Brief Exposure to Female Odors “Emboldens” Male Mice by Reducing Predator-Induced Behavioral and Hormonal Responses

Hormones and Behavior 40, 497–509 (2001) doi:10.1006/hbeh.2001.1714, available online at http://www.idealibrary.com on

Brief Exposure to Female Odors “Emboldens” Male Mice by Reducing Predator-Induced Behavioral and Hormonal Responses Martin Kavaliers, 1 Elena Choleris, and Douglas D. Colwell* Department of Psychology, University of Western Ontario, London, Ontario, N6A 5C2 Canada; and *Agriculture and Agri-Food Canada Lethbridge, Alberta T1J 4B1, Canada Received March 19, 2001, revised May 30, 2001, accepted June 4, 2001

In rodents, where chemical signals play a particularly important role in determining intersexual interactions, various studies have shown that male behavior and physiology is sensitive to female odor cues. Here we examined the effects of brief (1 min) and more prolonged (60 min) preexposure to the odors of a novel estrous female on the behavioral and hormonal responses of sexually experienced and inexperienced male mice, Mus musculus, to subsequent predator (cat and weasel) odor exposure and potential predator risk. Brief, but not prolonged, preexposure to the odors of an estrous female decreased the aversion and avoidance responses of male mice to cat odor in a Y-maze preference test, with the extent of responses being affected by a males prior sexual experience. Similarly, brief, but not prolonged, preexposure to female odors markedly attenuated the analgesic responses elicited in male mice by weasel odor. Brief exposure to a novel estrous female by itself had no significant immediate effects on either corticosterone or testosterone levels in the males. However, brief, but not prolonged, preexposure to the odors of an estrous female attenuated the marked increase in corticosterone and decrease in testosterone that were induced in males by exposure to weasel odor. The decreases in aversive responses to, and effects of, predator odor exposure that are induced by brief exposure to a novel estrous female may reflect a greater risk taking and boldness in males that could directly facilitate access to an immediately, and possibly transiently, available novel sexually receptive female. © 2001 Elsevier Science

Key Words: predator threat; corticosterone; testosterone; predator odor; analgesia; sexual experience; male sexual behavior.

1 To whom correspondence and reprint requests should be addressed. Fax: (519) 661-6391. E-mail: [email protected].

0018-506X/01 $35.00 © 2001 Elsevier Science All rights reserved.

Chemical signals play a significant role in determining the intraspecific behavior of animals influencing affiliative, reproductive, aggressive, and social behaviors (e.g., Brown, 1979; Hurst, 1987, 1990; Barnard, Hurst, and Aldhous, 1991; Humphries et al., 1999; Drickamer, 1992; Coopersmith and Lennington, 1992; Kavaliers and Colwell, 1995a). As well, the roles of chemical signals in interspecific behaviors such as predator detection and subsequent avoidance have been highlighted (Kats and Dill, 1998; Lima, 1999). Female odors in particular have been reported to have dramatic effects on male behavior. Exposure of male mice, Mus musculus, to either females or the odor cues of females has been shown to elicit a number of behavioral changes that are associated with shifts in intra- and intersexual responses, including that of aggressiveness (e.g., Hayashi and Kimura, 1974; Dixon and Mackintosh, 1975; Mainardi, 1978; Brown, 1979; Nyby and Whitney, 1980; Hurst, 1987, 1990; Smith, Barnard, and Behnke, 1996; Kavaliers, Colwell, and Choleris, 1998). The behavioral changes evident in males after exposure to a female may involve an overall reduced fearfulness and greater risk taking. Results of studies with guppies, Poecilia reticulatus, have provided evidence suggesting that the presence of a female is directly associated with greater risking taking, increasing male boldness in the presence of a predator (Godin and Dugatkin, 1996). This raises the possibility that the responses of male mice to predators may also be affected by preexposure to a female or her associated cues. Animals generally respond to the threat of predation and predator risk with a number of defensive behaviors, including immobilization or freezing and risk assessment (i.e., decision making as to when and

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how to feed etc. in the presence of a predator), increased wariness, and the suppression of nondefensive behaviors (Blanchard et al., 1990; Lima, 1998). Results of a variety of laboratory and seminatural studies have shown that rodents display aversive responses to, and avoidance of the odors of, predators such as the domestic cat (e.g., File et al., 1993; Jedrzejewski, W., Rychilk, L., and Jedrzdjewska, 1993; Kavaliers, Wiebe, and Galea, 1994; Kavaliers and Colwell, 1995b; Blanchard et al., 1998; Morrow et al., 2000). An additional consequence of exposure to a predator or predator odors is a reduction in the reactivity of rodents to a noxious thermal stimulus, i.e., pain inhibition or analgesia. Mice and other rodents display pronounced analgesic responses following exposure to either predators or odors of specific predators such as a cat or weasel (e.g., Lester and Fanselow, 1985; Kavaliers, 1990; Lichtman and Fanselow, 1991; Kavaliers and Colwell, 1991). Analgesia is advantageous in aversive situations such as predator exposure, in which responding to noxious stimuli might compromise effective defensives behaviours. Along with these behavioral effects, exposure to predators or their odor cues has been shown to elicit alterations in the levels of hormones that are involved in stress responses. The presence of a cat or the odor of a cat or other potential predators can activate the adrenal-hypothalamic-pituitary (HPA) axis and increase corticosterone levels in rodents (e.g., File et al., 1993; Blanchard et al., 1998; Perrot-Sinal, Kavaliers, and Ossenkopp, 1998; Morrow et al., 2000). Prior considerations of the effects of exposure to females or their odors on male behavior and physiology have been primarily concerned with relatively long-term exposures with little consideration of the possible immediate effects and responses. In addition, the nature of a male’s response to females and their odor cues has been reported to be sensitive to his prior sexual experience (Lydell and Doty, 1972; Mainardi, 1978; Nyby and Whitney, 1980; Wysocki 1986; Hurst, 1990; Barnard, Hurst, and Aldhous, 1991). This has been further suggested to depend on the nature of the female odor cues, with the responses of males to the low-volatility urinary odors of females being innate, while the responses to more volatile components are determined by prior sexual experience (Nyby and Whitney, 1980). Accordingly, in the present study we directly examined the effects of both brief and more prolonged preexposures to the odors of an estrous female on the subsequent responses of sexually experienced and inexperienced male mice to predator odor.

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Using an odor preference test (Coopersmith and Lenington, 1992) we examined the effects of brief (1min) and relatively prolonged (60-min) preexposure to the odors of female mice on the responses of sexually experienced and inexperienced male mice to predator (cat) and nonpredator (guinea pig) odors. We also considered the effects of preexposure to female odors on predator (weasel) odor-induced analgesia. In parallel, we examined the effects of a preexposure to female odor on corticosterone levels in male mice exposed to weasel odor. In addition, as exposure to females has been indicated to influence male testosterone levels (Marsden and Bronson, 1965; Macrides, Bartke, and Dalterio, 1975; Batty, 1978; Wysocki, Katz, and Bernhard, 1983), we also examined the effects of preexposure to a female and presentation of weasel odor on testosterone levels in male mice.

METHOD Animals Male and female mice (CF-1, 2–3 months of age) were housed either in mixed-sex pairs or singly, all groups in separate rooms, in clear polyethylene cages with wood-shavings bedding. Mice were held at 20 ⫾ 2°C under a 12-h light:12-h dark cycle (light 08:00 – 20:00 h). Food (Mouse Breeder Blox; Wayne Laboratory Diets, Madison WI) and water were available ad libitum. The males consisted of three groups: One group (sexually inexperienced isolated) were virgin males that had no prior exposure to the odors of unrelated females; another group (sexually experienced isolated) were paired with an adult female for 2 weeks before being housed singly for 5–7 days prior to the experiment; and the third group (sexually experienced paired) were housed for a minimum of 2 weeks with an adult female prior to experimentation.

EXPERIMENT 1: EFFECTS OF PREEXPOSURE TO A FEMALE ON THE RESPONSES OF MALES TO PREDATOR ODOR IN AN ODOR PREFERENCE TEST Apparatus Odor (predator vs nonpredator) responses of individual male mice (inexperienced isolated, experienced paired, and experienced isolated) were tested in a

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translucent Plexiglas Y-maze apparatus (5-cm diameter) with 30-cm arms (Coopersmith and Lenington, 1992). The stimulus compartments in the two arms of the Y, in which odor cues were placed, and the start box, in which a mouse was placed, were each 14 cm long. A solid Plexiglas barrier restricted the mouse to the start box, while perforated Plexiglas barriers at the ends of the two stimulus arms prevented contact with the odor sources while allowing detection of the odor. Removeable solid Plexiglas barriers were also present at “seams” 8 cm into each of the stimulus arms. The latter barriers prevented exposure of the mice to the odor cues until the designated test times. Procedures To minimize novelty responses, male mice were placed in the apparatus and allowed to explore the various arms (after being held in the star box for 5 min) for 30 min on 3 consecutive days prior to the testing day. On the test day a male mouse was placed in the start box of the apparatus for 15 min after which the solid barrier was removed allowing the mouse access to the two arms of the Y-maze. Two minutes later the solid Plexiglas barriers in the arms were removed, exposing the mouse to the stimulus odors in each arm. During the subsequent 5 min the duration of the time spent by a mouse in each arm within 8 cm of an odor source was recorded. “Preference,” as used hereafter, is defined as the duration of time the mouse spent in the one stimulus arm of interest divided by the total time spent in the two stimulus arms. Three different groups of mice were tested in one of three stimulus odor choice conditions: (1) predator (cat) odor vs nonpredator (control guinea pig) odor; (2) predator (cat) odor vs blank (no odor); and (3) nonpredator (guinea pig) vs blank (no odor). Mouse bedding (wood shavings) upon which a cat (female, 10 years of age and a proficient predator of mice) was placed for 90 min, was used as the source of cat odor, while guinea pig (1 year of age, laboratory-reared single housed) bedding of similar composition was the nonpredator odor source for all of the experiments. Clean bedding was used as the blank odor source. The same animals were used as odor sources for all of the experiments. Testing of male mice in the Y-maze was carried out after preexposure for either 1 or 60 min to the odors of either an estrous or nonestrous (anestrous, 1 min only) female. Freshly deposited urine and associated odors were obtained from single females that were placed for 1 h in a clean cage lined with blank filter paper (Whatman No. 4, England). Examination under an

ultraviolet light confirmed that the filter papers were scent marked with the females urinating generally within 5 min of arriving into the cage. The filter paper that lined a single cage was cut into strips and inserted into the tubes. Each male was presented with the odor of a single different female. In the odor preexposures male mice were individually placed in a Plexiglas-partitioned area (12.5 ⫻ 15 ⫻ 10 cm) that was provided with a vented Plexiglas tubes (10 cm in length and 3 cm in diameter and sealed at each end with fine plastic mesh through which the mice could not reach) containing the urine and associated odorous secretions of either (i) an individual estrous female mouse (unfamiliar female with inexperienced isolated, experienced paired, and experienced isolated males, or a familiar female paired previously with experienced isolated males; n ⫽ 10 in all cases) or (ii) nonestrous female mice (n ⫽ 5 males in all cases). Each male was presented with the odor of only one female and the odor of a different female was used with each male. Isolated females were primed with substrate from cages of other males (except those housed with an experimental male) to stimulate estrous cycling (Allen, 1922; Marsden and Bronson, 1986). Other females were noncycling and anestrous (nonestrous). Wet-mount vaginal smears taken in the afternoon of the day of testing were used to determine the estrous state of the females according to the following guidelines (Allen, 1922; Snell, 1941). Estrous was indicated by predominately cornified epithelial cells; proestrous by primarily nucleated epithelial cells; metestrous by a mixture of nucleated epithelial cells, cornified cells, and leucocytes; and diestrous by predominately leukocytes. Data Analyses All preference ratios were transformed to natural log (ln) values prior to analysis using analysis of variance (ANOVA) with mean comparisons planned a priori. ANOVA were run with the SuperAnova statistical package with a 0.05 significance level.

EXPERIMENT 2: EFFECTS OF PREEXPOSURE TO FEMALE ODOR ON PREDATOR-INDUCED ANALGESIA IN MALES PROCEDURES During the mid-to-late light period in a room separate from their holding rooms male mice were individually placed in clean cages (25 ⫻ 15 ⫻ 20 cm) and

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preexposed for either 1 or 60 min to the odors of either a nonestrous or estrous female (inexperienced isolated males– unfamiliar female; experienced paired males– unfamiliar female; and experienced isolated males– familiar and unfamiliar females) that was presented in a vented Plexiglas tube as described under Experiment 1. After the female odor source was removed the males were exposed to a second vented Plexiglas tube (10 cm long and 3 cm diameter and sealed at each end with plastic mesh) containing filter paper impregnated with either 2-propylthietane, the principal component of weasel anal gland odor (Crump, 1980), or butyric acid. Butyric acid is a novel aversive odor of approximately the same molecular weight and volatility as 2-propylthietane. Prior to any odor exposure, immediately after exposure to the odor of a female, and after exposure to the predator odor for either 30 s or 15 min, the nociceptive responses of individual mice were determined using the “hot-plate” test. Animals were placed individually on a warmed surface (“hot-plate”; AccuScan Instruments, Columbus, OH) maintained at 50 ⫾ 0.5°C and the latency of a foot lift or lick, whichever came first, was recorded. After this response was displayed or 60 s occurred the mouse was quickly removed from the surface and returned to his cage. Results of previous investigations have shown that repeated handling procedures and exposure to clean, unmarked (blank) filter paper have no significant effects on the thermal response latencies of the mice (Kavaliers and Colwell, 1991). Data Analyses Data were analyzed with a mixed-design repeatedmeasures ANOVA with mean comparisons planned a priori. A 0.05 level of significance was used.

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sure, (3) 30-s predator odor exposure, (4) 15-min control odor (butyric acid) exposure, (5) 60-min estrous female odor exposure, or (6) 1-min estrous female odor exposure. Other groups of male mice (n ⫽ 10 in each case) received one of a number of female odor preexposures followed by either predator odor or control exposures: (1) 60-min estrous female odor preexposure followed by 15 min of no odor exposure, (2) 1-min estrous female odor exposure followed by 15 min of no odor exposure, (3) 60-min estrous female odor exposure followed by a 15-min weasel odor exposure, (4) 1-min estrous female odor preexposure followed by a 15-min weasel odor exposure, or (5) 1-min estrous female odor preexposure followed by a 30-s weasel odor exposure. Male mice were exposed to the female and predator odors in a manner identical to that described under Experiment 2. Immediately following the odor exposures male mice were killed by cervical dislocation and trunk blood was collected on ice and then centrifuged at 14,000 rpm for 10 min and the resulting plasma was stored at ⫺50°C until time of assay. Plasma samples (100 ␮l) from the male mice were assayed in duplicate for corticosterone and testosterone using commercially available I 125-labeled RIA kits (Coat-a-Count, Diagnostic Products, Los Angeles, CA). For the corticosterone assay, the sensitivity of the assay was calculated to be 10 ng/ml and the intraassay coefficient of variation, measured in triplicate from low, medium, and high pools, ranged from 4 to 12%. For the testosterone assay, the antiserum had a cross-reactivity with 5␣-dihydrotestosterone of 5% and the sensitivity of the assay was 0.05 ng/ml as calculated from the standard curve. The intraassay coefficient of variation was measured in triplicate from low, medium, and high pools and ranged from 8 to 16%. Data Analyses

EXPERIMENT 3: EFFECTS OF PREEXPOSURE TO FEMALES AND PREDATOR ODOR ON CORTICOSTERONE AND TESTOSTERONE LEVELS

All hormonal values were transformed to natural log (ln) values prior to analysis by ANOVA with Fisher’s PLSD test run post hoc on main significant effects with a 0.05 significance level.

RESULTS Procedures Sexually inexperienced male mice (n ⫽ 10, in all cases) received one of a number of female odor preexposures or weasel (predator) odor exposure combinations: (1) basal-no odor exposures, (2) 15-min predator odor expo-

Experiment 1: Odor Preferences Predator–nonpredator odors. Sexually experienced and inexperienced male mice displayed a marked overall avoidance of the predator odor when

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presented a predator and nonpredator (guinea pig control odor) stimulus odor combination, with only 16 –20% of their time being spent in the arm holding the predator odor (Fig. 1A). This avoidance was affected by preexposure to female odor with a significant main effect of female preexposure [F(6, 18) ⫽ 67.19; P ⫽ 0.0001] and significant interaction of male experience and female preexposure [F(6, 18) ⫽ 2.23; P ⫽ 0.045]. Brief (1-min) preexposure to the odors of a novel estrous female significantly reduced the avoidance of the predator odor in all of the groups of male mice (all Ps ⫽ 0.001), though, in all cases the mice still showed a significant, but highly attenuated, avoidance of the predator odor, spending now 28 to 35% of their time in the predator arm. A 60-min preexposure to the odors of an estrous female had significant effects (P ⫽ 0.008) only on the predator avoidance displayed by the experienced isolated males. The avoidance that the males displayed to the predator odor here was, however, still significantly (P ⫽ 0.01) greater than that displayed after preexposure to the a female odor for 1 min. In the sexually experienced isolated male mice there was also significant difference (P ⫽ 0.0001) between the effects of preexposure to the odors of a familiar and unfamiliar (novel) estrus female (Fig. 2A). Brief (1-min) exposure to the odors of an unfamiliar female significantly attenuated the avoidance of the predator odor while preexposure to the odors of a familiar female that a male had been previously paired with had no significant effects on the avoidance of predator odor. Brief (1-min) preexposure to the odors of an nonestrous female had no significant effects on the predator odor avoidance displayed by any of the male mice, though the sexually inexperienced males did display a lower avoidance of predator odor than the experienced isolated (P ⫽ 0.22) and the experienced paired males (P ⫽ 0.016). Predator– blank odors. Male mice displayed a marked overall avoidance of the predator odor when presented a predator and blank stimulus odor combination, spending only between 16 and 18% of their time in the arm holding the predator odor (Fig. 1B). There was a significant main effect of female preexposure [F(2, 41) ⫽ 95.62, P ⫽ 0.0001]. A brief (1-min) preexposure to the odors of a novel estrous female significantly reduced this avoidance response in all of the groups of male mice (P ⫽ 0.0001). In all cases the mice still showed a significant, though highly attenuated, avoidance of the predator odor, spending between 34 and 38% of their total time in the predator

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FIG. 1. Effects of preexposure to the odors of either a novel estrous (1- or 60-min exposure) or nonestrous (1-min exposure) female on the subsequent responses of male mice in a Y-maze odor preference apparatus to (A) predator (cat)-and-nonpredator (guinea pig), (B) predator (cat)-and-blank (clean bedding), or (C) nonpredator– blank stimulus odor combinations. The responses of mice receiving no prior odor exposures (control) are also shown. Male mice were sexually naive (inexperienced isolated), had been previously paired with a female (experienced isolated), or were presently paired with a female (experienced paired). Responses are given as preference ratios (e.g., time spent in the vicinity of the predator odor:time spent in the vicinity of the predator odor ⫹ time spent in the vicinity of the nonpredator odor). Preferences were determined over a 5-min period; N ⫽ 10 in all cases. Vertical lines denote a standard error of the mean.

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arm with no significant difference between the various groups of mice. A 60-min preexposure to the odors of an estrous female also reduced the predator avoidance displayed by the inexperienced males (vs no female: P ⫽ 0.0012; vs nonestrous female: P ⫽ 0.032). This reduction was, however, significantly less than that elicited by the 1 min preexposure (P ⫽ 0.012). In all of the groups of male mice a 1 min preexposure to the odors of an nonestrous female had no significant effects on their avoidance of the predator odor. In the sexually experienced isolated male mice there was also a significant difference between the effects of preexposure to the odors of a familiar and unfamiliar (novel) estrous female (Fig. 2B). Whereas brief (1-min) exposure to the odors of an unfamiliar female significantly attenuated the avoidance of the predator odor, preexposure to the odors of a familiar female that a male had been previously paired with had no significant effect on the avoidance of the predator odor. Nonpredator– blank odors. Overall, male mice showed a slight avoidance of the novel odor of guinea pig when presented a nonpredator-and-blank-stimulus-odors combination, spending between 42 and 43% of their time in the arm holding the novel odor (Fig. 1C). There were no significant main effects of male experience and preexposure or their interaction. However, the factor of preexposure was significant for the inexperienced males [F(3, 16) ⫽ 6.26, P ⫽ 0.0051]. A 1-min preexposure to an estrous female eliminated the slight aversion for the novel odor, with the inexperienced males spending 49% of their time in the arm holding the guinea pig odor after a 1-min exposure to the odors an estrous female. The 60-min preexposure to the odors of an nonestrous female had no significant effects on the novel odor avoidance displayed by inexperienced male mice. There were also no significant differences between, or effects of, preexposure to the odors of either a familiar or unfamiliar female (Fig. 2C). FIG. 2. Effects of preexposure to the odors of either a familiar or unfamiliar (novel) estrous (1- or 60-min exposure) or nonestrous (1-min exposure) female on the subsequent responses of sexually experienced isolated male mice in a Y-maze odor preference apparatus to (A) predator (cat)-and-nonpredator (guinea pig), (B) predator (cat)-and-blank (clean bedding), or (C) nonpredator– blank stimulus odor combinations. The responses of mice receiving no prior odor exposures (control) are also shown. Male had been previously paired with the familiar female before being isolated for 5–7 days prior to exposure. Responses are given as preference ratios (e.g., time spent in the vicinity of the predator odor:time spent in the vicinity of the predator odor ⫹ time spent in the vicinity of the nonpredator odor). Preferences were determined over a 5-min period; N ⫽ 10 in all cases. Vertical lines denote a standard error of the mean.

Experiment 2: Predator Odor-Induced Analgesia Effects of predator odor exposure. Mice that were exposed to the odor of a predator (weasel) for either 30 s or 15 min showed increased thermal response latencies, indicative of the induction of analgesia as compared with mice that were exposed to either control (buytric acid) odor or no odor (Figs. 3A–3C). Overall, there were significant main effects for the factors of predator exposure [F(1, 148) ⫽ 1199, p⬍0.0001] duration of exposure [F(1, 148) ⫽ 23.53, p ⫽ 0.0001] and male experience with females [F(2, 148) ⫽

FIG. 3. Nociceptive responses of (A) sexually naive (inexperienced isolated), (B) sexually experienced and presently paired with a female (experienced paired), and (C) previously sexually experienced and paired with a female (experienced isolated) male mice that were exposed for either 1 or 60 min to the odors of a novel estrous or nonestrous (1 min only) female and then exposed for either 30 s or 15 min to weasel odor. Responses of mice (control) receiving no female odor exposure are also shown. Nociceptive sensitivity, as measured by the latency of response to a 50°C thermal surface, was determined before any odor exposures (baseline), after exposure to a female (postfemale), and after exposure to the predator odor (postpredator); N ⫽ 10 in all cases. Vertical lines denote a standard error of the mean.

504 3.84, p ⫽ 0.024] as well as an interaction of predator exposure ⫻ duration of exposure [F(9, 148 ⫽ 30.37, P ⫽ 0.0001]. Male mice that were exposed to a predator odor for 30 s showed significant (P ⫽ 0.0001) analgesic responses with no significant differences between the levels of analgesia in the three groups of mice. Similarly, male mice exposed to a predator odor for 15 min showed significant (P ⫽ 0.0001) analgesic responses with the latencies of the experienced paired males being significantly (P ⫽ 0.0089) lower than those of the experienced isolated males. In all of the groups of mice the levels of analgesia induced by the 30-s exposure to weasel odor were significantly (Ps ⬍ 0.002) lower than that induced by the 15-min exposure. There were no significant effects on, or differences between, the postexposure latencies of male mice that were exposed to the control odor. Effects of female odor exposure. Mice that were exposed to the odors of either an estrous or nonestrous female for either 1 min or 60 min showed slight, but in a few cases significant, analgesic responses [F(6, 285) ⫽ 14.92, P ⫽ 0.0001] with the magnitude of these effects being dependent on the prior sexual experience of the male (interaction of male experience ⫻ female preexposure: F(6, 285) ⫽ 2.15, P ⫽ 0.048] (Figs. 3A– 3C). Sexually inexperienced isolated male mice displayed significant analgesic responses to the odors of both estrous and nonestrous females (nonestrous: F(1, 19) ⫽ 27.21, P ⫽ 0.0001; estrous 1 min: F(1, 19) ⫽ 34.55, P ⫽ 0.0001; estrous 60 min: F(1, 19) ⫽ 44.71, P ⫽ 0.0001], with no significant differences between the analgesia induced by the various categories of female odors (Fig. 3A). In all cases these responses were markedly lower than those elicited by predator odor exposure. Sexually experienced paired males displayed significant (Ps ⬍ 0.01) analgesic responses after exposure to the odors of a novel estrous females for either 1 or 60 min, with no significant difference between the analgesia induced by the two exposures (Fig. 3B). Exposure to the odors of nonestrous females had no significant effect on the response latencies of the experienced paired males. Sexually experienced isolated males displayed significant (Ps ⬍ 0.01) analgesic responses after exposure to the odors of an estrous female (Fig. 3C). The increases in response latencies elicited by exposure to the odors of an estrous female were not significantly greater than those elicited by exposure to nonestrous females. However, exposure to the odors of a nonestrous female per se had a significant analgesic

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effect only if the female was unfamiliar [familiar female: F(1, 19) ⫽ 3.15, P ⫽ 0.077; unfamiliar female: F(1, 19) ⫽ 5.29, P ⫽ 0.022]. There were no significant differences between the analgesic responses induced by exposure to a familiar or unfamiliar nonestrous female as well as by 1-min or 60-min exposure to a familiar or unfamiliar estrous female. Effects of preexposure to female odor on the responses to predator odor. There were significant effects of the two main factors of female preexposure [F(6, 270) ⫽ 34.48, P ⫽ 0.0001] and duration of predator exposure [F(1, 270) ⫽ 32.04, P ⫽ 0.0001], their two-way interaction [F(6, 270) ⫽ 4.78, P ⫽ 0.0001] as well as the three-way interaction of the three main factors of male experience ⫻ female preexposure ⫻ duration of predator exposure [F(6, 270) ⫽ 3.16, P ⫽ 0.0052]. Experienced paired, experienced isolated, and sexually inexperienced males that were preexposed for 1 min to the odor of an estrous female and then exposed for 30 s to the predator odor no longer displayed any analgesia. Their response latencies were significantly (Ps ⬍ 0.02) lower than those of males that were not preexposed or that had been preexposed to the odors of a nonestrous female before being exposed to the predator odor for 30 sec. (Figs. 3A–3C). Males that were preexposed for 1 min to the odor of an estrous female and then exposed for 15 min to the predator odor displayed significantly (P s ⬍0.01) lower response latencies than males that had not been preexposed to a female or that had been preexposed to the odors of a nonestrous female before being exposed to the predator odor for 15 min. There was, however, still a significant predator odor-induced analgesia. The longer 60-min preexposure to the odors of an estrous female induce no significant attenuation of predator odor-induced analgesia, with the responses latencies of the experienced and inexperienced male mice of these groups being significantly higher than those of the males that were exposed to the odor of an estrous female for 30 s [all F(1, 19) ⬎ 13.50 , all P ⬍ 0.0003]. Preexposure for 1 or 60 min to the odors of a nonestrous female had no significant effect on the level of predator odor induced analgesia. The effects of preexposure to the odor of a female on the level of predator odor-induced analgesia were also dependent on a male’s prior experience with a female (Fig. 4). Although a 1 min preexposure to the odors of the familiar estrous female did reduce the level of analgesia induced by the 15-min exposure to predator odor in the experienced isolated males (vs no female, P ⫽ 0.0001; vs nonestrous familiar, P ⫽ 0.0001) this reduction was significantly less than that elicited by

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FIG. 4. Mean plasma levels of (A) corticosterone and (B) testosterone in sexually inexperienced isolated male mice as assessed immediately following exposure to weasel odor for either 30 s or 15 min, exposure to control odor (butyric acid) for 15 min, exposure to the odors of a novel estrous female for either 1 or 60 min, exposure to the odors of a novel estrous female for 1 or 60 min followed by a 15-min delay before measurement, exposure to the odors of a novel estrous female for 1 min and then exposure to weasel odor for 30 s or 15 min; or exposure to the odors of a novel estrous female for 60 min followed by exposure to weasel odor for 15 min. Basal hormonal values of mice receiving no odor exposures are also shown; N ⫽ 10 in all cases. Vertical lines denote a standard error or the mean.

preexposure to the odor of novel estrous female. The latencies of males exposed to a familiar estrous female were significantly higher than those of males exposed to an unfamiliar estrous female after 30 s (1-min female exposure, P ⫽ 0.0001; 60-min female exposure, P ⫽ 0.093 ) but not 15-min exposure to the weasel odor. Experiment 3: Corticosterone and Testosterone Levels Corticosterone. There was a significant main effect of the factor of odor exposure [F(10, 77) ⫽ 15.31, P ⬍

0.0001] on corticosterone levels. Post hoc comparisons showed that male mice that were exposed to weasel odor for 15 min (P ⬍ 0.0001) or 30 s (P ⫽ 0.025) displayed significant increases in corticosterone, with the 15-min exposure eliciting a significantly greater increase than the 30-s exposure (P ⬍ 0.0001) (Fig. 4A). Exposure to the control odor of butyric acid had no significant effects on corticosterone. Exposure to the odors of a novel estrous female for either 1 or 60 min had no significant effects on corticosterone either directly after exposure or 15 min later. Preexposure to the odors of a novel female for 1 min blocked the rise in corticosterone elicited by either a

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30-s or 15-min exposure to weasel odor. The corticosterone levels were not significantly different from either one another or basal levels. The 60-min female preexposure had no significant effects on the significant (P ⬍ 0001) predator odor-induced rises in corticosterone. Testosterone. There was a significant main effect of the factor of odor exposure [F(10, 77) ⫽ 25.65, P ⬍ 0.0001] on testosterone levels. Post hoc comparisons showed that male mice that were exposed to weasel odor for 15 min showed a significant decrease in testosterone levels (P ⬍ 0.0001) (Fig. 4B). Neither a 30-s weasel odor exposure nor a 15-min exposure to the control odor of butyric acid had any significant effects on testosterone. Exposure to the odors of a novel estrous female for 60 min caused a significant increase in testosterone (P ⬍ 0.0001), which was still evident 15 min later (P ⬍ 0.0001). A 1 min exposure to the odors of a novel estrous female had no significant effects on testosterone either immediately after exposure or 15 min later. Preexposure to the odors of a novel estrous female for 1 min blocked the fall in testosterone elicited by the 15-min exposure to weasel odor. The testosterone levels were not significantly different from the basal levels. In contrast, a 60-min preexposure to the odors of a novel estrous female had no significant effect on the decrease in testosterone levels elicited by the 15-min exposure to predator odor (P ⫽ 0.007). Predator odor exposure completely eliminated the increase in testosterone induced by the 60-min exposure to the novel estrous female.

DISCUSSION The results of the present study show that brief exposure to the odors of a novel estrous female mouse not only reduces the aversive responses displayed by male mice to a potential predator but also attenuates the behavioral and hormonal stress responses elicited in the males by the predator threat. Male mice that received a brief (1 min) preexposure to the odors of a novel estrous female displayed (i) a reduced aversion to, and avoidance of, the odors of a predator (cat) and a complete elimination of the avoidance of a novel odor (guinea pig) in a Y-maze preference test; (ii) decreased behavioral stress response, as shown by the lower analgesic responses elicited by exposure to the odor of a predator (weasel); and (iii) an attenuated hormonal stress response as evidenced by both the blunting of the rise in corticosterone and fall in testos-

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terone levels elicited by exposure to weasel odor. Neither a more prolonged (60 min) preexposure to the odors of a novel female nor preexposure to the odors of either familiar or nonestrous females consistently affected the behavioral and hormonal responses of sexually experienced male mice to predator odor. As such, male mice can be considered to be “emboldened” following brief exposure to a novel estrous female, displaying markedly reduced fear, stress, and avoidance responses to predator odor and the implied risk of predation. Consistent with the results of prior investigations control male mice that were exposed to predator odor displayed marked fear, anxiety, and stress responses. In the odor preference test these male mice displayed an intense aversion to, and avoidance of, predator (cat) odor and a slight, but significant, avoidance of a novel odor (guinea pig). This predator, and to a lesser extent novelty avoidance response, involves a heightened anxiety and/or fear which has been previously reported to be attenuated by anxiolytic agents (Blanchard et al., 1993; Kavaliers, Wiebe, and Galea, 1994a; Kavaliers and Colwell, 1995b; Dielenberg, Arnold, and MacGregor, 1999). Similarly, both prolonged (15 min) and brief (30 s) exposures to predators or their odors have been previously shown to elicit marked decreases in nociceptive sensitivity indicative of the induction of analgesia (e.g., Lester and Fanselow, 1985; Kavaliers, 1990; Kavaliers and Colwell, 1991; Lichtman and Fanselow, 1991). These analgesic responses involved stress-induced activation of endogenous opioid peptide mediated and nonopioid mediated neuromodulatory systems with the opioid-mediated responses elicited by 15-min exposure being of a greater magnitude and duration than the non-opioid-mediated responses elicited by the 30-s exposures (Kavaliers and Colwell, 1991). Stress-induced analgesia is adaptive in these situations, facilitating the expression of various active and passive antipredator defensive behavioral responses. It should be noted that exposure of experienced males to the odors of a novel estrous female and inexperienced males to estrous and nonestrous females also elicited a low-level analgesic response. This likely arises from, and incorporates, preparatory mechanisms for a sexual interaction that may encompass some aggressive components. The magnitude and duration of this analgesic response is, however, markedly lower than that elicited by the predator odors. In parallel to these predator-induced behavioral responses, exposure to weasel odor also elicited rises in corticosterone levels as well as decreases in testoster-

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one levels which are consistent with the results of prior investigations examining the effects of predator and predator odor exposures (File et al., 1993; Blanchard et al., 1998; Perrot-Sinal, Kavaliers, and Ossenkopp, 1999; Morow et al., 2000). These hormonal alterations most likely elicit motivational shifts that facilitate the expression of appropriate active and passive defensive responses. Brief exposure to female odors had no significant effects on corticosterone levels in males, consistent with the minimal analgesic responses elicited. Brief, but not prolonged, preexposure to a novel female reduced all of the predator odor and novelty elicited anxiety, fear, and stress responses in both sexually experienced and inexperienced males. Similarly, brief preexposure to the novel female eliminated the avoidance of the novel odor of guinea pig. This increased “boldness” elicited by brief exposure to female odors may involve short-term neuromodulatory shifts in the males that reduce the predator odorinduced anxiety and stress, increasing fearlessness and facilitating both mate search and aggressive interactions with other males competitors. Possible mediating candidates for these effects include neuroactive steroids whose levels, metabolism, and relative ratios are subject to rapid behavioral and environmental modulation (Compagnone and Mellon, 2000). Neuroactive steroids can have rapid anxiolytic effects, including decreasing avoidance of predator threat (Kavaliers, Wiebe, and Galea, 1994b). They can also rapidly enhance male sexual interest and intermale aggression, which would be adaptive in the present context (Guillot and Chapouthier, 1996). In this regard, it is of interest that the main olfactory regions, which detect and/or process both predator odors (Funk and Amir, 2000) and female odors (Nyby, 1983), are a particularly rich area of neuroactive steroid activity (Guillot and Chapouthier, 1996). Other central neurochemical changes are also likely. Evidence from studies with male rats indicates that exposure to the odors of estrous females can elicit rapid changes in central dopamine and possibly oxytocin levels, which could also contribute to the increased boldness (Pfaus, 1999; Sachs, 1999). The effects of preexposure to a female on responses to a predator were also affected by a males sexual experience and history. In the case of sexually experienced males exposure to the odors of an estrous female that a male was either presently or previously paired with elicited minimal modifications in the behavioral and hormonal responses to predator odor. Likewise, exposure to the odors of a nonestrous fe-

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males had minimal effects. This further supports the contention that it is the presence of a novel, sexually available female, possibly indicated by volatile odor cues, that elicits an apparent greater boldness in males. It is not just the presence of the odors of a female per se that elicits a reduced responsiveness to predator odor and greater boldness and risk taking by the male. This dependence on the presence of the odors of a novel female is reminiscent of the “Coolidge Effect” reported for the effects of a novel female on male sexual behavior (Brown, 1979). The situation was somewhat different for sexually inexperienced males. Here preexposure to the odors of nonestrous females, as well as more prolonged preexposure to the odor cues of estrous females, had some significant effects on the predator odor-elicited responses. This likely reflects the lack of prior experience with, and access to, females that results in augmented interest in responsiveness to the cues associated with any female. It may also result from lack of formation of a complete odor template of what is the most sexually receptive female (Barnard, Hurst, and Aldhous, 1991). This is reflected by the low-level analgesic response elicited in the sexually inexperienced males by exposure to the odors of either an estrous or nonestrous female by herself. These behavioral and neuromodulatory changes in the male mice may be elicited by either relatively short-lived highly volatile odor cues or relatively nonvolatile odor cues (Hayashi and Kimura, 1974; Brown, 1979; Nyby and Whitney, 1980; Nyby, 1983; Wysocki, Katz, and Berhnard, 1983; Sipos et al., 1995; Humphries et al., 1999; Mucignat-Caretta and Caretta, 1999a, b). Volatile odor cues appear to be ovarian steroid dependent and signal the sexual status of females. These chemical signals are perceived by the main or primary olfactory system, with the responses elicited being affected by a male’s sexual experience. In contrast, the low-volatility odor cues, which are detected by the accessory olfactory bulb and and vomeronasal organ, appear to be relatively independent of a female’s sexual state and are affected by a male’s prior sexual experience (Nyby and Whitney, 1980). The increased “boldness” and decreased responsiveness to predator risk in the male mice briefly exposed to female odors was not directly associated with an immediate increase in testosterone levels. Brief exposure to a novel estrous female had no apparent significant effects on male testosterone levels at either 1- or 15-min postexposure. However, due to the pulsatile reflexive release of testosterone in mice (Coque-

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lin and Desjardins, 1982) a possible peak in testosterone may not have been detected here. Whether a rise in testosterone would be elicited either at different or later time points still remains to be determined. The more prolonged 60-min exposure to a female, which did not ameliorate the predator response, did by itself result in a significant rise in plasma testosterone similar to that reported in prior investigations (Macrides, Bartke, and Dalterios, 1975; Batty, 1978; Smith, Barnard, and Behnke, 1996). This rise was shown to be blunted by exposure to the predator odor. Whether this fall in testosterone is associated with, or dependent on, the dramatic rise in corticosterone evident following predator odor exposure remains to be determined. As well, the relative impact of the pulsatile nature to testosterone release remains to be evaluated. Brief exposure to a novel female may signal the likelihood of an immediate, though temporally limited, availability of a sexually responsive estrous female. This could elicit a rapid, though of brief duration, motivational shift in the males away from defensive, anxiety-related responses to a possible predator threat to a search for, and possible sexual interactions with, a female. The more prolonged exposure to female odors is less likely to be indicative of the presence a transient female of limited availability. As such, it would not be adaptive for an experienced male to display any protracted reductions in antipredator responses. There is also evidence from investigations with guppies that females have a preference for bolder males (Godin and Dugatkin, 1996). Male guppies and potentially other species (Dill, Hedrick, and Fraser, 1999) utilize this preference, displaying greater boldness and predator inspection when a female is in their immediate vicinity. Male mice may be employing a similar strategy in regard to predator risk, with the brief preexposure to a female odor signaling the likelihood of there being a female in the immediate vicinity. The decreased responsiveness to predator odor may again either arise from, or incorporate, neuromodulatory shifts elicited by brief preexposure to female cues. As such, it is possible that the apparent greater risk taking or male boldness is a side effect of the lower fear and stress responses that are associated with a greater sexual motivation and “searching” for the briefly available novel female. The results of this study show that antipredator responses of males are condition or context dependent. Brief preexposure to the odors of a novel estrous female shifts the condition and likely motivational state of males such that they display greater “bold-

Kavaliers, Choleris, and Colwell

ness” and less behavioral and hormonal sensitivity to the risk of predation.

ACKNOWLEDGMENTS We thank Phero-Tech for supplying the weasel odor. We also thank two reviewers for their useful comments. This research was supported by Agriculture and Agri-Food Canada and the Natural Sciences and Engineering Research Council of Canada.

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