Cues that Elicit Ultrasounds from Adult Male Mice

Transcription

1 AMER. ZOOL., Cues that Elicit Ultrasounds from Adult Male Mice GLAYDE WHITNEY Department of Psychology Florida State University Tallahassee, Florida AND JOHN NYBY Department of Psychology Lehigh University Bethlehem, Pennsylvania SYNOPSIS. Adult male mice (Mm musculus) which have a prior history of experience with other adult male and adult female mice readily produce 70 khz ultrasonic vocalizations in the presence of urine from adult females but not in the presence of urine from adult males. Urine from immatures of either sex does not elicit ultrasounds from socially experienced adult males. The ultrasound eliciting potency of adult male urine was not improved substantially following castration of adult males, injection of testosterone propionate to castrated adult males, administration of estradiol benzoate to castrated adult males, or neonatal castration. Ovarian hormones do not appear to be necessary for either the appearance at puberty, or the maintenance during adulthood, of the ultrasound eliciting cues of female urine. Stage of estrus did not have a major modulating effect on urinary cues eliciling male ultrasounds. Treatments that did not substantially reduce the signal value of adult female urine include ovariectomy before or after puberty, ovariectomy with adrenalectomy, and neonatal administration of testosterone. The administration of testosterone to ovariectomized adult females, and hypophyseclomy, virtually eliminated the ability of urine from adult females to elicit ultrasounds from socially experienced adult males. The implication of pituitary hormones in the modulation of female urinary cues thai elicit ultrasounds is particularly interesting since pituitary factors are also implicated in the proximal causation of postparturient maternal aggression, which adult male ultrasounds may function to moderate. INTRODUCTION There is great diversity and redundancy in the cues that can elicit ultrasonic vocalizations from adult male mice. This wealth of multiple cues resulted in some initially puzzling findings, and some apparently contradictory claims, from our laboratory when we began to investigate the context of ultrasonic communication among adult mice (Mus musculus). What was to us a puzzling result occurred in our very first series of experiments (Whitney et al., 1971, 1973). As part of our first experiments we looked at individual differences among our subjects. As expected, there was quite a lot of variation among male mice in the likelihood of their producing ultrasonic signals in various contexts. We subsequently reported genetic (Coble and Whitney, 1972; Coble et al., We gratefully acknowledge the assistance of Charles Wysocki, Jill Schneider, and Gerard Dizinno in many aspects of the summarized research. Preparation of this report and portions of the original work were supported by N.S.F. Gram BNS ; Nyby and Whitney, 1978; Whitney tf al., 1978), ontogenetic (Dizinno etal., 1978; Nyby et al., 1978), and physiological mediation (Dizinno and Whitney, 1977; Nyby et al., 1977a; Nyby and Whitney, 1978) of influences on male ultrasound production. The surprise was that there was no consistent variation among adult females in how effective they were in eliciting ultrasounds from males. Essentially all of the systematic variation among pairs and consistency across time in ultrasound production was related to the male of the pair (Whitney et al., 1973). That was puzzling. An apparent contradiction arose across a series of subsequent experiments. When we first systematically investigated the cues from adult females that were necessary or sufficient to elicit ultrasounds from adult males, the results were quite clear (Whitney et al., 1974). Visual, auditory, or movement cues from females were not necessary. Chemical cues from females, contained in soiled bedding from female cages, were sufficient to elicit ultrasounds from adult males. Although chemical cues were implicated, female urine did not appear to be an 457

2 458 GLAYDE WHITNEY AND JOHN NYBY important source of cues influencing male ultrasounds. Indeed, the low level of response to female urine, compared to the higher level of response to intact females or to soiled bedding from female cages, led us to cautiously suggest that the active chemical substance was not primarily carried by urine, and that the weak response to female urine may be due to its "occasional contamination with some sufficient secretion that is not a usual constituent of female urine" (Whitney et al., 1974, p. 17). The contradiction became apparent shortly thereafter when a series of experiments indicated that female urine was often a very potent elicitor of ultrasounds from adult male mice (Nyby et al., 19776). Thus the apparent contradiction involved great variation in that, in some experiments, female urine was not very effective in eliciting ultrasounds, while in other experiments female urine was very effective. The former puzzling outcome was an apparent lack of variation among adult females in their ability to elicit ultrasounds from adult males. ONTOGENY OF SICNAL VALUE Both the lack of expected variation and the presence of unexpected variation were largely accounted for by the results of later experiments dealing with the ontogeny of cue function in the elicitation of ultrasounds. Dizinno et al., (1978) demonstrated that male mice learn the signal value of female urine during ontogeny. When they separated male mice from females at weaning and then in adulthood exposed males to female urine, most males did not produce ultrasounds in the presence of the female urine. However, males that were similarly isolated after weaning, but were as adults provided with brief exposures to adult males and females, did produce ultrasounds when subsequently tested with female urine. Those males that produced ultrasounds in the presence of female urine after experience with females, later ceased to produce ultrasounds when repeatedly tested with female urine. Thus the ultrasonic response of adult males to cues provided by the urine of adult females appeared to be a learned response in demonstrating both acquisition and extinction as a function of experience during ontogeny (Dizinno et al., 1978). It is interesting that the learning may take place very rapidly since the results of another experiment suggested that as little as one 3-minute exposure to females in adulthood could be followed by a high level of ultrasound production in the presence of female urine (Nyby et al., 19776). When dealing with a social communicative function of conspecific chemicals, it appears to be very common to adopt as a working hypothesis a simple unconditioned reflex model in which some chemical, a presumptive pheromone, is assumed to have innate cue function for the species. Indeed, Beauchamp et al., (1976) pointed out that possible effects of experience had not been examined for any of the five mammalian pheromones which had been reportedly characterized chemically. In our work with the male mouse, ultrasonic response to female urine, it appeared that an innate unconditioned reflex model was clearly inappropriate; a more accurate model seemed to be to view the cue properties of female urine as conditioned stimuli, perhaps in a straightforward classical Pavlovian conditioning paradigm. The earlier, apparently contradictory, findings that female urine sometimes did and other times did not elicit ultrasounds from males can be attributed in large part to the prior experience of the males. Female urine is not a potent elicitor of ultrasounds for males subjected to postweaning isolation from females. However, if adult males experience brief exposure to adult females, then female urine acquires signal value as an elicitor of ultrasounds from males. Other aspects of male experience also affect their likelihood of producing ultrasounds to various cues. Social experience with other males that is extensive enough to establish social subordination will inhibit ultrasound production to females (Nyby et al., 1976). Learning seems to be very rapid and pervasive so that across repeated tests with female cues, males will often come to produce anticipatory ultrasounds upon initial transfer to the testing situation (unpublished observations). Relatively short-term

3 CUES ELICITING ULTRASOUNDS 459 carryover effects of prior experience are also common. For instance, when males are sequentially tested with adult males and with adult females, exposure to a male can inhibit subsequent responding to a female while exposure to a female can increase later responding in the presence of a male. Normal urinary products are not the only chemicals that can acquire ultrasound eliciting properties. Artificial chemicals that do not normally elicit ultrasounds from male mice can acquire female signal value and come to elicit ultrasounds when suitably experienced by males (Nyby et al., 1978). Quick learning of multiple cues associated with females may also be responsible to some extent for the initially puzzling lack of variation among females in their potency as stimuli to elicit ultrasounds from males. If males readily learn many cues that they experience in association with females, then in some experiments the resulting redundancy of ultrasound eliciting cues could obliterate any signs of individual differences among females on specific stimulus dimensions. SIGNAL VALUE OF MOUSE URINE From considerations of cue redundancy due to learning, it may be tempting to adopt a very general view of ultrasound elicitation in which cue specificity plays little or no part. However, such a completely general view is almost certainly incorrect. When adult male mice have some exposure to other mice of both sexes, they very quickly become quite precise in that they readily produce ultrasounds in the presence of female urine and rarely produce ultrasounds in the presence of male urine (Nyby et al., 19776). Thus there are readily perceived sex differences in the signal value of mouse urine. An important communicatory role for urine-based chemical cues under natural conditions is of course compatible with what we now know about the ontogeny of the signal value of urine. In nature, reproductively successful male mice are obviously never complete isolates, and probably often have some experience with other adult males as well as with at least one adult female. The acquisition by male mice of sex cue value of urinary substances may be similar to imprinting in some bird species in which sexual imprinting can occur at a substantially more advanced age than does filial imprinting. The pubertal or dispersal age establishment of sex pheromone signal value could be important in population dynamics, tending to reduce the amount of close inbreeding in natural populations (Whitney and Nyby, 1977; Bateson, 1978). In view of the pervasive effects of experience upon the cues that will elicit ultrasonic vocalizations from adult mice, it is obviously of central importance to specify the prior social experience of males in any attempts to investigate the signals to which males are responding. One interesting set of questions has to do with the nature of the cues by which isolated male mice identify females. Another set of questions which may be of more central relevance to naturalistic situations, has to do with the sex-specific cues which elicit ultrasonic vocalizations from adult mice that have some familiarity with conspecifics. We present below a summary of the results of a series of experiments which were concerned with the hormonal substrates of sex-specific urinary cues that elicit ultrasounds from adult males. Social experience The approximately 200 adult male mice which served as subjects in these experiments were all systematically exposed during adulthood to both male and female adult mice to provide a standardized prior history of experience with conspecifics. All male subjects were housed in same-sex groups at weaning. Then after reaching sexual maturity they were individually housed. After 2 or 3 weeks of isolation, each male subject was provided with a social exposure regime. Social exposure consisted of placing an adult male and an adult female stimulus animal into the subject's home cage for 3 min each day over 8 consecutive days. The order of presentation of the 2 sexes was counterbalanced across subjects and reversed each day. To ensure that our male subjects did not become socially sub-

4 460 GLAYDE WHITNEY AND JOHN NYBY ordinate to the social-exposure males, the male stimuli were presented during the first few exposures by dangling them by their tails in front of the subject males. Socialexposure females were simply placed in the male's home cage. After the 8 days of social exposure, each subject male was tested for ultrasound production in the presence of a female on one 3-min test. Only males from pairs that produced ultrasounds were used as subjects in subsequent experiments. However, this screening procedure led to the elimination of very few potential subjects. Testing for ultrasounds These socially experienced adult male mice were then tested to see if they would produce ultrasounds in the presence of various chemicals. The test chamber was a plastic mouse cage (29 x 18 x 13 cm) and the stimuli were presented on cotton swabs that were dropped into the test chamber. Testing always occurred during the light portion of a 12:12 light/dark cycle. Ultrasounds were monitored with a Holgate ultrasonic receiver adjusted to a frequency of 70 khz (Whitney et ai, 1973). Before the introduction of a chemically treated cotton swab, a subject was monitored for 1 min to ensure that it was not emitting ultrasounds to incidental aspects of the test situation. If ultrasounds were detected during this 1-min habituation period, then 2 min without an ultrasound was required before introducion of a cotton swab. Ultrasounds were rarely detected during habituation. A 3-min test then began by placing the cotton swab into ihe test chamber. The amount of ultrasound produced was quantified by dividing the 3-min trial into 36 5-sec intervals. The number of intervals containing ultrasound was recorded, resulting in possible scores ranging from 0 to 36. All of the over 200 subjects were not exposed to each of ihe 1 9 stimulus conditions summarized below. In some experiments different stimuli were presented to independent groups, with one stimulus presentation to each subject. In other experiments the subjects were each exposed io the same subset of stimuli. In such multiple exposure experiments, the order of stimulus presentation was counterbalanced across subjects and individual exposures were separated by 48 hr. In either case control stimuli were employed to permit some comparison among stimuli across experiments. Urinary stimuli Figure 1 presents a summary of results for 19 stimulus conditions. In the figure the stimulus condition labeled " 1" is set at 100% and represents the amount of ultrasound produced by adult male mice in the presence of a cotton swab containing urine from a normal adult female mouse. For comparison purposes the mean ultrasound scores for the other 18 stimulus conditions are expressed as percent of responses relative to normal female urine or, when indicated, another appropriate control. Stimulus condition 2 (S-2) is a control of distilled water on a cotton swab. As indicated in the figure, very few ultrasounds are emitted in the presence of distilled water in comparison to the amount of ultrasound emitted in the presence of normal female urine. Stimulus 3 is urine from normal adult male mice. Obviously, as previously reported (Nyby et al., 1977ft), after experience as adults with other adult, male and female mice, adult males are quite precise in that they produce much ultrasound in the presence of normal adult female urine (S-l) and not in the presence of urine from normal adult male mice (S-3). Male urine (S-3) was no more effective than was distilled water (S-2). Similarly, urine collected from prepubertal age male mice (S-4) was not an effective elicitor of ultrasounds from socially experienced adult males. The ultrasound eliciting property of adult male urine was not substantially improved following castration of adult males (S-5), injections of testosterone proprionate to castrated adult males (S-6). administration of estradiol benzoate to castrated adult males (S-7), or neonatal castration (S-8). Urine collected from prepubertal age females (S-9) also was relatively ineffective as an elicitor of ultrasounds from socialk experienced adult males. The inefft'cthe-

5 CUES ELICITING ULTRASOUNDS 461 ness of urine from prepubertal females indicates that the ultrasound eliciting properties of female urine probably develop around puberty. However, contrary to some expectations but consistent with earlier findings of adult female invariance (Whitney et al., 1973, 1974), the potency of adult female urine to elicit ultrasounds did not vary substantially with estrous cycle. Expressed relative to urine from estrous females, urine obtained during proestrous (S-10), metestrus (S-11), and diestrus (S- 12), all elicited quite substantial amounts of ultrasound from males. Consistent with the interpretation that ovarian steroid hormones are relatively unimportant in determining the sex difference in signal value of adult mouse urine, neither long-term ovariectomy of adult females (S-13) nor ovariectomy combined with adrenalectomy (S-14), substantially affected the potency of adult female urine to elicit ultrasounds from adult males. In addition, ovarian steroid hormones do not appear to be necessary for the initiation at puberty of female cues, since ovariectomy prior to puberty (S-15) did not prevent the adult female urine from eliciting ultrasounds. All of the above results are consistent in l20r CO 2 O Q. CO C 60 UJ O 40 Ul a. 20 w II URINE STIMULUS 0 n FIG. I. 70 khz ultrasonic vocalizations of socially experienced adult male mice were quantified by recording ihe number of 5-sec time intervals containing ultrasounds during a 3-min lest. Stimuli were presented by dropping a treated cotton swab into the test cage. Mean scores are plotted as percent relative to responses of males to urine from normal adult females. Stimulus conditions are: I) urine from normal adult female mice; 2) distilled water control swabs; 3) urine from normal adult male mice; 4) urine from prepubertal age male mice; 5 urine from males castrated when adult; 6 urine from adult male castrates injected with testosterone propionate; 7 urine from adult male castrates injected with eslradiol ben/.oate; 8 urine from adult males castrated as neonates. 9 urine from prepubertal age female mice; 10 urine of females in proestrus, expressed relative to urine of estrus females; 11 urine of females during metestrus, expressed relative to urine of eslrus females; 12 urine of females during diestrus, expressed relative to urine of estrus females; 13 urine from females ovariectomized when adults; 14 urine from females ovariectomized and adrenaleclomized when adults; 15~urine from adult females ovariectomized prior to puberty; 16 urine from adult females administered testosterone propionate when neonates; 17 urine from adult ovarieclomi/ed females during injections of testosterone propionate; 18 urine from hypophysectomized adult females; 19 soiled cage shavings from hypophysectomized adult females, expressed relative to soiled cage shavings from normal adult females.

6 462 GLAYDE WHITNEY AND JOHN NYBY leading to the suggestion that ovarian steroid hormones are of relatively little importance in either the initiation at puberty, or the maintenance during adulthood, of the ultrasound eliciting cues of female urine. However, the pattern of results across several experiments may suggest a minor modulating effect of ovarian hormones upon the ultrasound-eliciting signal value of urine. Stimulus condition 7, which was urine from castrated males given estradiol benzoate, appears slightly above the other male conditions, while metestrus (S- 11), adult ovariectomy (S-13), ovariectomy combined with adrenalectomy (S-14), and prepubertal ovariectomy (S-15), resulted in adult female urine which appears in Figure 1 to be slightly less effective than the other adult female urine conditions (S-10 to S-16). Although there may appear to be a pattern of ovarian hormone involvement, we wish to emphasize that in the individual experiments, appropriate comparisons were not consistently significant statistically, and that these ovarian hormone conditions can at best account for only a small proportion of the variability between normal female urine and male urine in ultrasound eliciting potency. Although castration of males in adulthood (S-5) or on the first day after birth (S-8) did not improve the ability of adult male urine to elicit ultrasounds, early masculinization of females might have reduced the potency of adult female urine. However, it did not. Females treated with testosterone when neonatal (S-16) produced urine in adulthood that was as effective as the urine of normal adult females. Finally with stimulus condition 17 we have a treatment condition which dramatically, and statistically significantly (X 2 (1) = 14.63, P <.0002), reduced the ultrasound eliciting potency of urine from adult females. The administration of testosterone propionate to ovariectomized adult females (S-17) virtually eliminated the ability of their urine to elicit ultrasounds. Exogenous androgen administration to females results in urine that is no more effective than distilled water (S-2) or male urine (S-3 to S-8) for eliciting ultrasounds. However, this effect of exogenous androgen is probably not a direct effect of circulating androgen on urinary potency because neither neonatal castration (S-8) nor adult castration (S-5) affected the potency of male urine. One possibility is that the effect of exogenous androgen is mediated by the inhibitory effect of testosterone on the release of hormones from the pituitary gland. If so, then the urine from adult hypophysectomized female mice should also not elicit ultrasounds from socially experienced male mice. The urine from hypophysectomized females (S-18) was much less potent in eliciting ultrasounds than was the urine from sham-operated control females (X 2 (1) = 13.40,P<.003). This effect of hypophysectomy on potency of female urine is probably not a trivial effect of urinary dilution resulting from the diabetes insipidus of hypophysectomized animals. Stimulus 19 in Figure 1 is the potency of soiled bedding from hypophysectomized females' cages relative to the potency of soiled bedding from the cages of control females. The soiled bedding from hypophysectomized females is relatively ineffective even though it might be expected to concentrate relatively nonvolatile constituents of urine. In another experiment not included in Figure 1 we diluted urine from normal females to equal the volume per unit body weight of the urine from hypophysectomized females. The resulting urine from normal females, although diluted by a factor of 6, was still more potent (X 2 (1) = 4.48, P <.04) for ultrasound elicitation than was the urine from hypophysectomized females. The above results again indicate that male mice, which have had experience during early adulthood with both other adult male and adult female mice, clearly discriminate between the urine of adult males and that of adult females. Adult males typically produce ultrasounds in the presence of urine from adult females but not in the presence of urine from adult males and not in the presence of urine from immatures of either sex. Urine from females acquires ultrasound eliciting signal value at around puberty. However, ovarian hormones do not appear to be necessary for cither die

7 CUES ELICITING ULTRASOUNDS 463 appearance at puberty, or the maintenance during adulthood, of ultrasound-eliciting urinary cues. For males, neither neonatal castration nor adult castration leads to an increase in the potency of male urine to elicit ultrasounds. Treatments of females which did reduce the ultrasound eliciting cue value of their urine were administration of exogenous testosterone to adults, and hypophysectomy. The effects of both of these treatments are consistent with the suggestion that pituitary hormones are important in regulating the ultrasound eliciting potency of mouse urine. These results implicating pituitary factors in the proximal modulation of female urinary cues that elicit ultrasounds from adult males are particularly interesting in view of their parsimonious relationship with the suggestion of an ultimate evolutionary role for sexual selection in the phylogeny of ultrasonic communication among adult mice. Pituitary factors have also been implicated in the proximal causation of postparturient maternal aggression, which adult male ultrasounds may function to moderate. REFERENCES Bateson, P Sexual imprinting and optimal outbreeding. Nature 273: Beauchamp, G. K., R. L. Doty, D. G. Moulton, and R. Mugford The pheromone concept in mammalian chemical communication: A critique. In R. L. Doty (ed.), Mammalian olfaction, reproductive processes and behavior, pp Academic Press, New York. Coble, J. R. and G. Whitney Genetics of ultrasound production by adult male Mus musculus. Amer. Zool. 12:89. (Abstr.) Coble, J. R., G. Whitney, and M. Alpern Genetics and evolutionary significance of ultrasound production in adult Mus musculus. Behav. Genet. 3: (Abstr.). Dizinno, G. and G. Whitney Androgen influence on male mouse ultrasounds during courtship. Hormones Behav. 8: Dizinno, G., G. Whitney, andj. Nyby Ultrasonic vocalizations by male mice (Mus musculus) to a female sex pheromone: Experiential determinants. Behav. Biol. 22: Nyby, J., G. Dizinno, and G. Whitney. 1977a. Sexual dimorphism in ultrasonic vocalizations of mice (Mus musculus): Gonadal hormone regulation. J. Comp. Physiol. Psychol.91: Nyby, J., G. A. Dizinno, and G. Whitney Social status and ultrasonic vocalizations of male mice. Behav. Biol. 18: Nyby.J., and G. Whitney Ultrasonic communication of adult myomorph rodents. Neurosci. Biobehav. Rev. 2:1-14. Nyby, J., G. Whitney, S. Schmitz, and G. Dizinno Postpubertal experience establishes signal value of mammalian sex odor. Behav. Biol. 22: Nyby, J., C. J. Wysocki, G. Whitney, and G. Dizinno. 1977A. Phermonal regulation of male mouse ultrasonic courtship (Mus musculus). Anim. Behav. 25: Whitney, G., M. Alpern, G. Dizinno, and G. Horowitz Female odors evoke ultrasounds from male mice. Anim. Learn. Behav. 2: Whitney, G.,J. R. Coble, M. Stockton, and E. F. Tilson Ultrasonic emissions: Do they facilitate courtship of mice? J. Comp. Physiol. Psychol. 84: Whitney, G. andj. Nyby Ontogeny of mammalian pheromone function: Adult experience is crucial for mice. Proceedings of XVth International Ethological Conference, Bielefeld, W. Germany, p. 71 (Abstr.) Whitney, G., J. Nyby, J. R. Coble, and G. A. Dizinno Genetic influences on 70 khz ultrasound production of mice (Mus musculus). Behav. Genet. 8:574. (Abstr.) Whitney, G. D., M. D. Stockton, and E. F. Tilson Possible social function of ultrasounds produced by adult mice (Mus musculus). Amer. Zool. 11:634. (Abstr.)