Vocal Signals of Sexual Motivation in Male and Female Rodents

Abstract

Purpose of the Review

Rodents produce ultrasonic vocalizations (USV) under different social contexts, including courtship and reproduction. The present review aims to summarize the behavioral, bioacoustical, and physiological evidence that USV are reliable signals of sexual motivation in both male and female rodents.

Recent Findings

USV are actively produced by both sexes during sexual interactions, contrary to earlier assumptions. Male-typical and female-typical vocal behaviors can be identified. Calling rates and acoustic parameters, such as call duration, frequency, and energy, can be modulated rapidly over time by motivational state and sexual context. USV produced in response to sexual context could be regulated by the brain on a moment-to-moment basis through non-classical mechanisms of steroid action. Finally, I provide some practical considerations for the acoustic and statistical analyses of these vocal signals.

Summary

USV can be used as signals of sexual motivation in both sexes to study brain and hormonal mechanisms underlying sexual behavior or sexual differentiation.

Introduction

The study of rodent sexual behavior has contributed to an understanding of the role of hormones in the organization and activation of sexual behaviors, sexual determination, and differentiation. In addition, a circuitry of brain areas and neurochemicals involved in the regulation of sexual motivation and copulatory behavior have been characterized particularly in males [1, 2]. Male sexual behaviors typically studied include ano-genital investigation, mounting, intromissions, penile reflexes, and ejaculation. Female sexual behaviors typically studied include stereotypical motor patterns (i.e., hops and darts), lordosis, approach, and avoidance of the male [1, 3]. Most of these behaviors are exhibited by one sex or the other and these behaviors may not be exhibited in all rodent species. An additional sexual behavior that has been understudied even though it is widespread among species and is exhibited by both sexes is the production of courtship vocalizations. The main reason these vocalizations have been understudied is due to the inconspicuous nature of the behavior. Most of courtship vocalizations are ultrasonic, well above the human hearing range, and rodents do not show any obvious movement of the mouth while emitting them. But, ultrasonic vocalizations (USV) are commonly produced in response to opposite-sex stimuli in a great number of rodents [4].

USV in rodents were discovered in the 1950s and described by many studies in the 1970s [5]. In the last two decades, there has been a steady mounting interest in the study of rodent USV, mostly for its potential to become a useful measure to behaviorally phenotype individuals in mouse models of neurodevelopmental disorders [6,7,8]. In rats, USV have been measured in studies of the neuroanatomical and pharmacological basis of motivation and emotion, including depression [9] and vocal impairment in the rat model of Parkinson’s disease [10, 11]. Finally, USV have been used to study the genetic basis and brain circuitry of vocal communication [12, 13].

Most of these studies have been conducted in laboratory species and there is a general understanding of the behavioral context under which these vocalizations are produced. Moreover, studies on wild populations have confirmed the production of these vocal signals in response to social stimuli [14,15,16]. Rodent ultrasonic behavior has been reported mostly during offspring-mother communication, juvenile play, territorial or aggressive behavior, and reproductive behavior [17,18,19,20,21,22,23,24]. Among the different social contexts in which USV have been recorded, opposite-sex interactions reliably elicit the production of USV in the majority of species and co-occur with other pre-copulatory and copulatory behaviors. USV produced by both sexes fit the description of an appetitive sexual response observed in vertebrates [25] and have been proposed to be a promising signal of sexual arousal and affective state [12, 26, 27]. This review provides a summary of the behavioral, bioacoustical, and physiological evidence supporting that these signals are sexually motivated in both males and females. Finally, some practical considerations are made on the recording and the acoustic and statistical analyses of these signals, with emphasis on Golden/Syrian hamster (Mesocricetus auratus) USV, a solitary species in which sexual and social motivation are less likely to be confounded.

USV as Behavioral and Acoustical Correlates of Sexual Motivation

Vocal Behavior and Sexual Context

The number of vocalizations produced in response to a social stimulus was the first measurement that was quantified during the early studies of rodent vocal behavior. Generally, during mating, not all sniffing or mounting is accompanied by USV in rodents, but when vocalizations occur, they occur in close temporal proximity to those behaviors [28,29,30]. The total number of USV can vary widely among individuals within a species [28, 31••]. Typically, high calling rates during intersexual encounters have been interpreted as evidence of high levels of sexual motivation and arousal in many species [29, 30, 32,33,34,35].

Usually both males and females emit USV in response to sexual stimuli or during interactions. However, the identity of the caller cannot be easily determined by a human observer. It used to be assumed that females were mostly silent in the presence of a male [33, 36,37,38]. When an intact female was paired with an anesthetized or devocalized male, no or few USV were detected from the pair [35, 36, 39,40,41,42,43]. In contrast, when an intact male was paired with an anesthetized or devocalized female, high numbers of USV were recorded [35, 36, 40, 43]. Thus, it was assumed that the main caller during a male-female interaction was the male. However, anesthetized adults of either sex have been reported to elicit poor vocal responses [40] and devocalized partners may simply not elicit calling in all intact subjects. For instance, early work showed that a female mouse would rarely vocalize with a devocalized male [43]. However, a recent study using a microphone array system able to identify the caller during a male-female interaction demonstrated that although males produce 82% of the USV recorded while interacting, both sexes are exchanging calls and about 61% of female calls occurred within 1 s of a male call [44••].

Although the study of the USV produced and exchanged during a male-female interaction may need additional technology to assure the caller’s identity, USV produced in response to sexual cues can be studied using a different experiment design. Vocal response to olfactory cues or post-interactions would assure the identity of the caller and avoid uncontrolled effects on a subject’s behavior caused by a live stimulus. Rodents vocalize extensively in response to opposite-sex odors vs. same-sex odors [16, 32, 34, 45, 46]. Additionally, both male and female golden hamsters, mice, and rats vocalize immediately after a heterosexual interaction [28, 31••, 32, 47•, 48, 49]. Another available option is to record male USV while the female is in lordosis position. In golden hamsters, the female is believed to remain silent while in position, but further confirmation is required [32, 50].

Based on the vocal behaviors observed after dyadic interactions or in response to odors, researchers have reported “male-typical” or “female-typical” vocal behaviors in some species. Rodents do not seem to produce sex-specific vocalizations, which are those produced by only one sex [51]. Instead, both sexes produce similar types of calls and may differ in calling rates, in the context in which the call is used or in the acoustic structure of shared calls. Sex differences have been mostly observed in the number of USV produced in a specific context, and with a weak to a varying degree of sex differences in acoustic structure [31••, 47•, 52,53,54, 55•]. Sex differences in vocal behavior, such as vocalizing a higher number of USV after interacting with an opposite-sex individual, but not with a same-sex individual, have been repeatedly found in golden hamsters [31••, 32, 48]. Higher calling rates in males after interacting with a female, but not with a male, can be considered a “male-typical behavior.” Similarly, significantly higher calling rates in females after interacting with a male, but not with a female, can be considered a “female-typical behavior” (Fig. 1a, b). This approach has been useful to characterize the effects of castration and hormone replacement on vocal behavior and the development of sex differentiation (see below) [48].

Fig. 1
figure1

Call production and call duration after opposite-sex interactions in golden hamsters. a, b Mean calling rates (± SE) produced before and after a 3-min interaction with a same-sex individual (N = 9 female subjects, N = 10 male subjects) or with an opposite-sex individual (N = 18 female subjects, N = 17 male subjects). Calling rates after an opposite-sex individual were significantly greater than after a same-sex individual. This “sex-typical” vocal behavior was consistent with early work by O. R. Floody. Post-interaction rates with an opposite-sex individual were not significantly different between males and females (P = 0.07). c There is a decrease in the mean number of USV (± SE) produced during the first, second, and third minute of acoustic recording in males but not in females. d Duration (ms) of calls produced by males (N = 14) and females (N = 12) after an opposite-sex interaction was positively correlated with calling rates (generalized linear model (GLM) F3,22 = 5.88, P = 0.0042 (effect of calling rates), effects of sex or the interaction between sex*calling rates were not significant). Call duration was significantly predicted by e energy (dB) and f inter-quartile frequency bandwidth (kHz) (generalized linear mixed model (GLMM) F1,1036 = 438.1, P < 0.001 (effect of energy), F1,1035 = 19.8, P < 0.001 (effect of bandwidth)). Effects of sex, sex*energy, or sex*bandwidth were not significant. Figures and updated statistical analyses were reproduced from [31••]

Acoustical Correlates of Sexual Motivation and Context

The advantage of working with USV produced in response to sexual stimuli is that, in addition to calling rates, the acoustic signal itself has characteristic properties. Sound frequency or pitch is the most common property of sound that can be measured. The frequency range of USV produced during or after sexual interactions can vary between species. USV can vary between 20 and 125 kHz in mice [28, 47•], 50–75 kHz in rats (except male post ejaculatory calls) [20, 39, 49, 56], and 20–55 kHz in golden hamsters [31••, 57]. Call length or duration is another characteristic that can be easily measured in USV. Open-access or commercially available software normally provides a long list of acoustic properties that can be measured in animal vocalizations. Interestingly, several of those acoustic properties have been found to correlate with motivation, arousal, and internal state in many species of mammals including humans [58, 59] and could even contained signals of prosody [26]. For instance, calls of low vs. high frequency have been repeatedly associated with different affective states in rats [60, 61]. Calls of constant low frequency (22 kHz) have been associated with aggression and anxiety, whereas calls of modulated high frequency (around 50 kHz) have been associated with appetitive behaviors during mating and playing [20, 60, 61]. More recently, associations between acoustic parameters and motivation have been found in other species. For example, female prairie voles in proximity of their same-sex cage mate produced USV in which acoustic variables such as frequency and duration covaried significantly with heart rate [62•]. Although the context of vocal elicitation was affiliative rather than sexual, this study offers preliminary data supporting that features of ultrasonic vocal prosody and autonomic regulation shared common neurophysiology [62•].

Additionally, recent studies in golden hamsters suggest that changes in call duration could be considered an acoustic correlate of sexual motivation. First, calling rates recorded after male-female interactions decreased with time of separation, specifically in males (Fig. 1c), and call duration is positively correlated with calling rates in both sexes (Fig. 1d). Second, longer call durations can be significantly predicted by higher call energy (Fig. 1e) and greater frequency bandwidth (Fig. 1f) [31••]. In this case, energy is the total energy computed from the spectrogram of each USV (including harmonics and resonances). Moreover, hamster USV can vary from tonal calls to atonal and nonlinear (aperiodic) calls. When the structure that produces a sound oscillates irregularly and out of synchrony, the frequencies of the sound (fundamental and harmonics) are also irregular or “nonlinear” [63]. Increased vocal effort can produce nonlinear sounds, like when an intense scream is produced [63]. This aperiodicity or chaotic nature may be reflected in measures of energy and bandwidth (frequency range containing most of the energy of the call) and these could be expected to increase with arousal [e.g., 64, 65]. Acoustic nonlinear phenomena in audible broadband vocalizations produced by female mice were recently shown to vary significantly with context and female estrous state, indicating varying motivational levels during an interaction with a male [66••] (see below).

Subsequent studies in male golden hamster showed that changes in social context and competition altered acoustic features such as call duration and energy over time. Specifically, the presence of a male competitor decreased the duration and energy of post-female calls significantly over time compared to post-female calls in the absence of a competitor [67••]. These parameters also decreased over time, if that competitor was a familiar winner (i.e., a male that defeated the subject in a fight), but not a familiar neutral male (i.e., a male that familiarized with the subject through a wire mess where fighting was not possible) [67••]. Interestingly, in male rats, duration of postejaculatory calls varied according to context as well. During the postejaculatory period, a male produces 22-kHz USV and if during this time of vocal production, the male is moved to a neighboring compartment; it can develop a preference for this new compartment [68••]. After conditioning to a new place associated to postejaculatory calls, odor cues from unfamiliar males (but not familiar males) decreased duration of the postejaculatory calls, while mating cues (e.g., copulatory chamber and presence of the female) increased duration of the postejaculatory calls [68••]. Thus, changes in social context that could be assumed to alter internal state of a subject can be reflected in changes in acoustic properties of sexual vocalizations produced by that subject.

Modulatory effects of context on USV have been studied mostly in males. However, females’ vocal behavior is likely to change across the estrous cycle and interact significantly with context. Female USV are suspected to increase during behavioral estrus to signal fertility. The standard method to induce behavioral estrus (sequential injections of estradiol (E2) followed by progesterone) was highly effective in activating high levels of 50-kHz calls in female rats [69]. Moreover, contact with male odors, or the male itself, provoked significantly higher rates of “post-male” ultrasound calling in estrous than in non-estrous female golden hamsters [32, 70, unpublished data]. In mice, female broadband squeaks, associated with physical rejection of males and lower incidence of mounting behavior, presented nonlinear segments that were significantly longer during estrus than during diestrus [66••]. In this case, nonlinear acoustic features may be used to express high motivation to avoid mating with a “non-desirable” male during estrus.

Thus far, I have provided evidence suggesting that acoustic features of USV could serve as an index of sexual motivation. This evidence has focused on the production rather than on the perception of USV. Evidence for a functional role of USV in sexual behavior has been contradictory in rats [39, 71,72,73] and therefore, the relationship between sexual motivation and USV has been questioned [3]. Based on playback experiments, female rats have been reported to exhibit high levels of social approach to male USV compared to a control sound [73], while others have found not significant effects of USV on eliciting approach by the opposite sex [39, 72]. Moreover, the presence or absence of vocal behavior does not appear to influence copulatory behavior in sexually naïve rats [71]. However, other studies in laboratory and wild mice have found that male USV may play a role in sexual selection [16, 74, 75]. Females have been observed to approach to the source of USV and to prefer complex vs. simple sequences of USV [16, 74, 75]. Because USV are not designed to travel far and to attract conspecifics from a distance [76], it is likely that USV are mostly involved in coordinating male-female interactions or maybe even related to post-copulatory mechanisms, such as female cryptic choice (female control of sperm use). Moreover, questions related to sexual selection may yield contradictory results in domesticated strains, in which “non-choosy” females may have been selected as colony breeders. Interestingly, however, adult hamster USV can prolong female lordosis in comparison to stress-elicited calls from young hamsters [77] and male mice USV can increase activation of kisspeptin neurons involved in follicular maturation in diestrous females [78•].

In summary, vocal behavior in several species of adult rodents is elicited by sexual stimuli and sexual context. Although it may vary by species, sex-typical vocal responses can be identified and clearly characterized in both males and females. Acoustic variability can change over time, context, and ultimately with internal state, providing an opportunity to study dynamic physiological changes within sexual context.

USV Are Organized and Regulated by Steroid Hormones via Classical and Non-classical Mechanisms

Exposure to steroid hormones during some period of developmental sensitivity permanently induces sex characteristics of reproductive and ultrasonic vocal behavior in rodents. A few studies have performed gonadectomy and hormone treatment before 10 days of age and examined vocal behavior in adults [40, 48, 79, 80]. A thorough set of experiments were performed in the golden hamster based on the established male- and female-typical vocal behaviors (summarized results from [48] in Table 1). As with other reproductive behaviors, vocal behavior can be masculinized or feminized. In particular, androgens can organize and activate male-typical vocal behaviors in female hamsters via androgen aromatization. In males, vocal behavior can be activated by testosterone (T) even in the absence of organizing effects of gonadal hormones caused by neonatal castration. However, estrogens will not activate male-typical vocal behavior unless androgens were present during neonatal development (Table 1).

Table 1 Effects of hormones on vocal and other sexual behaviors during perinatal development and adulthood in golden hamsters. Male-typical behavior refers to males calling more after females than after males. Female-typical behavior refers to females calling more after males than after females. Information summarized from [48]

After sex-typical behavior substrates have been organized by the presence or absence of the testes, different concentrations of gonadal hormones in circulation will activate further expression [81]. Effects of activating androgens in male vocal behavior have been shown in a number of species in which castration in adulthood significantly decreased calling behavior that is later restored with androgen replacement therapy (Table 2). In some species, USV can also be restored in males with estrogen, but not with dihydrotestosterone, suggesting that the androgenic effects may be mediated via local aromatization of T (Table 2). On the other hand, ovarian hormones activate the production of USV in female rodents. A decreased number of USV have also been observed in ovariectomized females (Table 2). Replacement therapy with either high or low doses of E2 followed by progesterone restored maximal levels of USV in female rats and hamsters (Table 2). Treatment with E2 alone was only partially effective in restoring female vocal behavior, whereas T alone was effective in restoring female USV in most species (Table 2).

Table 2 Example of the effects of hormone manipulations in adult vocal behavior. Vocal behavior was elicited in males during a brief interaction with a female (during m-f pairing), after an interaction with a female (post m-f pairing), or during the exposure to female olfactory cues. Vocal behavior was elicited in females during a brief interaction with a devocalized male (during f-m pairing), during an interaction with a female (during f-f pairing), or after an interaction with a male (post m-f pairing)

These organizational and activational effects of sex hormones on the production of USV are likely regulated via classical mechanisms. Gonadal steroids ultimately alter behavior by passing freely through cell membranes, targeting intracellular steroid receptors, and changing the expression of steroid-regulated genes [81, 90••]. Significant progress has been made in the study of a steroid-sensitive circuitry that regulates reproductive behaviors via classical mechanisms [90••]. However, steroid action is not limited to this type of slow, genomic mechanism. Steroids can also be synthetized in the brain, act as a neurotransmitter by binding to receptors at the cell membrane and initiating intracellular cascades that modulate behavior [91,92,93].

Accumulating evidence has demonstrated sexual behaviors can be acutely controlled by locally synthesized steroids, particularly estrogens, via the aromatization of androgens in the brain [27, 92,93,94,95]. Moreover, rapid fluctuations in plasma T are observed in response to sexual stimuli in many mammal species [96,97,98]. Those rapid surges in gonadal T could serve as an androgen source for the local production of brain estrogens, sustaining intense sexual arousal and copulatory behavior in mammals [27, 99]. Estrogens have been demonstrated to have rapid and transient effect on sexual behavior of gonadally intact male rodents [100, 101]. More recently, rapid effects of estrogens have been demonstrated to also modulate acoustic properties of USV produced by male golden hamsters after a sexual interaction with a female (i.e., post-female calls) [102•]. As mentioned above, many acoustic features can be measured in USV. Different parameters related to sound frequency or amplitude can be reduced using a principal of component analysis, which can help identify parameters that represent most of the variation in the calls. Call duration and a principal component factor represented mostly by center frequency (frequency with 50% the total energy) and energy decreased significantly over time after a single acute injection of T or E2 (30 and 15 min respectively) compared to a vehicle injection [102•]. Although a decrease over time in acoustic parameters due to T seems contradictory, other studies have also found rapid dampening effects of T on ultrasonic calling and urinary marking in two species of deer mice that was context dependent [103, 104]. Specifically, T decreased calling in paired but not in non-paired males (sexually naïve) and urinary marking in subordinate but not in dominant males [103, 104•]. Clearly, more work needs to be done to understand the time course, interaction with other factors, and specific behavioral effects of rapid steroid action on rodent communication. But, these mechanisms could explain how vocal behavior is regulated by the brain on a moment-to-moment basis in response to social context.

Brain Regulation of Vocal and Sexual Behaviors Overlap in Rodents

Although the neural circuitry and neuroanatomical substrates of social behaviors have been challenging to dissect, it is well established that sex-steroid-modulated social behaviors are likely controlled by an integrated and complex network of limbic areas including the medial extended amygdala, the lateral septum (LS), the medial preoptic area (MPOA), the anterior hypothalamus, the ventromedial hypothalamus (VMH), and the midbrain periaqueductal gray (PAG) [105] and that this network connects to the mesolimbic reward system to form a social decision-making network [106].

The MPOA and the VMH are two brain regions that contain high concentrations of androgen and estrogen receptors and consequently their role in the regulation of sexual behavior has been examined [2, 81]. Bilateral intracranial implants of T in the MPOA of castrated males effectively restored USV production in response to female urine (mice) or female bedding (rats) [107,108,109]. In contrast, T implants into the VMH, septum, anterior hypothalamus, ventral tegmental area (VTA), or medial nucleus of the amygdala of castrated male mice partially restored USV in response to female cues [108,109,110]. Although T implanted into the VMH did not restore 50-kHz vocalizations in castrated male rats either [111], electrolytic microlesions in the VMH impaired the ability of peripheral T to restore USV following castration [112] and an androgen receptor blocker implanted at the VMH also inhibited the restoration of calling [107]. In general, androgen stimulation of the MPOA has been the most effective in restoring USV production, and in some cases, MPOA in combination with other regions like the VTA [113]. Other androgen- and estrogen-sensitive areas and their integrated role in controlling the production of sexual USV deserve further investigation in male rodents.

In females, brain control of USV has been studied in detail in the golden hamster. Ultrasound rate in females is believed to depend on the integrity of brain areas including the MPOA, amygdala, lateral septum-bed nucleus of the stria terminalis (LS-BNST), VMH, and PAG [114,115,116,117,118]. Lordosis, a behavioral response that has contributed greatly to the understanding of the neural circuitry of female sexual receptivity, shares several physiological properties with vocal behavior. Both depend on gonadal hormones, with maximal levels of each (lordosis and USV) requiring the combined effect of E2 and P. Both USV and lordosis can be affected by lesions of MPOA, LS-BNST, VMH, and PAG [114, 117, 119,120,121]. The role of PAG has been suggested to be particularly central for ultrasonic vocal control in female hamsters [120]. In general, the production of vocalizations in mammals is controlled by two vocal pathways: the limbic cingulo-periaqueductal and the motorcortical [122]. The PAG is responsible for the initiation of nonverbal emotional vocal utterances and, in addition to the anterior cingulated cortex, mediates the voluntary control of vocal initiation human and non-human animals. Both pathways make contact with the phonatory motor neurons via the reticular formation [122, 123]. Therefore, both the PAG and the reticular formation could be critical to the production of sexually motivated USV in rodents.

In summary, evidence suggests that the regulation of USV production is mediated by brain mechanisms shared with other reproductive behaviors. The brain regions involved in vocal control suggest that male and female vocal behavior is one more social behavior regulated by the common neural network (the social behavior network) [105, 124]. The motivation to vocalize is likely regulated by a circuitry involving the MPOA and it be could be hypothesized to be regulated by the combined input from the VTA, nucleus accumbens, and prefrontal cortex as it has been demonstrated in other appetitive behaviors [2]. Ultrasonic calling by female golden hamsters in response to odors of conspecifics is mediated by both the olfactory and vomeronasal systems which are tightly connected to the medial amygdala [70]. Therefore, as in other copulatory behaviors, input from the medial amygdala to the MPOA could be expected to affect the production of USV [2].

Practical Considerations on the Recording and Analysis of USV

In the past, USV were recorded with bat detectors that convert the ultrasound signal into an audible one. This is an easy way to confirm the production of USV and bat detectors are relatively inexpensive. However, more sophisticated techniques are needed to accurately record rodent vocalizations. A number of technical issues can be addressed to improve the recording and analysis of rodent USV. For instance, it is necessary to use a directional microphone with a high frequency flat response that can capture an approximate equal and faithful output of all frequencies. Hamsters and rats vocalize between 20 and 60 kHz, whereas mice can vocalize close to 100 kHz. Rodents can also produce low- to medium-frequency sounds that have been related to motivational states of high sexual arousal or stress [66••, 125•]. Therefore, microphones with a broad frequency range provide a greater opportunity to capture a range of calls of interest. Data acquisition hardware coupled with recording software should digitize sounds at a sampling rate that is more than twice the highest frequency contained in the sound of interest [126]. For example, if vocalizations of interest are between 30 and 50 kHz, then the sampling rate should be at least 100 kHz. Recording sounds at a poor sample rate will generate digitized signals with “phantom frequencies” or frequencies that do not represent the original sound. This effect is called aliasing and should be avoided [126]. It is also important to use a recording system that allows continuous recording to preserve information about the time of sound occurrence, as well as sound activation triggers when long recordings are desired. In addition, special attention should be given to the place where recordings are made and the position of the microphones. Cage bedding and Plexiglas walls will interfere with the quality of the recordings. The use of sound attenuating materials and avoiding small cages with bedding is highly recommended.

Recording of USV can generate an extensive amount of data that may be difficult to analyze at first. For species in which USV have never been described, a general knowledge of the repertoire and incidence under different behavioral contexts is necessary. For most laboratory species, repertoires with the most commonly produced call types and acoustic features of interest have already been identified and established [e.g., 14, 28, 31•, 41]. The categorization of USV into call types has been used to characterized repertoires produced in different contexts or by different populations or strains [47•, 75, 127]. However, we still lack an understanding of the meaning of their content. Furthermore, the categorization of USV call types can be highly subjective and time consuming. The occurrence of different call types can be highly variable depending on the species. In the case of the golden hamster, this variability in call types yielded nonsignificant associations with sex or context [31••, 67••]. On the contrary, acoustic parameters can be objectively measured by sound analysis software that can even detect calls automatically [126, 128••]. Automated methods of call detection can work but reliability should be corroborated by a human observer.

Finally, the statistical analysis of vocal signals deserves careful consideration. First, the unit of acoustic analysis must be clearly defined and be consistent. The unit of acoustic analysis could be just a note, a syllable, a composite of syllables, a sequence, or a song. The actual definition of each varies across studies and taxon. Rodents produce vocalizations that consist on a single spectrographic continuous sound or frequency contour. This is technically a note, but some called this a syllable. A frequency contour can break and decrease, increase, or stop for a very small fraction of time. These are USV with frequency jumps (FJ). Because acoustic properties such as peak frequency may vary greatly between single and FJ calls, the spectral structure of these calls should be analyzed separately.

Once the unit of analysis has been defined, the intrinsic variability in acoustic parameters should be preserved per individual. Statistical methods such as generalized linear mixed models (GLMM) incorporate hierarchical structure of data into the analysis (e.g., calls within individuals, individuals within categories, or treatments) and allow direct estimation of between- and within-individual variances [129]. In classical analysis of variance approaches, between-individual correlations are based on an individual’s average value of the dependent variable that can often be biased due to within-individual variation [130]. For example, if one is interested in examining the effects of treatment groups in predicting call duration, instead of averaging call duration per individual prior to statistical testing, one can include duration of all vocalizations recorded in the GLMM. This way, the variation within individuals can be estimated and accounted in the model. GLMM incorporate two types of parameters: fixed and random. The combination of fixed and random parameters or effects can account for repeated measures within individuals (random effects) but also among treatments and interactions (fixed effects) [131]. Much of the variability in the data will reside in the random effects whose purpose is to measure the variability among individuals, populations, experimental blocks, or variables that are expected to vary randomly by undefined causes (e.g., individual differences) [129, 131]. Another advantage is that the normality of the data is tested in the residuals generated by the model and the model is flexible in handling non-normal data overall [131]. If the residuals are not normality distributed or if the variance is not homogeneous, a mathematical transformation of the dependent variable can often normalize the residuals. This type of analysis has been widely adopted in the field of ecology and evolution and it offers great potential in the field of behavioral sciences [130].

Final Concluding Remarks

The study of vocal behavior in a sexual context can complement the rich repertoire of pre-copulatory and post-copulatory behaviors. In summary, USV produced by male and female rodents are first organized and activated by gonadal hormones. Initiation of vocal sexual behavior could be acutely regulated by rapid local production of estrogen and the gonadal elevation of T that occur in response to sexual stimuli (Fig. 2). The underlying brain circuitry responsible for the production and modulation of rodent USV is still incomplete but some brain regions involved in the motivational aspects of other sexual behaviors (e.g., MPOA) are known to be implicated in ultrasonic vocal behavior (Fig. 2). USV contain acoustic features that can be quantified objectively and can vary in a moment-to-moment basis in response to sexual context and internal state (Fig. 2).

Fig. 2
figure2

Proposed mechanisms involved in the elicitation and modulation of vocal behavior produced in a sexual context. a During a male-female interaction, multisensory sexual cues are exchanged between animals and processed by the brain sensory systems. b In response to the sexual encounter, a rapid elevation of testosterone (T) is produced by the gonads (at least by the testes) and can be detected in circulation. Peripheral T will presumably cross the blood brain barrier and modulate brain function [96,97,98]. c The gonads have already organized and activated steroid-sensitive target tissue in the brain for the production of vocal behavior (Tables 1 and 2). d The excess of T could be converted into estrogen by the enzyme aromatase in the brain. A rapid increase of aromatase activity could also occur directly in response to sexual cues, and thus, increase the local production of estrogen and modulate vocal behavior [27, 99, 101]. The brain regions depicted in the diagram have been studied and implicated in the production and restoration of USV after gonadectomy in male or in female rodents [107, 108, 110, 120, 121]. e Spectrograms of USV recorded from golden hamsters during a male-female interaction in a neutral arena without barriers. Copulation started approximately 1 min after the first encounter and these USV sequences were recorded approximately 4 min after the first intromission. ei USV recorded while the male was investigating the female’s rear end. eii USV recorded 16 s before male started mounting the female. In both instances, the female was immobile in lordosis position. f Acoustic parameters can vary on a moment-to-moment basis in response to rapid changes in circulating hormonal levels [102, 104] or context [20, 28, 31••, 47•, 66••, 67••, 68••]. Acoustic analysis software, such as Raven Pro 1.4 (The Cornell Lab of Ornithology, Ithaca, NY, U.S.A., http://www.birds.cornell.edu/brp/raven/ravenversions.html), can provide information within a selection (dashed line square) about the length or duration of a call, center frequency (frequency containing 50% of the energy), peak frequency (frequency with the maximum amplitude), bandwidth (inter-quartile bandwidth or frequency range between the frequencies that exceeds 25% and 75% of the energy), and energy. Note the change of parameters from one note to the next and the increased energy in the frequency harmonics in call 2 compared to call 1. BNST bed nucleus of the stria terminalis, LS lateral septum, MeA medial amygdala, MPOA medial preoptic area, VMH ventromedial hypothalamus, PAG midbrain periaqueductal gray

The measurement of USV offers some advantages over other reproductive behaviors. First, the measurement of USV can contribute to the study of different components of sexual behavior, distinguishing between motivation (appetitive behaviors) and performance (consummatory behaviors). USV can be easily measured in the absence of other individuals that may affect the motivational state and behavior of the subject. USV can be measured in both sexes, particularly in females whose sexual behavior has been understudied [1]. The study of USV can improve our understating of female sexuality and cyclic changes in motivation. Golden hamsters offer a particularly great advantage to study sexual vocal behavior because their USV are emitted mostly under a sexual context rather than under an affiliative context. As a solitary species, animals interact with other conspecifics mostly to mate or to compete for resources [132, 133]. A same-sex stimulus is a poor stimulant of USV in this species, which is not the case in other highly social species, such as mice and rats. Finally, USV offer great opportunity to explore sex differences in brain and behavior, as well as the development of sexual differentiation. This can be especially relevant now that the effects of endocrine disruptors during development in adult sexual behavior are starting to emerge [134••].

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Acknowledgments

I would like to thank Robert E. Johnston, Elizabeth Adkins-Regan, and Andrew Bass for guidance and support in the writing of an earlier version of this manuscript. I thank Gregory J. Peters and Luke Remage-Healey for helpful comments on how to improve this manuscript. Finally, I would like to thank Fay Guarraci and Lesley Marson for constructive editorial and reviewer comments and for the invitation to contribute with this article.

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Correspondence to Marcela Fernández-Vargas.

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Fernández-Vargas, M. Vocal Signals of Sexual Motivation in Male and Female Rodents. Curr Sex Health Rep 10, 315–328 (2018). https://doi.org/10.1007/s11930-018-0179-9

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Keywords

  • Ultrasonic vocalizations
  • Sexual behavior
  • Sexual motivation
  • Sex steroids
  • Bioacoustics
  • Communication
  • Nongenomic steroid action
  • Rodents
  • House mouse
  • Rats
  • Golden hamsters
  • Syrian hamsters