BEHAVIORAL BIOLOGY, 14, 237-240 (1975), Abstract No. 4313
BRIEF REPORT Olfactory Perception of the Nonsocial Environment by Male House Mice 1 J. A. MARUNIAK, K. J. DARNEY, JR., and F. H. BRONSON
Department of Zoology, The University of Texas, Austin, Texas 78712 It has been previously shown that the most profound elicitor of urinary marking in male mice is biological or physical novelty. In the present study the frequency of urinary marking by anosmic or control males was tested when either alone or in the presence of a female. Anosmia was induced by perfusing the nasal epithelium with zinc sulfate solution and verified by a conditioned response to an odor. Marking rates of anosmic males were significantly lower in both test situations. The somewhat surprising depression of marking among isolated, anosmic males strongly suggests that olfaction is the primary modality for detecting even nonbiological novelty. A second experiment revealed that the depression of marking in anosmic males was not due solely to a reduction of locomotor activity. Recently we reported on factors that regulate the frequency of urinary marking by male house mice, the two most important being social rank (Desjardins et al., 1973) and environmental novelty (Maruniak et al., 1974). Regarding the latter factor, male mice mark at high rates in a strange test cage but habituate rapidly to repeated daily testing regardless of the presence or absence of a stimulus male. Exposure of male-habituated males to a strange male has no effect on marking frequency but exposure to the presence of a different type of stimulus animal, e.g., a deermouse or a female house mouse decidely elevates their marking. Thus novelty is a strong elicitor of urinary marking and may be defined for the mouse on the basis of either social cues or an unfamiliar physical environment. The present experiment was designed to study the effect of zinc sulfate-induced anosmia on urinary marking by previously isolated male mice when placed in a strange cage with or without a female present. The results confirm our previous conclusions regarding the 1This work was supported by U.S. Public Health Grant HD-03803 from the National Institute of Child Health and Human Development. 237 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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importance of novelty but, somewhat surprisingly, they also strongly suggest that the perception of the non-social environment may also be mediated via olfaction. Sixty CF-1 male mice were obtained from Carworth Farms at 8 weeks of age and isolated in 18 × 29 X 13 cm polypropylene cages until used in this experiment at 13 weeks of age. Animal rooms were maintained at 23 ° C on a 14:10 light:dark cycle (lights off at 1900) and given ad lib food and water until the start of the experiment. Males were first trained to respond to an odor cue and, following successful conditioning, were rendered anosmic or not. The odor training test was then used to verify success of the anosmic manipulation and finally, anosmic and control males were tested for urine marking in either a social or nonsocial situation. In more detail, all experimental males were trained to find one-sixth of an Oreo cookie buried in 2 to 4 cm of clean pine shavings. This procedure has been shown by Edwards et al., (1972) to result in a reliable test for anosmia. The daily training procedure consisted of 20 hr of food deprivation followed by a 5 rain session in a neutral cage in which the latency for each male to find the buried cookie was recorded. Each session was followed by 4 hr access to food. This cycle was repeated for 4 days. Only the 52 males that achieved latency scores of less than two rain by day 4 were considered sufficiently trained to be used as test subjects. Thirty-two of these males were anesthetized with ether; the blunted tip of a 25 gauge hypodermic needle was inserted 2 mm into each nostril and 0.02 ml of the 5% zinc sulfate solution was injected (Alberts, 1974; Edwards, 1974). The mice were held nose down for 2 rain to prohibit ingestion, and the back of the throat was aspirated. The same procedure was performed on the remaining 20 males using physiological saline. All males were food deprived for the following 20hr,!then tested for latency to find the cookie. All saline treated males found the cookie well within the accepted time with a group mean latency of 29 + 3 sec (range 15-55 sec) while 24 zinc sulfate treated males failed to find the cookie within the 5 min test period and were considered anosmic. At 2200 on day 5 (the day following zinc sulfate treatment), after 8 hr access to food, the males were tested for urinary marking. Each male was placed into one side of a bottomless 30 × 30 X 15 cm test cage divided into two equal-sized chambers by a 0.2 cm wire mesh barrier. The four combinations of test and stimulus animals were anosmic males across from (a) an empty chamber or (b) an adult female, and saline treated males across from (c) an empty chamber or (d) an adult female. Adult females were used at random stages of their estrous cycle since this factor has been previously shown to be unimportant in the elicitation of marking (Maruniak e t al., 1974). During the 1 hr tests the test cages were placed on large, clean sheets of Whatman No. 2 filter paper. Urine marks on the filter paper were then later counted under ultraviolet light (3666 A, 15 W bulb; Desjardins et al., 1973).
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The zinc sulfate treated males were again taken off food; a cookie test performed on the following day verified continued anosmia in all cases. The mean urinary marking rate for anosmic males when there was no stimulus animal in the second chamber was 103 + 20, and with an adult female in the second chamber was 123 + 25. Saline treated males with no stimulus animal in the second chamber marked at a mean rate of 318 + 48, and with an adult female in the second chamber, 408 + 16. Analysis of variance revealed significance associated with the depression of marking among anosmic animals ( P < 0 . 0 1 ) ; the presence of the female, however, failed to significantly alter marking rates and there was no significant interaction. A second group of 15 males, matched in age and experience t o t h e first group, were divided so that 10 received zinc sulfate treatment and five received saline treatment. These were experimentally treated the same as the first group except that they were tested for locomotor activity instead of urine marking on the day following zinc sulfate or saline treatment. Seven confirmed anosmic and five saline treated males were placed in the type of cage described above. The filter paper floor was divided into six equal-sized areas by pencil lines. Line crossings were recorded for the first 3 min of exposure and again after 50 rain of exposure to the test cage. The mean numbers of lines crossed by anosmic males was 61 + 5 during the first 3 min in the cage and 30 +- 7 after 50 rain. Saline treated males crossed 90 + 17 lines in the first 3 rain test and 37 + 10 in the second test. The differences between the activity of anosmic and saline treated males were not significant at either time period (P ~ .05) but the small sample size must be noted. Two conclusions regarding olfaction and urinary marking emerge from these experiments. First, the data show the overriding importance of nonsocial environmental novelty in eliciting urinary marking. The addition of a strange female to the strange cage elevated marking only by an additional and nonsignificant 25%. Strange females effectively and dramatically elicit marking in males previously habituated to a test cage (Maruniak et al., 1974). Secondly, and surprisingly, the mouse's recognition of nonsocial environmental novelty (at least as defined in this simple test cage) seems to be almost entirely mediated by olfaction. Previous work has shown that males react to their first exposure to the test cage by marking at rates of 300-400/hr; repeated exposures result in habituated rates of about 100/hr (Maruniak et al., 1974). Such frequencies of marking are consistent with the present results. Intact males marked an average of 318 times an hour when placed in the strange test cage. In contrast, anosmia males, when first exposed to the test cage, marked at a mean rate of only 103 marks/hr, i.e., about the same baseline as previously established for habituated intact males. Importantly, the action of zinc sulfate-induced anosmia to decrease urinary marking seems to be traceable only in a relatively small part to depressed locomotor activity. Anosmia depressed marking by an average of 70-80% in the social and
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non-social test situations but the possible locomotor-depressing effect of anosmia was statistically insignificant and averaged only about 26% (32% in the first 3 min and 19% after 50 min). Nonsignificance of these data may have been traceable to a small sample size. The present results, nevertheless, when considered in t o t o , certainly imply that the nonsocial novelty of these test cages was almost entirely perceived via olfactory cues, and that the depressed marking was due more to a motivational change than to a change in locomotor activity. On the basis of this and previous experiments with urinary marking, one can define a familiar environment for the male mouse as one in which he marks at a low rate because he is not perceiving novel olfactory cues. Undoubtedly, there are limits to the types of novel odors that would be able to elicit high marking rates (e.g., the novel odor of predators?). As a hypothesis, then, a male mouse in nature is probably familiar with a certain number of odors: his own, the normal odors of his home range and/or territory, and those of any cohabitants and neighbors. These would all be familiar and should elicit only basal maintenance marking rates (100 marks/hr). One should then expect more marking at the boundaries of a home range and/or territory or during penetration into an unfamiliar area and its new odor field, social rank permitting.
REFERENCES Alberts, J. R. (1974). Producing and interpreting experimental olfactory deficits. Physiol. Behav. 12, 657-670. Desjardins, C., Maruniak, J. A., and Bronson, F. H. (1973). Social rank in house mice: differentiation by ultraviolet visualization of urinary marking patterns. Science 182, 939-941. Edwards, D. A. (1974). Non-sensory involvement of the olfactory bulbs in the mediation of social behaviors. Behav. Biol. 11, 287-302. Edwards, D. A., Thompson, M. L., and Burge, K. G. (1972). Olfactory bulb removal vs. peripherally induced anosmia: differential effects on the aggressive behavior of male mice. Behav. Biol. 7, 823-828. Maruniak, J. A., Owen, K., Bronson, F. H., and Desjardins, C. (1974). Urinary marking in male house mice: responses to novel environinental and social stimuli. Physiol. Behav. 12, 1035-1039.