Ultrasonic Courtship Vocalizations of Adult Male Mice: A Laboratory Exercise Illustrating Comparable Activation by either Estradiol or Testosterone.

For the past ten years, I have employed rodent ultrasonic vocalizations (USVs; i.e., vocalizations above the upper frequency limits of human hearing) as one of several behavioral measures of hormone and drug effects. In my experience this simple behavior is robust, inexpensively quantified, and of inherent interest to students. Although several different rodent species produce USV in various social/environmental contexts, the majority of published studies have examined rats and house mice. In this paper I focus on the vocalizations of adult mice, provide relevant background on factors that mediate their USV production, describe a sample laboratory exercise I use in my biopsychology course, and discuss several variations on this exercise that may be of interest to other instructors. The first published reports of mammalian USV appeared in the late 1930s (reviewed by Sales and Pye, 1974). In these earliest reports, Howard Griffin and his collaborators demonstrated that bats produced ultrasonic pips to detect, locate, and track their insect prey. Subsequent investigations of these bat vocalizations resulted in a vast literature spanning numerous sub-disciplines of biology. Among the more intriguing findings was the discovery that to evade capture many insects also produce ultrasounds to “jam” the bat USVs (Spangler, 1988). The neural mechanisms that bats use while in flight to locate prey and navigate have also been extensively investigated. Present knowledge regarding the neural mechanisms of sound localization are largely based upon this work and similar research with owls (e.g., Konishi, 1993). The earliest reports that rodents produce ultrasonic vocalizations began to appear in the 1950s. Noirot’s pioneering work focused on calls made by infants (Sales and Pye, 1974). Anderson (1954) was first to report that the adult rats also produced ultrasonic calls. 22 kHz calls by rats were subsequently identified in both males and females when isolated, injured or stressed, when confronted by a predator, during drug withdrawal, by a defeated male during intermale aggression, and by copulating males during the post-ejaculatory interval (Barfield and Geyer, 1972, 1975; Lore et al., 1976; Blanchard et al., 1991; Van derPoel and Miczek, 1991; Brudzynski and Ociepa, 1992; DeVry et al., 1993). Adult male and female rats also emit a ∼50 kHz vocalization during socio-sexual interactions with conspecifics (Thomas and Barfield, 1985; White and Barfield, 1987). Pharmacological studies have begun to examine the neurotransmitter systems that mediate the adult USV (Fernandez-Gausti, 1986; Brudzynski and Barnabi, 1996; Knutson et al., 1999; Bialy et al., 2000; Brudzynski, 2001; Wintink and Brudzynski, 2001; Beck et al., 2002; Brudzynski and Pniak, 2002;). Adult house mice (Mus musculus) also produce ultrasonic vocalizations (40–70 kHz; Sales, 1972). Both adult male and female mice emit these calls, but during different social contexts. Vocalizations during heterosexual encounters are made almost exclusively by the male and appear correlated with his level of sexual arousal (Nyby 1983). Females also vocalize, but much less often and typically only during same-sex encounters (Maggio and Whitney, 1985; Moles and D’Amato, 2000; D’Amato and Moles, 2001). In contrast to rats, adult mice appear not to produce USV during any contexts other than reproduction. However, the possibility that mice may produce ultrasonic calls unique to other social and environmental circumstances has not been thoroughly explored. It may be possible that detailed spectrographic and temporal examination of calls in different contexts may reveal distinctive features of calls that occur in specific contexts and/or during specific motivational states. The availability of a wide variety of laboratory mouse strains as well as spontaneous and engineered mutants, provides an exceptional opportunity for comparative studies of USV, the genes that mediate these vocalizations and their underlying motivational state(s). Several types of olfactory cues from females (i.e., urine, saliva, vaginal fluids) elicit USV from sexually experienced male mice (Whitney et al., 1974; Nyby et al., 1977; Byatt et al., 1986). The vomeronasal (Jacobson’s) organ and the accessory olfactory system play a particularly important role in detecting the chemosignals that elicit USV (Bean, 1982; Wysocki et al., 1982). Indeed, the involvement of the vomeronasal and accessory olfactory systems responding to several other putative mouse pheromones suggests that male USV may be elicited by one or more pheromones (Halpern, 1987). However, the pheromone(s) that elicit(s) USV from males has not been isolated or identified. In mice, as in many mammalian species, full expression of male reproductive behavior depends upon sufficient levels of circulating androgens. Castration significantly impairs male mouse copulation and ejaculation. USVs are also similarly affected, but may be restored in castrates by testosterone (Dizinno and Whitney, 1977; Nunez et al., 1978, 1984; Bean et al., 1986). These activating effects could be mediated directly via testosterone (T) action. Alternatively, T could also be converted to other neuroactive metabolites via two metabolic pathways. The first pathway involves the conversion of T to dihydrotestosterone (DHT) by the enzyme 5α-reductase, the second involves T being converted to estradiol (E2) via the enzyme aromatase. Consistent with the importance of T’s conversion to E2, many regions within the brain of males and females are rich in aromatase. Moreover, the brain of males and females possess estrogen as well as androgen receptors. Therefore, circulating T could activate male reproductive behavior in several ways. T could act directly on androgen receptors, or it could do so following conversion to DHT. Alternatively, T could activate male reproductive behaviors by activation of estrogen receptors (ER) following conversion to E2. This aromatization hypothesis, in fact, accounts for many effects of T in a number of mammalian species. In this paper, I describe a laboratory exercise employed in my biopsychology course that may be accomplished in three weekly three-hour laboratory periods. This exercise illustrates the important activational effects of T on masculine copulatory behavior and is useful in demonstrating that T’s effects within the CNS may sometimes be dependent upon conversion to E2. This exercise is extremely useful in illustrating why the common characterization of hormones as either masculine or feminine is erroneous. Moreover, this exercise demonstrates the type of empirical evidence that serves as the basis for the aromatization hypothesis. This hypothesis has played a significant role in guiding research into the organizational effects of gonadal hormones on the developing CNS and the resulting structural and functional CNS sex differences (McEwen, 1992). The laboratory exercise has also proven to be a useful means for providing students with their first experience employing an invasive surgical procedure with a laboratory rodent.