Physiology & Behavior, Vol. 24, pp. 737-740. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A. Elicitation of Male Mouse Ultrasounds: Bladder Urine and Aged Urine from Females' JOHN NYBY AND DAVID ZAKESKI Department of Psychology, Lehigh University, Bethlehem, PA 18015 Received 24 August 1979 NYBY, J. AND D. ZAKESKI. Elicitation of male mouse ultrasounds: Bladder urine and aged urine from females. PHYSIOL. BEHAV. 24(4)737-740, 1980.--Male mice (Musmusculus) emit ultrasonic courtship vocalizations in response to female mouse urine. Experiment 1 demonstrated that female bladder urine is an effective cue for ultrasound elicitation. Thus the ultrasound-eliciting factors in female urine appear to be produced prior to kidney filtration or during the filtration process itself. Experiment 2 demonstrated that externally voided female urine remained an effective cue for ultrasound- elicitation after one week of open-air ageing but appeared to lose some activity after one month. Thus the ultrasound- eliciting substance appears quite nonvolatile and environmentally stable. Age Bladder Chemocommunication Courtship Mouse Pheromone Ultrasound Urine MALE mice will under some circumstances emit 70 kHz courtship vocalizations in response to female urine [14, 18, 20]. Recent work [17] indicates that the ultrasound-eliciting substance in female urine is water soluble, heat resistant, and nonvolatile. The substance is first produced by females around puberty although its production appears to be hor- monally regulated by pituitary rather than ovarian hormones [17,21]. However, neither the site of production nor the chemical nature of this ultrasound-eliciting substance is known. One method of gaining information as to how chemosig- nals enter urine involves comparing the effectiveness of bladder urine and externally voided urine. In some species and experimental paradigms, for example, bladder urine ap- proximates the effectiveness of externally voided urine in bringing about the observed behavioral or physiological changes [1, 3, 7, 8, 12]. Such a finding suggests that the active urinary substance is produced by the body prior to kidney filtration or perhaps during the filtration process it- self. In yet other work voided urine appears more effective than bladder urine [2, 4, 5, 11]. This sort of finding would suggest that the active urinary component appears to be added to the urine or becomes activated after leaving the bladder possibly by various sexual accessory glands. Fi- nally, a third possibility is that the active substance is pres- ent in bladder urine but under certain circumstances may be deactivated as it passes through the urethra [13]. Variability also appears to exist between species and be- havioral paradigms in how long externally voided sex signals retain their behavior-eliciting qualities. For example, the normal male preference for female urine was found to cease after the urine had been voided for 24 hours in rats [12] and by 48 hours in guinea pigs [1]. In contrast, the aggression reducing and sex-eliciting abilities of female mouse urine did not appear to be reduced after one week of aging [8], while male hamster flank marks retained high levels of behavior- eliciting activity as long as 10 days [10] and female hamster vaginal scent marks as long as 50 days [9,10]. In the following 2 experiments we attempt to gain addi- tional information concerning the site of production and the long-term stability of the female ultrasound-eliciting sub- stance. The ultrasound eliciting qualities of female bladder urine as well as the fade-out time for behavior-eliciting ac- tivity are examined. METHOD The design features common to both experiments will be described prior to presenting the experiments. Additional information will be presented in the experiments themselves. Animals Subjects consisted of adult DBA/2J male mice whose ul- trasonic vocalizations were experimentally measured in re- sponse to urine. Social-Experience Animals consisted of adult male and adult female C57BL/6J mice that were systematically placed in the adult subjects' home cages prior to using the subjects in experiments. Urine donors, consist- ing of C57BL/6J and Swiss Webster mice, provided urine as stimuli for ultrasound elicitation. Apparatus Ultrasounds were monitored with a Holgate ultrasonic Mk V receiver [19,20]. The test chamber consisted of the subject's home cage, a translucent plastic cage measuring 29× 18x13 cm with wood shavings for bedding and a wire tWe thank Tom Cheng for generously supplying some of the female mice, Cathy Hazlehurst for helping in data collection, and Judy Anderson for helping with graphics. This research was supported by NSF grant BNS77-15265 and NIH grant 1507 IRR07173. Copyright © 1980 Brain Research Publications Inc.--0031-9384/80/040737-04502.00/0