic behavior. PHYSIOL. BEHAV. 27(1) 19-25, 1981.--Intact and castrate male mice were subjected to avoidance training using the urine of intact and castrate donors as stimuli. Both intacts and castrates learned to discriminate between the odors, with such learning modifying their spontaneous responsiveness to such odors in an open field in the predicted direction. This result suggests that the previously reported lack of responsiveness in castrates [15] was not due to a simple sensory deficit. A second experiment determined that increasing the relative aversion exhibited by intact subjects to the odors of intact donors also increased the subject's aggression directed at a castrate opponent swabbed with such odors. Modification of castrates' responsivity had no such effect on their aggression. This indicated that replacing the responsivity to aggression-related odors which is lost following castration, is not equivalent to replacing the hormone. The results are discussed with regard to possible hormonal effects on the social significance of olfactory cues to the recipient animal. Aggression
Aversion
Mice
Olfaction
Urine
A N U M B E R of investigators have found the urine of aggressive male mice to be aversive to male conspecifics [7, 9, 14]. Also, a recent study found that castration of subjects eliminated their tendency to avoid the side of an open field spotted with urine, while testosterone treatment replaced the response, clearly demonstrating the androgen-dependent nature of the aversion [ 15]. Analogous evidence concerning the responsiveness of male rats to the odors of females showed castrate males to exhibit less investigation of the odors than did intacts, as well as failing to display the normal preference for the odors of receptive over nonreceptive females [3,4]. However, it appears that the effects of castration do not generalize to another type of odor excreted by mice, namely, alarm or stress-related odors [15]. It was found that both intact and castrate male mice avoided the urine from severely stressed castrate donors. These results suggest that male gonadal hormones may play an important role in sensitizing the particular systems concerned with responsivity to socially relevant stimuli, such as those involved in agonistic and sexual interactions. And furthermore, these results suggest that the mechanism behind the effects of gonadal hormones on rodent aggression and sexual behavior may lie in part in their influence on the response to the relevant olfactory stimuli. The failure of an organism to exhibit a spontaneous response to a stimulus or a spontaneous discrimination between stimuli does not necessarily imply the organism is incapable of detection or discrimination [2]. Indeed, castrate rats failed to spontaneously discriminate between the odors of receptive and nonreceptive females [3,4], despite the fact
that thirsty castrates have been found to do so for water reward [2]. The first experiment of the present study sought to determine if castrate male mice are able to learn a discrimination between the urine of intact and castrate donors, and if so, whether such training would influence their subsequent spontaneous responsiveness to the odors.
EXPERIMENT 1 In this experiment a group of intact and a group of castrate subjects were trained to avoid shock by avoiding the side of a modified T-maze in which the urine of intact males was present, and approaching the side which contained the odor of castrate males. Another group of intacts and one of castrates were trained with the opposite requirements. The importance of using the urine of both intact and castrate donors should be noted. While it is quite possible that castrate males are able to detect the odors of the urine of intact donors, it is conceivable that they are unable to detect that quality that distinguishes it from that of castrates, which has previously been found not to be aversive [9,14]. That particular quality was suggested to be the secretion of the coagulating gland [8], an androgen-dependent gland that secretes directly into the urine. Thus, requiring a discrimination between the odors of intact and castrate donors would suggest any change in performance to be due to the discrimination of that characteristic of the urine that gives it its aversive quality. However, the two urine types may differ in other discriminable ways.
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 Inc.--0031-9384/81/070019-07502.00/0
20
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METHOD
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Respondill,q SIt/~je~l,~ The subjects were 60 Swiss-Webster male mice. After weaning at 25_+ 1 days of age they were housed in groups of four under a partially reversed 12:12 hr light-dark cycle with lights on at 01:00 hr and off at 13:00 hr. The cages were 31.4x 19.7x 12.7 cm mouse boxes with wire tops. Food and water were available ad lib.
Urine Dum)rs and Urine Collection The donors were 24 males obtained from the same stock as the responders. At 70 days of age 12 of the donors were castrated via scrotal incision under ether anesthesia; the remaining 12 were sham-operated. Urine was collected using a method similar to one previously described [15]. Briefly, donors were housed individually in a metabolism cage for three weeks following surgery. Urine was obtained over a 16-18 hr period during which time food was removed to prevent contamination of the urine. The urine was stored in airtight glass receptacles and was used within 7 hr of collection. A minimum of 72 hr elapsed between each occasion of urine collection.
1
Training Procedure At approximately 70 days of age two subjects from each box of four were chosen at random and castrated. The remaining subjects were sham-operated. Twelve of the intact subjects were subsequently trained to avoid the intact urine and approach the castrate urine (intact avoid group, or IAV), while 12 were trained with the opposite requirements (intact approach, or lAP). Likewise, 12 castrates were trained to avoid (CAV) and 12 to approach (CAP) intact urine. The remaining intacts and castrates were " t r a i n e d " with no stimuli present (NS). Sixteen days after surgery the avoidance training began. This training consisted of 80 trials distributed across four sessions. Within a session, a subject
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Training Apparattts Avoidance training took place in an apparatus designed for use with olfactory stimuli. As pictured in Fig. I, it consisted of a start box (11 x 14x 15 cm) which opened out to two goal boxes (each 18x 14x 15 cm). The wall between the goal boxes contained a hole permitting movement of the subject from one side to the other, and yet could be closed off through the use of a guillotine door. Likewise, pushing the movable back wall of the start box closed this section off from the goal boxes, as well as forcing a subject in the start box to move from it. Located on either side of the start box were removable stimulus boxes (1 l x 7 x 15 cm) within which stimulus odors were placed. Holes at the end of each goal box provided output of both air and odors to an adjacent enclosure (30x30x30 cm). The air contained in this enclosure was drawn out via an exhaust blower and exited the room through tubing attached to the exhaust vent in the ceiling of the room. Thus, while the exhaust blower was on, air passed through the stimulus boxes, into the goal boxes, out of the test chamber, and finally out of the room. Auxiliary exhaust fans provided further replacement of air to the apparatus between trials. The apparatus was made of Plexiglas and rested on a grid of 0.158 cm diameter rods through which shock was delivered. The shock source was a BRS/Foringer, Model No. SG-901.
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was given 10 trials with an intertrial interval of 60 sec, followed one hr later by another 10 trials. Forty-eight hr elapsed between each session. The intact urine stimuli were obtained from sets of four intact donors, with each animal contributing 0.20 ml to the pool. Castrate stimulus odors were obtained from analogous sets of castrate donors. Pooling of the urine prevented subjects from discriminating between odors on the basis of individual differences [5], and has previously been determined not to effect the aversive quality of the urine [10]. The urine stimuli were presented by applying 0.25 ml of one type to an absorbent cotton ball in one of the stimulus boxes. The other type was presented in a similar fashion in the other stimulus box.
L E A R N E D AVERSION AND AGONISTIC BEHAVIOR Each trial began by placing the subject in the start box. If after 15 sec a choice (defined as the entire body, excluding the tail, entering a goal box) was not made, one-half of the start box was closed off, which forced the subject to the choice point, but did not necessarily force a choice. If another 15 sec elapsed, the subject was forced into a goal box by completely closing off the start box. Entry into the correct side resulted in removal from the apparatus, while entry into the incorrect side resulted in a two-sec 0.4 mA scrambled shock. The guillotine door separating the goal boxes was then raised and the subject was required to escape the shock by moving to the correct side. Unforced entry into a goal box resulted in closing of the start box and either removal or shock as in forced entry. Essentially, the procedure was a forced choice, correction procedure. Following each trial the stimulus boxes were removed and the auxiliary fans were turned on for 45 sec. Also, absorbent paper beneath the apparatus was replaced with a clean piece. Approximately 15 sec before the next trial the stimuli were replaced and the auxiliary fans turned off. The location of the stimuli was determined by a Gellerman series in which strategies such as alternation or win-stay, lose-shift were unsuccessful in producing performance above 50% correct. Subjects in the NS group were "trained" using an identical procedure except for the use of distilled water in place of the urine.
21
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Aversion Testing Aversion testing was conducted 48 hr after the last training session. The apparatus and procedure has been described and used successfully in previous studies [ 14,15]. Briefly, ten spots of urine from the intact pool were distributed approximately equidistant throughout one-half of a 45 x 45 cm open field, and 10 spots of urine from the castrate pool were placed on the other half. The subject was placed in the center of the field and the time spent on the side to which the castrate urine had been applied was measured over a 300 sec period. The testing took place in a dark room under red light illumination 0.5-5 hr into the dark part of the cycle. RESULTS AND DISCUSSION
The results of a 5x8 (treatment group x trial block) Analysis of Variance (ANOVA) for the training data revealed a highly significant treatment group by trial block interaction, F(28,385)=5.38, p<0.001. This demonstrates that the trend in the mean number of choices toward the castrate odor across the blocks varied as a function of treatment group. As plotted in Fig. 2, the NS group exhibited a flat function at about 50% toward the castrate side, while the AV groups (IAV and CAV) exhibited a consistent increase in the number of responses toward the castrate odor across blocks, and the AP (lAP and CAP) showed a consistent decrease. Analyses of the simple effects of treatment group at the various trial blocks using the Satterthwaite correction for repeated measures [18] indicated no difference at the first block, F(4,413) = 2.21, p >0.10, while there was a substantial effect at all other blocks, all F's>7.25, all p's<0.001. Post hoc analyses using Scheffe's S-method [11] indicated that while IAV and CAV did not differ from one another at any block, their mean differed from that of the NS group at blocks three through eight (critical mean difference = 1.37, c~=0.05). Likewise, while IAP and CAP were not found to differ at any block, they differed from that of the NS group, and hence the more extreme IAV and CAV groups, on
FIG. 2. Mean number of choices toward the side to which castrate urine was applied as a function of treatment group and trial block (chance response is approximately 5 per block).
blocks four through eight. Thus, both AV groups learned to avoid the intact odor, while both AP groups avoided the castrate odor, with no evidence of any hormonal effect. An ANOVA performed on the number of correct choices, rather than the number toward the castrate side, for the subjects in the four trained groups indicated only a significant main effect of trial block, F(7,308)= 19.56, p<0.001. Trend analysis revealed a significant linear trend, F(1,44)= 123.32, p<0.001, with no higher order trends reaching significance. That is, all groups learned the discrimination, and did so approximately equally well. It is interesting that it was no more difficult to train the lAP males to approach the odor of an intact, an odor which is itself somewhat aversive, than it was to train the IAV males to avoid the odor. This result is likely due to the extreme aversiveness of the shock relative to the mild aversiveness of the odor.
Aversion Random movement by the subjects during aversion testing would result in approximately 150 sec spent on each side of the field. However, if one urine type was more aversive one would expect deviation from this value. The amount of time spent on the castrate side of the field as a function of shock contingency and hormonal status is provided in Table 1. An ANOVA revealed a pronounced main effect of shock contingency, F( 1,44) = 137.04, p <0.001. That is, the training was found to generalize to the aversion test with both AV groups showing an aversion and both AP groups an attraction to the intact odors.
22
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TABLE I MEAN TIME SPENTON THE CASTRATESIDE OF 3HE ()PEN FIELD AS A FUNCTIONOF SHOCK CONTINGENCYAND HORMONE STATUS" Shock Contingency
AV AP
Hormone Status Intact
Castrate
204.32 _+ 6.49+ 108.93 _+ 8.33+
191.28 ± 9.66+ 107.16 ± 5.51;
Mean time=sec _+ standard error. *p values refer to comparisons with random response value of 150 sec and were determined by t-tests. +p<0.001.
Thus, Experiment 1 was successful in answering two questions. First, castrates are able to learn a discrimination between the odors of intacts and castrates, which suggests that the lack of spontaneous responsiveness in castrates is not due to a sensory deficit. And second, the responsivity is easily modified via training, with the change persisting for at least 48 hr. EXPERIMENT 2 Previous research has determined that varying the nature of the odor of an opponent animal can modify the agonistic behavior of a subject [12]. Likewise, gonadal hormones may have an effect on the nature of an animal's response to urine odors and thereby influence agonistic interactions. Implicit in this suggestion is the notion that variability in the responsivity of male mice to aggression-related odors should cause variation in their agonistic behavior just as varying the odor does. Evidence supportive of such a relationship, but not conclusive, consists of: the finding of a differential responsivity to odors by dominant and subordinate mice [9] which are also known to differ in agreesiveness [16], the lack of responsiveness in castrates [15] which also generally lack aggressiveness [1], and the finding of individual differences in responsivity that were predictive of the outcome of an agonistic encounter [14]. The second experiment was designed tb determine the effect of experimental manipulation of the responsiveness of males to a standard urine odor on their agonistic behavior when confronted with an animal having the odor. METHOD
Responding, Subjects The subjects were 112 male mice obtained from the same stock and housed in groups of four under the same conditions as in Experiment I.
Donor and Opponent Animals The donors were 24 males housed individually in a metabolism cage for a minimum of three weeks prior to the onset of urine collection. The opponents used during aggression testing were 112 males housed under the same conditions as the responders. At 35 days of age each opponent was castrated. They were used when 65-75 days of age.
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At approximately 70 days ot" age 56 of the subjects ~erc castrated, while the remaining 56 were sham-operated. The intacts were assigned to one of four groups, three of which were treated as the lAP, IAV, and NS groups of Experimenl I. A fourth group (IS'F) was ' t r a i n e d " with the odor present, but no shock. That is, a subject from the [ST group was allowed to make a choice and was then removed from the goal box as if the choice were correct. The 56 castrates were assigned to analogous groups designated CAP, CAV, CNS, and CST. Approximately 20 days after surgery, training began. It was conducted as previously described, except that only the urine from intact donors was used. The other stimulus box contained an equivalent amount of distilled water. Each subject received a total of 80 trials as described in Experiment I. Aversion and aggression testing began 48 hr after the last training session. Half of the subjects in each group were given the aversion test first, followed 80-90 rain later by the aggression test. The remaining subjects received the tests in the opposite order. Aversion testing proceeded as described in Experiment I. During the aggression phase, each subject was tested for aggression directed toward an opponent swabbed with 0.06 ml of urine. A different opponent was used during each test in order to eliminate possible effects of prior use on the aggression of a subject. A particular subject was confronted with urine taken from the same pool for both behavioral tests. The procedure for aggression testing consisted of initially placing a subject in a mouse box with clean bedding on the floor. A Plexiglas cover was placed over the box, and a barrier was inserted through a slit in the top, dividing the box into two sections. The opponent was then swabbed with urine and placed in the box on the side opposite that of the subject. The barrier was then lifted and the test began. Frequency and total time the subject engaged in the following behaviors were recorded for 10 rain: investigating nosing, vigorous nosing, chasing, tail-rattling, and biting attack. Immediately after the session the subject was raled on the following scale, which is similar to one previously used [6]: 0---little or no nosing; l--occasional nosing; 2--frequent nosing; 3--fiequent vigorous nosing: 4--chasing and tailrattling; 5--biting attack, including wrestling: 6--frequent fierce attack, including biting and wrestling. All aggression testing took place 1-5 hr into the dark period under red light illumination. RESULTS AN[) DISCUSSION
Training,, The analysis for this phase was a 2 × 4 x 8 (hormone status × training condition x trial block) ANOVA. As expected, a pronounced training by trial block interaction was found, F(21,728)=6.87, p<0.001, which renders the main effects of training condition, F(3,104)= 86.44, p <0.001, and trial block, F(7,728)= 15.10, p<0.001, somewhat obscure. It is obvious from inspection of Fig. 3, which plots correct choices as a function of group and trial block, that the interaction was due to the fact that the AV and AP groups had learned the task, while the ST and NS groups had not. These results essentially replicate those of Experiment I.
A version A 2 x 4 x 2 ANOVA (hormone status x training condition
LEARNED AVERSION AND AGONISTIC BEHAVIOR
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× test order, i.e., before or after aggression test) revealed a significant hormone status by training condition interaction, F(3,96)=2.74, p<0.05. The data depicting this interaction are plotted in Fig. 4. Analyses of the simple effects of hormone status at different levels of training condition indicated that the interaction was due to differences between the intact and castrate subjects of the ST groups, F( 1,96) = 14.68, p <0.001, and NS groups, F(1,96) =9.37, p <0.005, while no differences were found between the AV groups, F(1,96)=0.59, p>0.20, or the AP groups, F(1,96)=0.05, p>0.20. Further analyses of the effects of training and hormone status were accomplished via post hoc comparisons. The two intact control groups (IST and INS) were found not to differ from one another, nor were the castrate controls (CST and CNS). Comparisons of IAV with the average of the intact controls indicated that training intacts to avoid the odor was effective in increasing the time spent on the clean side of the field, F(1,96)= 15.06, p<0.01. Likewise, the CAV group was found to exhibit a substantially greater aversion response than the castrate controls, F(1,96)=48.59, p<0.01. Also, the IAP group was found to exhibit a significantly lower aversion response than did intact controls, F(1,96)= 38.78, p<0.01, as did the CAP group when compared with the castrate controls, F(1,96)=9.18, p<0.01.
Aggression
FIG. 3. Mean number of correct choices as a function of hormone status, training condition, and trial block (chance response is approximately 5 per block).
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23
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AP
CONDITION
FIG. 4. Mean time (sec + standard error) spent on the clean side of the open field as a function of hormone status and training condition (random response is approximately 150 sec).
The analyses of the aggression test data were performed separately for the rating measure and the component behaviors. The mean aggression rating, as well as the number attacking, for each group is provided in Table 2. A 2 × 4 x 2 (hormone status x training condition × test order) A N O V A on the rating scale data revealed a significant effect of hormone status, F(1,96)=26.60, p<0.01, with intacts more aggressive than castrates. This result, which replicates the well-documented effect of castration on aggression [1], is further substantiated by the finding that 21 intacts attacked their opponent, while only 5 castrates did, X2(1)=8.65, p<0.01. An interesting effect of test order was also found, F( 1,96) = 6.49, p <0.05. While the difference was small, it was quite consistent, as those subjects receiving the aversion test first received higher scores in all but two groups (CAP and CNS), and in those there was no difference. The basis for such an effect cannot be determined at this time, but it is suggested that pre-exposure to the odors in the aversion test may have served a priming function, perhaps initiating a hormone release, which sensitized the subjects to such aggression-related odors. It should be noted such a priming effect could not have involved gonadal hormones, as the order effect was quite large in the CAV group. The effect of primary interest, namely training condition, was not found to be significant, F(3,96)= 1.76, p>0.10. However, a planned comparison between the two AV groups (average of 1AV and CAV) and the AP groups (average of IAP and CAP) revealed the AV subjects to exhibit a higher rating (3.00) than the AP subjects (2.14), F(1,96)=4.45, p<0.05. Further planned comparisons revealed that while the IAV group (4.14) had a higher rating than the IAP (2.57), F(1,96)=7.48, p<0.01, the same was not true for CAV (1.86) compared to CAP (1.71), F(i,96)=0.14, p>0.20. Thus, experimentally manipulating olfactory responsivity affects agonistic behavior in intacts but not castrates. Finally, a multivariate analysis of variance was performed on the frequencies and times for the various component be-
24
S A W Y b. R TABLE 2 MEAN AGGRESSION RATING AND THE NUMBER OF SUBJECTS THA 1 ATTACKED AS A FUNCTION OF HORMONE STATUS. TRAINING CONI)ITI()N, AND TEST ORDER ~ Hormone Status
Training Condition
"lest Order Aversion First + + ± +
Attacked
Aggression First
Attacked
Intact Intact Intact Intact
AV AP ST NS
4.71 3.29 3.00 3.71
0.77 0.80 0.74 0.93
5/7 3/7 2/7 4/7
3.57 1.86 2.86 2.57
± 0.84 + 11.59 + 0.59 +: 11.51
3/7 1"7 27 1/7
Castrate Castrate Castrate Castrate
AV AP ST NS
2.57 _+ 0.46 1.71 ± 0,46 1.86 + 0.59 1.71 ± 0,61
1/7 0/7 1/7 1/7
1.14 1.71 1.29 1.71
+ ± ± ±
0/7 1/7 11/7 1/7
0.15 0.15 0.20 0.61
Aggression rating: mean _+ standard error; n=7. *Ratings range from a possible low of 0.00 to a possible high of 6.00.
h a v i o r s , including the a t t a c k l a t e n c y (600 sec if no attack). A s u m m a r y o f t h e s e m e a s u r e s is p r o v i d e d in T a b l e 3. J u s t as for the rating m e a s u r e , a significant effect o f h o r m o n e s t a t u s was f o u n d , F ( 1 1 , 8 6 ) = 5 . 4 8 , p < 0 . 0 0 1 . U n i v a r i a t e A N O V A s r e v e a l e d t h a t h o r m o n e s t a t u s affected e a c h of the c o m p o n e n t b e h a v i o r s in t h e e x p e c t e d f a s h i o n , all p ' s < 0 . 0 5 . T h e multiv a r i a t e a n a l y s i s also i n d i c a t e d a n effect o f t r a i n i n g c o n d i t i o n , F(33,254)=2.09, p <0.01. U n i v a r i a t e A N O V A s r e v e a l e d t h a t t r a i n i n g i n f l u e n c e d i n v e s t i g a t i v e n o s i n g time a n d f r e q u e n c y as well as c h a s i n g t i m e a n d f r e q u e n c y , all p ' s < 0 . 0 5 , while its
effect on a t t a c k time a p p r o a c h e d but did not r e a c h c o n v e n tional levels o f significance, p < 0 . 1 0 . M u l t i v a r i a t e c o m p a r i s o n s indicated the c o m b i n e d A V g r o u p s were significantly different from the A P g r o u p s , F ( 1 1 , 8 6 ) = 2 . 0 3 , p < 0 . 0 5 , and the control g r o u p s , F(11,86)=3.87, p < 0 . 0 1 , o n n o s i n g time a n d f r e q u e n c y , c h a s i n g time and f r e q u e n c y , a n d a t t a c k time, all p ' s < 0 . 0 5 , while the A P g r o u p s did not differ from the c o n t r o l s . Lastly, while the effect o f test o r d e r a p p r o a c h e d significance, F(11,86)= 1.66, p < 0 . 1 0 , n o n e o f the i n t e r a c t i o n effects did so.
TABLE 3 MEAN VALUES (FREQUENCIES AND TIMES*) FOR EACH COMPONENT AGGRESSIVE BEHAVIOR Treatment Group
IAV lAP IST INS CAV CAP CST CNS
Component Behaviors Nose
Vig. Nose
Chase
Tail Rattle
Attack
Freq. Time Freq. Time Freq. Time Freq. Time
25.63 79.67 17.04 62.99 20.50 61.19 17.64 49.96
8.36 23.29 2.88 7.12 3.93 9.65 5.93 12.37
1.21 1.52 0.16 0.19 0.14 0.15 0.21 0.13
5.79 6,32 1,13 2,80 2.64 3,09 2.71 2.51
7.00 15.06 0.82 2.51 2.29 2.27 2.79 3.43
Freq. Time Freq. Time Freq. Time Freq. Time
19.71 53.14 16.29 56.29 13.64 48.75 10.29 24.44
1.57 2.59 0.57 2.04 0.64 1.51 1.00 2.80
0.14 0.07 0.00 0.00 0.00 0.00 0.07 0.04
0.36 0.30 0.00 0.00 0.21 0.51 0.29 0.33
0.21 0.19 0.29 0.33 0.50 0.84 0.86 1.32
*Time in seconds.
Attack Latency
381.40 558.80 479.50 459.21) 559.60 583.40 592.90 552.90
L E A R N E D AVERSION AND AGONISTIC BEHAVIOR G E N E R A L DISCUSSION The present study served to answer two primary questions. Experiment 1 demonstrated that the lack of responsivity of castrate mice to the odors of intact mice as previously reported [15] is not due to a simple sensory deficit, as castrates were able to learn a discrimination between such odors and the unaversive odors of castrates. Experiment 2 examined the extent to which manipulations that modify responsiveness to aggression-related odors also act to modify agonistic behavior. The results showed that modification upward in the aversion response of intact males was related to a higher aggression rating, and a greater incidence of attack when compared to intact males subjected to a downward modification in their response. This finding supports the notion that threat or fear of attack may be an important component of aggression in rodents [14,16]. However, with regard to castrate subjects, modification in the responsiveness to urine odors did not appear to be related to their agonistic behavior. That is, subjecting castrates to training that resulted in an "intact-like" aversion response to male odors, did not result in "intact-like" agonistic behavior. Thus, in terms of the effects of manipulating olfactory responsiveness on aggression, hormone status appears to have a pronounced effect. A possible explanation for the failure to influence the agonistic behavior of castrate subjects concerns what might be labeled the social significance of urine odors. Perhaps male gonadal hormones effect brain mechanisms that inter-
25 pret the significance of socially relevant stimuli [17]. Desensitizing such mechanisms via removal of the hormone could presumably alter spontaneous social responsiveness to various stimuli without affecting an animal's ability to learn responses to such stimuli. In other words, castration may have made neutral stimuli out of what were previously socially significant odors. And, while the avoidance training was able to replace the aversiveness of the odor to the castrate, it was not able to replace the social significance of the odors, and hence, could not replace the agonistic behavior. Another similar explanation for the findings of Experiment 2, is that removal of the gonads may have disrupted the normal activity of another "olfactory system." This suggestion is stimulated by recent evidence demonstrating the presence of a system, namely the vomeronasal system, that is sensitive to socially relevant odors, particularly sexrelated odors [13, 19, 20]. Assuming an effect of gonadal hormones on this system, as well as its sensitivity to aggression-related odors, can explain the failure of castrates to show intact-like aggression. Again, the integrity of the olfactory system could have mediated the learning of the aversion to the odor without influencing the social significance, and hence the agonistic responses toward an animal having the odor. Future research is needed to determine the adequacy of these explanations, as well as other unresolved issues, such as the basis for the test order effect on aggression as found in Experiment 2.
REFERENCES
1. Beeman, E. A. The effect of male hormone on aggressive behavior in mice. Physiol. Zool. 20: 373-405, 1947. 2. Carr, W. J. and W. F. Caul. The effect of castration in the rat upon the discrimination of sex odours. Anita. Behav. 10: 20-27, 1962. 3. Carr, W. J., L. S. Loeb and M. L. Dissinger. Responses of rats to sex odors. J. comp. physiol. Psychol. 59: 370-377, 1965. 4. Carr, W. J., L. S. Loeb and N. R. Wylie. Responses to feminine odors in normal and castrated male rats. J. comp. physiol. Psycho1. 62: 336-338, 1966. 5. Hahn, M. E. and E. C. Simmel. Individual recognition by natural concentrations of olfactory cues in mice. Psyehon. Sci. 12: 183-184, 1968. 6. Hyde, J. S. and T. F. Sawyer. Estrous cycle fluctuations in aggressiveness of house mice. Hormones Behav. 9" 290-295, 1977. 7. Jones, R. B. and N. W. Nowell. Aversive and aggressionpromoting properties of urine from dominant and subordinate male mice. Anim. Learn. Behav. 1: 207-210, 1973. 8. Jones, R. B. and N. W. Nowell. The coagulating gland as a source of aversive and aggressive-inhibiting pheromone(s) in the male albino mouse. Physiol. Behav. 11: 455-462, 1973. 9. Jones, R. B. and N. W. Nowell. Effects of androgen on the aversive properties of male mouse urine. J. Endocr. 60: 19--25, 1974. 10. Jones, R. B. and N, W. Nowell. A comparison of the aversive and female attractant properties of urine from dominant and subordinate male mice. Anim. Learn. Behav. 2: 141-144, 1974.
11. Kirk, R. E. Experimental Design: Procedures for the Behavioral Sciences. Belmont, CA: Wadsworth, 1968. 12, Mugford, R. A. and N. W. Nowell. Pheromones and their effect on aggression in mice. Nature, Lond. 226: 967-968, 1970. 13. Powers, J. B. and S. S. Winans. Vomeronasal organ: Critical role in mediating sexual behavior of the male hamster. Science 187: 961-963, 1975. 14, Sawyer, T. F. Aversive odors of male mice: Castration and experiential effects, and the predictability of the outcomes of agonistic encounters. Aggr. Behav. 4: 263-275, 1978. 15, Sawyer, T. F. Androgen effects on responsiveness to aggression and stress-related odors of male mice. Physiol. Behav. 25: 783787, 1980. 16. Scott, J. P. and E. Fredericson. The causes of fighting in mice and rats. Physiol. Zool. 24: 273-309, 1951. 17. Soares, M, J., W. D. Kalberer and M. J. Erpino. Progesterone: Effects on investigatory preferences, aggression, and olfaction in orchidectomized, testosterone-treated mice. Behav. Biol. 23: 260-266, 1978. 18. Winer, B. J. Statistical Principles in Experimental Design, (2nd ed.), New York: McGraw-Hill, 1971. 19. Wysocki, C. J. Neurobehavioral evidence for the involvement of the vomeronasal system in mammalian reproduction. Neurosci. Biobehav. Rev. 3: 301-341, 1979. 20. Wysocki, C. J., J. L. Wellington and G. K. Beauchamp. Access of urinary nonvolatiles to the mammalian vomeronasal organ. Science 207: 781-783, 1980.
Learned Aversion to the Odors of Male Mice: Effects on Agonistic Behavior T H O M A S F. S A W Y E R
Psychology Department, North Central College N a p e r v i l l e , I L 60566 R e c e i v e d 24 D e c e m b e r 1980 SAWYER, T. F. Learned aversion to the odors of male mice: Effects on agonistic behavior. PHYSIOL. BEHAV. 27(1) 19-25, 1981.--Intact and castrate male mice were subjected to avoidance training using the urine of intact and castrate donors as stimuli. Both intacts and castrates learned to discriminate between the odors, with such learning modifying their spontaneous responsiveness to such odors in an open field in the predicted direction. This result suggests that the previously reported lack of responsiveness in castrates [15] was not due to a simple sensory deficit. A second experiment determined that increasing the relative aversion exhibited by intact subjects to the odors of intact donors also increased the subject's aggression directed at a castrate opponent swabbed with such odors. Modification of castrates' responsivity had no such effect on their aggression. This indicated that replacing the responsivity to aggression-related odors which is lost following castration, is not equivalent to replacing the hormone. The results are discussed with regard to possible hormonal effects on the social significance of olfactory cues to the recipient animal. Aggression
Aversion
Mice
Olfaction
Urine
A N U M B E R of investigators have found the urine of aggressive male mice to be aversive to male conspecifics [7, 9, 14]. Also, a recent study found that castration of subjects eliminated their tendency to avoid the side of an open field spotted with urine, while testosterone treatment replaced the response, clearly demonstrating the androgen-dependent nature of the aversion [ 15]. Analogous evidence concerning the responsiveness of male rats to the odors of females showed castrate males to exhibit less investigation of the odors than did intacts, as well as failing to display the normal preference for the odors of receptive over nonreceptive females [3,4]. However, it appears that the effects of castration do not generalize to another type of odor excreted by mice, namely, alarm or stress-related odors [15]. It was found that both intact and castrate male mice avoided the urine from severely stressed castrate donors. These results suggest that male gonadal hormones may play an important role in sensitizing the particular systems concerned with responsivity to socially relevant stimuli, such as those involved in agonistic and sexual interactions. And furthermore, these results suggest that the mechanism behind the effects of gonadal hormones on rodent aggression and sexual behavior may lie in part in their influence on the response to the relevant olfactory stimuli. The failure of an organism to exhibit a spontaneous response to a stimulus or a spontaneous discrimination between stimuli does not necessarily imply the organism is incapable of detection or discrimination [2]. Indeed, castrate rats failed to spontaneously discriminate between the odors of receptive and nonreceptive females [3,4], despite the fact
that thirsty castrates have been found to do so for water reward [2]. The first experiment of the present study sought to determine if castrate male mice are able to learn a discrimination between the urine of intact and castrate donors, and if so, whether such training would influence their subsequent spontaneous responsiveness to the odors.
EXPERIMENT 1 In this experiment a group of intact and a group of castrate subjects were trained to avoid shock by avoiding the side of a modified T-maze in which the urine of intact males was present, and approaching the side which contained the odor of castrate males. Another group of intacts and one of castrates were trained with the opposite requirements. The importance of using the urine of both intact and castrate donors should be noted. While it is quite possible that castrate males are able to detect the odors of the urine of intact donors, it is conceivable that they are unable to detect that quality that distinguishes it from that of castrates, which has previously been found not to be aversive [9,14]. That particular quality was suggested to be the secretion of the coagulating gland [8], an androgen-dependent gland that secretes directly into the urine. Thus, requiring a discrimination between the odors of intact and castrate donors would suggest any change in performance to be due to the discrimination of that characteristic of the urine that gives it its aversive quality. However, the two urine types may differ in other discriminable ways.
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 Inc.--0031-9384/81/070019-07502.00/0
20
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METHOD
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Respondill,q SIt/~je~l,~ The subjects were 60 Swiss-Webster male mice. After weaning at 25_+ 1 days of age they were housed in groups of four under a partially reversed 12:12 hr light-dark cycle with lights on at 01:00 hr and off at 13:00 hr. The cages were 31.4x 19.7x 12.7 cm mouse boxes with wire tops. Food and water were available ad lib.
Urine Dum)rs and Urine Collection The donors were 24 males obtained from the same stock as the responders. At 70 days of age 12 of the donors were castrated via scrotal incision under ether anesthesia; the remaining 12 were sham-operated. Urine was collected using a method similar to one previously described [15]. Briefly, donors were housed individually in a metabolism cage for three weeks following surgery. Urine was obtained over a 16-18 hr period during which time food was removed to prevent contamination of the urine. The urine was stored in airtight glass receptacles and was used within 7 hr of collection. A minimum of 72 hr elapsed between each occasion of urine collection.
1
Training Procedure At approximately 70 days of age two subjects from each box of four were chosen at random and castrated. The remaining subjects were sham-operated. Twelve of the intact subjects were subsequently trained to avoid the intact urine and approach the castrate urine (intact avoid group, or IAV), while 12 were trained with the opposite requirements (intact approach, or lAP). Likewise, 12 castrates were trained to avoid (CAV) and 12 to approach (CAP) intact urine. The remaining intacts and castrates were " t r a i n e d " with no stimuli present (NS). Sixteen days after surgery the avoidance training began. This training consisted of 80 trials distributed across four sessions. Within a session, a subject
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Training Apparattts Avoidance training took place in an apparatus designed for use with olfactory stimuli. As pictured in Fig. I, it consisted of a start box (11 x 14x 15 cm) which opened out to two goal boxes (each 18x 14x 15 cm). The wall between the goal boxes contained a hole permitting movement of the subject from one side to the other, and yet could be closed off through the use of a guillotine door. Likewise, pushing the movable back wall of the start box closed this section off from the goal boxes, as well as forcing a subject in the start box to move from it. Located on either side of the start box were removable stimulus boxes (1 l x 7 x 15 cm) within which stimulus odors were placed. Holes at the end of each goal box provided output of both air and odors to an adjacent enclosure (30x30x30 cm). The air contained in this enclosure was drawn out via an exhaust blower and exited the room through tubing attached to the exhaust vent in the ceiling of the room. Thus, while the exhaust blower was on, air passed through the stimulus boxes, into the goal boxes, out of the test chamber, and finally out of the room. Auxiliary exhaust fans provided further replacement of air to the apparatus between trials. The apparatus was made of Plexiglas and rested on a grid of 0.158 cm diameter rods through which shock was delivered. The shock source was a BRS/Foringer, Model No. SG-901.
/ /
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FIG. 1. Olfactory avoidance training apparatus. The flow of air is depicted by the dashed lines (SB = stimulus box, GB = goal box. MW = movable wall, GD = guillotine door, AF - auxiliary fan, EB = exhaust blower).
was given 10 trials with an intertrial interval of 60 sec, followed one hr later by another 10 trials. Forty-eight hr elapsed between each session. The intact urine stimuli were obtained from sets of four intact donors, with each animal contributing 0.20 ml to the pool. Castrate stimulus odors were obtained from analogous sets of castrate donors. Pooling of the urine prevented subjects from discriminating between odors on the basis of individual differences [5], and has previously been determined not to effect the aversive quality of the urine [10]. The urine stimuli were presented by applying 0.25 ml of one type to an absorbent cotton ball in one of the stimulus boxes. The other type was presented in a similar fashion in the other stimulus box.
L E A R N E D AVERSION AND AGONISTIC BEHAVIOR Each trial began by placing the subject in the start box. If after 15 sec a choice (defined as the entire body, excluding the tail, entering a goal box) was not made, one-half of the start box was closed off, which forced the subject to the choice point, but did not necessarily force a choice. If another 15 sec elapsed, the subject was forced into a goal box by completely closing off the start box. Entry into the correct side resulted in removal from the apparatus, while entry into the incorrect side resulted in a two-sec 0.4 mA scrambled shock. The guillotine door separating the goal boxes was then raised and the subject was required to escape the shock by moving to the correct side. Unforced entry into a goal box resulted in closing of the start box and either removal or shock as in forced entry. Essentially, the procedure was a forced choice, correction procedure. Following each trial the stimulus boxes were removed and the auxiliary fans were turned on for 45 sec. Also, absorbent paper beneath the apparatus was replaced with a clean piece. Approximately 15 sec before the next trial the stimuli were replaced and the auxiliary fans turned off. The location of the stimuli was determined by a Gellerman series in which strategies such as alternation or win-stay, lose-shift were unsuccessful in producing performance above 50% correct. Subjects in the NS group were "trained" using an identical procedure except for the use of distilled water in place of the urine.
21
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Aversion Testing Aversion testing was conducted 48 hr after the last training session. The apparatus and procedure has been described and used successfully in previous studies [ 14,15]. Briefly, ten spots of urine from the intact pool were distributed approximately equidistant throughout one-half of a 45 x 45 cm open field, and 10 spots of urine from the castrate pool were placed on the other half. The subject was placed in the center of the field and the time spent on the side to which the castrate urine had been applied was measured over a 300 sec period. The testing took place in a dark room under red light illumination 0.5-5 hr into the dark part of the cycle. RESULTS AND DISCUSSION
The results of a 5x8 (treatment group x trial block) Analysis of Variance (ANOVA) for the training data revealed a highly significant treatment group by trial block interaction, F(28,385)=5.38, p<0.001. This demonstrates that the trend in the mean number of choices toward the castrate odor across the blocks varied as a function of treatment group. As plotted in Fig. 2, the NS group exhibited a flat function at about 50% toward the castrate side, while the AV groups (IAV and CAV) exhibited a consistent increase in the number of responses toward the castrate odor across blocks, and the AP (lAP and CAP) showed a consistent decrease. Analyses of the simple effects of treatment group at the various trial blocks using the Satterthwaite correction for repeated measures [18] indicated no difference at the first block, F(4,413) = 2.21, p >0.10, while there was a substantial effect at all other blocks, all F's>7.25, all p's<0.001. Post hoc analyses using Scheffe's S-method [11] indicated that while IAV and CAV did not differ from one another at any block, their mean differed from that of the NS group at blocks three through eight (critical mean difference = 1.37, c~=0.05). Likewise, while IAP and CAP were not found to differ at any block, they differed from that of the NS group, and hence the more extreme IAV and CAV groups, on
FIG. 2. Mean number of choices toward the side to which castrate urine was applied as a function of treatment group and trial block (chance response is approximately 5 per block).
blocks four through eight. Thus, both AV groups learned to avoid the intact odor, while both AP groups avoided the castrate odor, with no evidence of any hormonal effect. An ANOVA performed on the number of correct choices, rather than the number toward the castrate side, for the subjects in the four trained groups indicated only a significant main effect of trial block, F(7,308)= 19.56, p<0.001. Trend analysis revealed a significant linear trend, F(1,44)= 123.32, p<0.001, with no higher order trends reaching significance. That is, all groups learned the discrimination, and did so approximately equally well. It is interesting that it was no more difficult to train the lAP males to approach the odor of an intact, an odor which is itself somewhat aversive, than it was to train the IAV males to avoid the odor. This result is likely due to the extreme aversiveness of the shock relative to the mild aversiveness of the odor.
Aversion Random movement by the subjects during aversion testing would result in approximately 150 sec spent on each side of the field. However, if one urine type was more aversive one would expect deviation from this value. The amount of time spent on the castrate side of the field as a function of shock contingency and hormonal status is provided in Table 1. An ANOVA revealed a pronounced main effect of shock contingency, F( 1,44) = 137.04, p <0.001. That is, the training was found to generalize to the aversion test with both AV groups showing an aversion and both AP groups an attraction to the intact odors.
22
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TABLE I MEAN TIME SPENTON THE CASTRATESIDE OF 3HE ()PEN FIELD AS A FUNCTIONOF SHOCK CONTINGENCYAND HORMONE STATUS" Shock Contingency
AV AP
Hormone Status Intact
Castrate
204.32 _+ 6.49+ 108.93 _+ 8.33+
191.28 ± 9.66+ 107.16 ± 5.51;
Mean time=sec _+ standard error. *p values refer to comparisons with random response value of 150 sec and were determined by t-tests. +p<0.001.
Thus, Experiment 1 was successful in answering two questions. First, castrates are able to learn a discrimination between the odors of intacts and castrates, which suggests that the lack of spontaneous responsiveness in castrates is not due to a sensory deficit. And second, the responsivity is easily modified via training, with the change persisting for at least 48 hr. EXPERIMENT 2 Previous research has determined that varying the nature of the odor of an opponent animal can modify the agonistic behavior of a subject [12]. Likewise, gonadal hormones may have an effect on the nature of an animal's response to urine odors and thereby influence agonistic interactions. Implicit in this suggestion is the notion that variability in the responsivity of male mice to aggression-related odors should cause variation in their agonistic behavior just as varying the odor does. Evidence supportive of such a relationship, but not conclusive, consists of: the finding of a differential responsivity to odors by dominant and subordinate mice [9] which are also known to differ in agreesiveness [16], the lack of responsiveness in castrates [15] which also generally lack aggressiveness [1], and the finding of individual differences in responsivity that were predictive of the outcome of an agonistic encounter [14]. The second experiment was designed tb determine the effect of experimental manipulation of the responsiveness of males to a standard urine odor on their agonistic behavior when confronted with an animal having the odor. METHOD
Responding, Subjects The subjects were 112 male mice obtained from the same stock and housed in groups of four under the same conditions as in Experiment I.
Donor and Opponent Animals The donors were 24 males housed individually in a metabolism cage for a minimum of three weeks prior to the onset of urine collection. The opponents used during aggression testing were 112 males housed under the same conditions as the responders. At 35 days of age each opponent was castrated. They were used when 65-75 days of age.
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At approximately 70 days ot" age 56 of the subjects ~erc castrated, while the remaining 56 were sham-operated. The intacts were assigned to one of four groups, three of which were treated as the lAP, IAV, and NS groups of Experimenl I. A fourth group (IS'F) was ' t r a i n e d " with the odor present, but no shock. That is, a subject from the [ST group was allowed to make a choice and was then removed from the goal box as if the choice were correct. The 56 castrates were assigned to analogous groups designated CAP, CAV, CNS, and CST. Approximately 20 days after surgery, training began. It was conducted as previously described, except that only the urine from intact donors was used. The other stimulus box contained an equivalent amount of distilled water. Each subject received a total of 80 trials as described in Experiment I. Aversion and aggression testing began 48 hr after the last training session. Half of the subjects in each group were given the aversion test first, followed 80-90 rain later by the aggression test. The remaining subjects received the tests in the opposite order. Aversion testing proceeded as described in Experiment I. During the aggression phase, each subject was tested for aggression directed toward an opponent swabbed with 0.06 ml of urine. A different opponent was used during each test in order to eliminate possible effects of prior use on the aggression of a subject. A particular subject was confronted with urine taken from the same pool for both behavioral tests. The procedure for aggression testing consisted of initially placing a subject in a mouse box with clean bedding on the floor. A Plexiglas cover was placed over the box, and a barrier was inserted through a slit in the top, dividing the box into two sections. The opponent was then swabbed with urine and placed in the box on the side opposite that of the subject. The barrier was then lifted and the test began. Frequency and total time the subject engaged in the following behaviors were recorded for 10 rain: investigating nosing, vigorous nosing, chasing, tail-rattling, and biting attack. Immediately after the session the subject was raled on the following scale, which is similar to one previously used [6]: 0---little or no nosing; l--occasional nosing; 2--frequent nosing; 3--fiequent vigorous nosing: 4--chasing and tailrattling; 5--biting attack, including wrestling: 6--frequent fierce attack, including biting and wrestling. All aggression testing took place 1-5 hr into the dark period under red light illumination. RESULTS AN[) DISCUSSION
Training,, The analysis for this phase was a 2 × 4 x 8 (hormone status × training condition x trial block) ANOVA. As expected, a pronounced training by trial block interaction was found, F(21,728)=6.87, p<0.001, which renders the main effects of training condition, F(3,104)= 86.44, p <0.001, and trial block, F(7,728)= 15.10, p<0.001, somewhat obscure. It is obvious from inspection of Fig. 3, which plots correct choices as a function of group and trial block, that the interaction was due to the fact that the AV and AP groups had learned the task, while the ST and NS groups had not. These results essentially replicate those of Experiment I.
A version A 2 x 4 x 2 ANOVA (hormone status x training condition
LEARNED AVERSION AND AGONISTIC BEHAVIOR
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× test order, i.e., before or after aggression test) revealed a significant hormone status by training condition interaction, F(3,96)=2.74, p<0.05. The data depicting this interaction are plotted in Fig. 4. Analyses of the simple effects of hormone status at different levels of training condition indicated that the interaction was due to differences between the intact and castrate subjects of the ST groups, F( 1,96) = 14.68, p <0.001, and NS groups, F(1,96) =9.37, p <0.005, while no differences were found between the AV groups, F(1,96)=0.59, p>0.20, or the AP groups, F(1,96)=0.05, p>0.20. Further analyses of the effects of training and hormone status were accomplished via post hoc comparisons. The two intact control groups (IST and INS) were found not to differ from one another, nor were the castrate controls (CST and CNS). Comparisons of IAV with the average of the intact controls indicated that training intacts to avoid the odor was effective in increasing the time spent on the clean side of the field, F(1,96)= 15.06, p<0.01. Likewise, the CAV group was found to exhibit a substantially greater aversion response than the castrate controls, F(1,96)=48.59, p<0.01. Also, the IAP group was found to exhibit a significantly lower aversion response than did intact controls, F(1,96)= 38.78, p<0.01, as did the CAP group when compared with the castrate controls, F(1,96)=9.18, p<0.01.
Aggression
FIG. 3. Mean number of correct choices as a function of hormone status, training condition, and trial block (chance response is approximately 5 per block).
~
23
NS
AP
CONDITION
FIG. 4. Mean time (sec + standard error) spent on the clean side of the open field as a function of hormone status and training condition (random response is approximately 150 sec).
The analyses of the aggression test data were performed separately for the rating measure and the component behaviors. The mean aggression rating, as well as the number attacking, for each group is provided in Table 2. A 2 × 4 x 2 (hormone status x training condition × test order) A N O V A on the rating scale data revealed a significant effect of hormone status, F(1,96)=26.60, p<0.01, with intacts more aggressive than castrates. This result, which replicates the well-documented effect of castration on aggression [1], is further substantiated by the finding that 21 intacts attacked their opponent, while only 5 castrates did, X2(1)=8.65, p<0.01. An interesting effect of test order was also found, F( 1,96) = 6.49, p <0.05. While the difference was small, it was quite consistent, as those subjects receiving the aversion test first received higher scores in all but two groups (CAP and CNS), and in those there was no difference. The basis for such an effect cannot be determined at this time, but it is suggested that pre-exposure to the odors in the aversion test may have served a priming function, perhaps initiating a hormone release, which sensitized the subjects to such aggression-related odors. It should be noted such a priming effect could not have involved gonadal hormones, as the order effect was quite large in the CAV group. The effect of primary interest, namely training condition, was not found to be significant, F(3,96)= 1.76, p>0.10. However, a planned comparison between the two AV groups (average of 1AV and CAV) and the AP groups (average of IAP and CAP) revealed the AV subjects to exhibit a higher rating (3.00) than the AP subjects (2.14), F(1,96)=4.45, p<0.05. Further planned comparisons revealed that while the IAV group (4.14) had a higher rating than the IAP (2.57), F(1,96)=7.48, p<0.01, the same was not true for CAV (1.86) compared to CAP (1.71), F(i,96)=0.14, p>0.20. Thus, experimentally manipulating olfactory responsivity affects agonistic behavior in intacts but not castrates. Finally, a multivariate analysis of variance was performed on the frequencies and times for the various component be-
24
S A W Y b. R TABLE 2 MEAN AGGRESSION RATING AND THE NUMBER OF SUBJECTS THA 1 ATTACKED AS A FUNCTION OF HORMONE STATUS. TRAINING CONI)ITI()N, AND TEST ORDER ~ Hormone Status
Training Condition
"lest Order Aversion First + + ± +
Attacked
Aggression First
Attacked
Intact Intact Intact Intact
AV AP ST NS
4.71 3.29 3.00 3.71
0.77 0.80 0.74 0.93
5/7 3/7 2/7 4/7
3.57 1.86 2.86 2.57
± 0.84 + 11.59 + 0.59 +: 11.51
3/7 1"7 27 1/7
Castrate Castrate Castrate Castrate
AV AP ST NS
2.57 _+ 0.46 1.71 ± 0,46 1.86 + 0.59 1.71 ± 0,61
1/7 0/7 1/7 1/7
1.14 1.71 1.29 1.71
+ ± ± ±
0/7 1/7 11/7 1/7
0.15 0.15 0.20 0.61
Aggression rating: mean _+ standard error; n=7. *Ratings range from a possible low of 0.00 to a possible high of 6.00.
h a v i o r s , including the a t t a c k l a t e n c y (600 sec if no attack). A s u m m a r y o f t h e s e m e a s u r e s is p r o v i d e d in T a b l e 3. J u s t as for the rating m e a s u r e , a significant effect o f h o r m o n e s t a t u s was f o u n d , F ( 1 1 , 8 6 ) = 5 . 4 8 , p < 0 . 0 0 1 . U n i v a r i a t e A N O V A s r e v e a l e d t h a t h o r m o n e s t a t u s affected e a c h of the c o m p o n e n t b e h a v i o r s in t h e e x p e c t e d f a s h i o n , all p ' s < 0 . 0 5 . T h e multiv a r i a t e a n a l y s i s also i n d i c a t e d a n effect o f t r a i n i n g c o n d i t i o n , F(33,254)=2.09, p <0.01. U n i v a r i a t e A N O V A s r e v e a l e d t h a t t r a i n i n g i n f l u e n c e d i n v e s t i g a t i v e n o s i n g time a n d f r e q u e n c y as well as c h a s i n g t i m e a n d f r e q u e n c y , all p ' s < 0 . 0 5 , while its
effect on a t t a c k time a p p r o a c h e d but did not r e a c h c o n v e n tional levels o f significance, p < 0 . 1 0 . M u l t i v a r i a t e c o m p a r i s o n s indicated the c o m b i n e d A V g r o u p s were significantly different from the A P g r o u p s , F ( 1 1 , 8 6 ) = 2 . 0 3 , p < 0 . 0 5 , and the control g r o u p s , F(11,86)=3.87, p < 0 . 0 1 , o n n o s i n g time a n d f r e q u e n c y , c h a s i n g time and f r e q u e n c y , a n d a t t a c k time, all p ' s < 0 . 0 5 , while the A P g r o u p s did not differ from the c o n t r o l s . Lastly, while the effect o f test o r d e r a p p r o a c h e d significance, F(11,86)= 1.66, p < 0 . 1 0 , n o n e o f the i n t e r a c t i o n effects did so.
TABLE 3 MEAN VALUES (FREQUENCIES AND TIMES*) FOR EACH COMPONENT AGGRESSIVE BEHAVIOR Treatment Group
IAV lAP IST INS CAV CAP CST CNS
Component Behaviors Nose
Vig. Nose
Chase
Tail Rattle
Attack
Freq. Time Freq. Time Freq. Time Freq. Time
25.63 79.67 17.04 62.99 20.50 61.19 17.64 49.96
8.36 23.29 2.88 7.12 3.93 9.65 5.93 12.37
1.21 1.52 0.16 0.19 0.14 0.15 0.21 0.13
5.79 6,32 1,13 2,80 2.64 3,09 2.71 2.51
7.00 15.06 0.82 2.51 2.29 2.27 2.79 3.43
Freq. Time Freq. Time Freq. Time Freq. Time
19.71 53.14 16.29 56.29 13.64 48.75 10.29 24.44
1.57 2.59 0.57 2.04 0.64 1.51 1.00 2.80
0.14 0.07 0.00 0.00 0.00 0.00 0.07 0.04
0.36 0.30 0.00 0.00 0.21 0.51 0.29 0.33
0.21 0.19 0.29 0.33 0.50 0.84 0.86 1.32
*Time in seconds.
Attack Latency
381.40 558.80 479.50 459.21) 559.60 583.40 592.90 552.90
L E A R N E D AVERSION AND AGONISTIC BEHAVIOR G E N E R A L DISCUSSION The present study served to answer two primary questions. Experiment 1 demonstrated that the lack of responsivity of castrate mice to the odors of intact mice as previously reported [15] is not due to a simple sensory deficit, as castrates were able to learn a discrimination between such odors and the unaversive odors of castrates. Experiment 2 examined the extent to which manipulations that modify responsiveness to aggression-related odors also act to modify agonistic behavior. The results showed that modification upward in the aversion response of intact males was related to a higher aggression rating, and a greater incidence of attack when compared to intact males subjected to a downward modification in their response. This finding supports the notion that threat or fear of attack may be an important component of aggression in rodents [14,16]. However, with regard to castrate subjects, modification in the responsiveness to urine odors did not appear to be related to their agonistic behavior. That is, subjecting castrates to training that resulted in an "intact-like" aversion response to male odors, did not result in "intact-like" agonistic behavior. Thus, in terms of the effects of manipulating olfactory responsiveness on aggression, hormone status appears to have a pronounced effect. A possible explanation for the failure to influence the agonistic behavior of castrate subjects concerns what might be labeled the social significance of urine odors. Perhaps male gonadal hormones effect brain mechanisms that inter-
25 pret the significance of socially relevant stimuli [17]. Desensitizing such mechanisms via removal of the hormone could presumably alter spontaneous social responsiveness to various stimuli without affecting an animal's ability to learn responses to such stimuli. In other words, castration may have made neutral stimuli out of what were previously socially significant odors. And, while the avoidance training was able to replace the aversiveness of the odor to the castrate, it was not able to replace the social significance of the odors, and hence, could not replace the agonistic behavior. Another similar explanation for the findings of Experiment 2, is that removal of the gonads may have disrupted the normal activity of another "olfactory system." This suggestion is stimulated by recent evidence demonstrating the presence of a system, namely the vomeronasal system, that is sensitive to socially relevant odors, particularly sexrelated odors [13, 19, 20]. Assuming an effect of gonadal hormones on this system, as well as its sensitivity to aggression-related odors, can explain the failure of castrates to show intact-like aggression. Again, the integrity of the olfactory system could have mediated the learning of the aversion to the odor without influencing the social significance, and hence the agonistic responses toward an animal having the odor. Future research is needed to determine the adequacy of these explanations, as well as other unresolved issues, such as the basis for the test order effect on aggression as found in Experiment 2.
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