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Quantitative and Qualitative Features of Neonatal Vocalizations in Mice
C H A P T E R
13 Quantitative and Qualitative Features of Neonatal Vocalizations in Mice Angela Caruso1,2, Mara Sabbioni1, Maria Luisa Scattoni1,*, Igor Branchi3,* 1
Research Coordination and Support Service, Istituto Superiore di Sanità, Rome, Italy 2 Sapienza University of Rome, Rome, Italy 3 Center for Behavioral Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy
I AUDIBLE AND ULTRASONIC VOCALIZATIONS EMITTED BY MOUSE PUPS
II FUNCTIONAL ROLE OF PUP ISOLATION-INDUCED USVs
Mus musculus pups are born blind and deaf with overall limited sensory-motor abilities; they are also incapable of feeding or thermoregulation. Therefore, during the first two postnatal weeks, their survival is strictly dependent on the mother. It is well known that at this earliest stage, mouse pups are highly vocal, emitting a variety of calls in different motivationally relevant contexts that elicit adequate maternal care (Branchi, Santucci, Vitale, & Alleva, 1998; Ehret & Bernecker, 1986). For instance, they can produce vocalizations in the audible range when cleaned by the mother, in response to pain or roughhandling, or when accidentally pinched. They can also emit low-frequency wriggling calls when they push for the mother’s nipples or when crawling over each other inside the nest (Hahn & Lavooy, 2005). Mouse pups also produce abundant ultrasonic vocalizations (USVs), whistle-like sounds within the 30–90 kHz frequency range above the human hearing threshold of 20kHz (Sales & Pye, 1974; Sales & Smith, 1978; Zippelius & Schleidt, 1956). A large amount of data indicate that, although both audible sounds and USVs are generated in the larynx by air passing over the vocal cords, the mechanisms underlying the production of these two types of sounds markedly differ (for further details of the mechanisms of USV production, see Chapter 5 in this volume). The focus of the present chapter is on the emission of USVs because these have been shown to play a highly relevant role in mother-pup communication, allowing a fine-tuning of pup behaviors with maternal responses. *Co-last senior authors Handbook of Ultrasonic Vocalization, Volume 25 ISSN: 1569-7339 https://doi.org/10.1016/B978-0-12-809600-0.00013-5
Neonatal USVs are emitted by mice as well as by other altricial rodent species. The USVs are produced by pups when they are outside the nest and far from the mother and littermates. Therefore, these calls are defined as “isolation-induced USVs” (Branchi, Santucci, & Alleva, 2001; Hofer, Brunelli, Masmela, & Shair, 1996; Sales & Pye, 1974; Sales & Smith, 1978; Sewell, 1970; Smith & Sales, 1980; Zippelius & Schleidt, 1956). Upon hearing the USVs from an isolated pup, the mother rapidly investigates the sound, identifies the pup’s location, and retrieves it to the nest (Ehret & Bernecker, 1992; Noirot, 1972; Fig. 13.1). Similar to other behavioral responses, USV features change with the age of the pups. Indeed, the calling rate emitted by mouse pups follows an ontogenetic profile: it increases during the first 6–7 days of life, reaches a peak around postnatal day 8, and then starts to decrease until the total disappearance of calls when pups are two weeks old (Hahn et al., 1998; Noirot, 1966; Thornton, Hahn, & Schanz, 2005). The reduction of isolation call emission parallels the emergence of pups’ ability to thermoregulate, move, and see (eye opening), and with the consequent capability to forage for themselves or search for their mother (Elwood & Keeling, 1982). The notion that USVs are emitted by pups when isolated from the nest to gain their mother’s attention suggests a communicative role for USVs. In fact, neonatal USVs emitted under social isolation are considered distress calls for help and therefore interpreted as essential communicative signals. However, several authors have challenged this interpretation, opening a controversial debate about the functional role of pup USVs.
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Frequency (kHz)
Maternal response
Isolation condition
120 90 60 30 300
0 Time (ms) Ultrasonic emission
FIG. 13.1 The communicative role of ultrasonic vocalizations. After social isolation, mouse pups emit USVs that elicit in the dam a prompt response concerning care-giving behaviors. The sonogram illustrates three two-component ultrasonic calls of approximately 50–70 ms in duration and 60–90 kHz in frequency.
Blumberg and Alberts (1990) supported the idea that the ultrasonic emission is an acoustic by-product of a respiratory maneuver that physiologically increases oxygen delivery to metabolically active tissues (Blumberg, 1992; Blumberg & Alberts, 1990). Later, Blumberg and Sokoloff (2001) affirmed that USV production is caused by an abdominal compression reaction that increases blood flow return to the heart in response to decreased temperature when the pup falls out of the nest trying to right themselves or is handled roughly by adult mice or human experimenters (Blumberg & Sokoloff, 2001). These hypotheses were, however, later disproved (Shair & Jasper, 2003). In order to elucidate the functional role of neonatal USVs, the emission pattern has been extensively studied by monitoring several crucial factors such as physiological or environmental parameters, and by characterizing maternal response to USV production (Blumberg & Sokoloff, 1998; Branchi et al., 1998; Farrell & Alberts, 2002; Noirot, 1972). Another approach that provided insight into the role of ultrasonic signals, but has been so far rarely investigated, is the accurate evaluation of motor behavioral response in conjunction with the ultrasonic emission. It was, however, clearly demonstrated that a connection between USV emission and pup motor activity exists. The high calling rate of USVs during locomotor behavior is an active response to isolation, helping the pups to rejoin the mother (Branchi, Santucci, Puopolo, & Alleva, 2004).
In conclusion, regardless of the origin of USVs, as a result of adaptive pressure for expression of an emotional state or in association with motor activity, pup USVs evolved as biologically meaningful signals communicating a state of negative arousal that results in promoting maternal care (Ehret, 2005; Portfors, 2007).
III FACTORS ELICITING MOUSE PUP USVs Although mouse pups are in a highly immature state at birth, they are already responsive to tactile and olfactory stimuli and able to process them. Mouse pups are indeed able to modulate their USV production according to several environmental conditions such as thermal and olfactory stimuli or as a result of a combination of these factors (Elwood & McCauley, 1983). A great deal of evidence suggests exposure to a cold environment as a potent eliciting stimulus for neonatal USVs because newborn mice depend upon the mother for keeping their body temperature constant. During this poikilothermic period, pups encourage maternal care by calling the dam through the USVs. Experiments that demonstrated a relationship between USVs and the development of homeothermy have been performed by Okon (1970). He reported a limited vocalizing behavior at birth, its increase during the transition to homeothermy, and finally its decrease after homeothermy has been fully achieved (Okon, 1970). A narrow
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IV THE IMPORTANCE OF USVs IN THE MOUSE MOTHER-OFFSPRING RELATIONSHIP
correlation between decreased environmental temperature and increased USV calling rate has also been described in a large number of later studies (Blumberg, Efimova, & Alberts, 1992; Elwood & Keeling, 1982; Noirot, 1972). In addition to the thermal condition, another important factor affecting USV production is the presence of olfactory cues. Oswalt and Meier (1975) found that infant rats vocalized less when tested under soiled bedding conditions with home cage material as compared to clean bedding or no bedding conditions (Oswalt & Meier, 1975). A few years later, Conely and Bell (1978) reported that newborn rats produce more USVs in the presence of olfactory stimuli from adult females than in the presence of that from adult males (Conely & Bell, 1978). Similar findings in isolated mouse pups confirmed that the odors of littermates and mothers led to a calming response with a reduction in USV emission. By contrast, the exposure to a novel odor such as clean bedding induced a stress response that, in turn, increased USV production orgyi, (D’Amato & Cabib, 1987; Kapusta & Szentgy€ 2004; Marchlewska-Koj, Kapusta, & Olejniczak, 1999; W€ ohr, van Gaalen, & Schwarting, 2015). Overall, infant USVs are emitted in response to stressful conditions that generate aversive arousal in the pup. This observation is in agreement with the hypothesis that USVs represent communicatory signals.
IV THE IMPORTANCE OF USVs IN THE MOUSE MOTHER-OFFSPRING RELATIONSHIP USV emission plays an important communicative role in the adult-pup relationship, and is critical, in particular, for the well being and survival of the pups (Smith & Sales, 1980). A great number of studies have shown that USVs emitted by newborns affect various aspects of adult behavior. In particular, they are able to stop maternal ongoing activities and switch to specific responses such as retrieving a lost infant and nest building (Hennessy, Li, Lowe, & Levine, 1980; Nitschke, Bell, & Zachman, 1972; Sewell, 1970; Smotherman, Bell, Starzec, Elias, & Zachman, 1974). The first study in this field, demonstrating an early mother-pup relationship induced by USVs, has been performed by Zippelius and Schleidt (1956). They reported that USVs produced by scattered pups (1–12 days old) elicited maternal activities aimed at searching for the pups and guiding them to the nest. In addition, although they did not control for confounding factors, Zippelius and Schleidt found that pups unable to emit USVs (e.g., being dead or anesthetized) were not retrieved by the mother. Furthermore, the cessation of ultrasonic emission by older pups (two weeks old) coincided with a considerable decrease in the mother’s
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retrieving and nest-building behaviors and an increased probability of being attacked or killed by adults (Noirot, 1966; Noirot & Pye, 1969). Further evidence confirming the effects of isolation calls on the maternal response has been provided by Sewell (1970). Mouse females were exposed to a purely acoustic stimulus (recorded calls emitted by a 5-day-old isolated mouse litter) or control signals (recorded background noise or artificial 45-kHz pulses). The females responded by going toward the speaker replaying the infant calls. This and other playback studies have shown that vocalizing pups can be replaced by a loudspeaker emitting pup calls, confirming the idea that acoustic signals elicit maternal response and that visual and olfactory cues are not always necessary (Ehret & Haack, 1982; Okabe et al., 2013; Uematsu et al., 2007). Moreover, it has been demonstrated that a variety of ultrasonic sounds, even if they contain acoustic features never found in the pup USVs, can be attractive to the dams (Ehret, 2005; Ehret & Haack, 1981). Taken together, these findings suggest that ultrasonic emission could be considered as an arousal-producing stimulus for the mother, who has the natural tendency to approach any type of ultrasonic sound although they significantly prefer those with acoustic features similar to pup calls. In the literature, a considerable amount of data indicate that both the physiological and emotional states of the mother affect ultrasonic emission by the offspring (D’Amato & Populin, 1987; D’Amato, Scalera, Sarli, & Moles, 2005; Weller et al., 2003; W€ ohr et al., 2008). Female genotype or hormonal changes affect maternal response to pup USVs (Curry et al., 2013; Greene-Colozzi, Sadowski, Chadwick, Tsai, & Sahin, 2014). For instance, lactating mothers respond faster than nonlactating mothers (Ehret & Haack, 1984; Koch & Ehret, 1989). Moreover, D’Amato et al. (2005) found that mouse pups raised by mothers with higher maternal responsiveness emitted lower call rates than pups raised by mothers with low maternal responsiveness, suggesting these maternal states or behaviors are negatively correlated with the neonatal USV calling rate (D’Amato et al., 2005). A phenomenon called “maternal potentiation” confirmed the importance of maternal cues for the emission of pup USVs (Shair et al., 2015). In 1994, Hofer and colleagues showed that the calling rate of rat pups reisolated after a brief reunion with the mother increases approximately two- or three-fold over the rate shown during the first isolation. Interestingly, this USV potentiation does not develop until postnatal day 7–9, reaches a peak around day 13, and declines when all USV responses cease (Hofer, Brunelli, & Shair, 1994; Hofer, Masmela, Brunelli, & Shair, 1998; see also Chapter 15 in this volume). More recently, the maternal potentiation of USVs has been shown also in mouse pups. In addition, altered levels of maternal potentiation have been associated with
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social deficits in genetic mouse models of neurodevelopmental disorders, such as autism spectrum disorders (ASD) (Scattoni, Crawley, & Ricceri, 2009).
V RECORDING AND ANALYSIS OF MOUSE USVs While a great number of studies have been focused on the circumstances leading to pup USV production, fewer have explored the acoustic features of USVs. Given that USVs, by definition, are well beyond the human hearing threshold, their recording and analysis are dependent on computerized electronic equipment. The first studies aimed at investigating USVs used bat detectors, which transform sounds by lowering their frequency to the human hearing range and allowing the estimation of the vocalization rate (Brudzynski, 2009). Over the years, due to progress in equipment technology, advanced measurement systems have been developed providing the opportunity for detailed analyses of USV structure, thus adding an accurate qualitative insight to the common quantitative one (Branchi et al., 2001; Scattoni, Gandhy, Ricceri, & Crawley, 2008). USVs are characterized by acoustic properties of sound waves, which are sound frequency, wavelength, period, amplitude, intensity, and direction of propagation. Indeed, the parameters commonly considered for describing each ultrasonic call are peak frequency (in kHz), duration (in ms) and amplitude (expressed in dB as a relative unit). The digital analysis of the spectrogram has allowed us to visualize these sounds as sonograms that are threedimensional images in which the x-axis represents time, the y-axis represents frequency, and the color represents the amplitude. The spectrographic analyses performed in a large number of studies confirmed that USVs are whistle-like sounds emitted within the 30–90kHz frequency band, with a great variation in duration (approximately 5–200 ms per call). Also, they are separated by breaks (inter-call intervals) and emitted in bouts at rates of about 10 calls per second (Brudzynski, 2009; for further details, see Chapter 5 in this volume). Recently, Scattoni and colleagues classified mouse calls into 10 categories defined according to the internal frequency changes, duration, and spectrographic shape, in line with previously published categorizations (Branchi et al., 1998; Brudzynski, Kehoe, & Callahan, 1999; Panksepp et al., 2007; Sales & Smith, 1978; Scattoni et al., 2008). USVs are classified as complex calls if one call element contains two or more directional changes in pitch, each 6.25 kHz. Harmonic calls are characterized by one main call element, resembling the complex call, and additional call fragments of different frequencies surrounding the main call. A two-component call consists of two sound components: a main call
component (flat or downward) with an additional punctuated fragment toward the end. An upward call exhibits a continuous increase in pitch that is 12.5 kHz with a terminal dominant frequency at least 6.25 kHz higher than the frequency at the beginning of the vocalization. A downward call exhibits a continuous decrease in pitch that is 12.5 kHz with a terminal dominant frequency at least 6.25 kHz lower than the frequency at the beginning of the vocalization. A flat call displays a constant frequency from the beginning to the end of the call (with changes not more than 3 kHz). A chevron call resembles an inverted-U shape, which is identified by a continuous increase in pitch 12.5 kHz followed by a decrease that is 6.25 kHz. Short calls are punctuated and shorter than 5 ms in duration. Composite calls are formed by two harmonically independent components, emitted simultaneously. Frequency-step calls are characterized by instantaneous frequency changes appearing as a vertically discontinuous “step” on a spectrogram, but with no interruption in time (see Fig. 13.2). The 10 categories described above are used to analyze the call distribution (the relative number of calls emitted within each category), and their probability of occurrence. The experimenter determines the call category of each emitted USV based on the visual analysis of the spectrogram. The development of new techniques for automatic identification of the acoustic features of USVs is warranted for a faster and more detailed analysis of large data sets (Burkett, Day, Penagarikano, Geschwind, & White, 2015; Grimsley, Gadziola, & Wenstrup, 2012). Overall, a quantitative and qualitative characterization, rather than a quantitative count only, helps in better appreciating subtle differences in mouse USV emission. Further studies are needed to investigate the potential biological significance of the differences among the call categories.
VI DEVELOPMENTAL CHANGES OF MOUSE PUP USVs FEATURES The structural features of sound production that occur during the first two postnatal weeks have been extensively examined (Ehret, 1980; Grimsley, Monaghan, & Wenstrup, 2011; Noirot, 1966, 1968; Noirot & Pye, 1969). The increase in call rate during the first days of life is primarily due to a higher number of bouts produced, whereas its subsequent decline is due to a lower number of USVs within each bout. The reduction with age of the interval between calls (the inter-call interval) has been hypothesized to reflect an increased lung capacity allowing a higher emission rate (Elwood & Keeling, 1982). The study of call categories within a bout has shown that ultrasonic patterns vary extensively over development
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VII STRAIN AND GENOTYPE DIFFERENCES IN MOUSE PUP USVs
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FIG. 13.2 Typical sonograms of USVs of mouse pups (C57BL/6J strain) illustrating five distinct call categories. From left to right: USV representing the short, chevron, complex, two-components, and frequency-steps call categories. Time (in ms) is indicated on the x-axis, frequency (in kHz) is indicated on the y-axis, and relative intensity or loudness is indicated by color (see color-coded bar on the right side of the figure).
with old pups emitting a larger vocal repertoire compared to young ones. Usually all neonates start repeatedly emitting the same vocal component. However, when they are able to produce more complex vocal sequences, each pup will have a unique and individual ultrasonic profile different from those of littermates. An interesting example of developmental changes is the increased proportion of harmonic calls over the initial days (Grimsley et al., 2011). This phenomenon could be interpreted as extremely advantageous for pups under isolation from the evolutionary standpoint. First, harmonic calls, as broadband sounds, are easier to locate than tonal sounds. Second, because harmonic calls are characterized by nonlinear components, they may convey negative emotional states that are appropriate to isolation condition. Taken together, these considerations support the hypothesis that harmonic calls may enhance the probability of isolated pups to be found and retrieved by the mother. In addition, changes in the spectral and temporal parameters of the mouse pup USVs during the first postnatal weeks have been studied in detail. Even though sound frequency usually increases with age, resulting in a higher peak frequency that is above the dominant frequency of adult calls, and call duration declines, it has been reported that each call type can show a specific pattern of developmental changes (Ey et al., 2013; Hahn et al., 1998; Liu, Miller, Merzenich, & Schreiner, 2003; Motomura et al., 2002; Noirot & Pye, 1969; Scattoni et al., 2008). On one hand, accurate analysis suggests that developmental changes that are reflected in the acoustic features and vocal repertoire may have a highly communicative value because they may be used by the mother to recognize pups on the basis of their age. On the other, the age-
dependent changes in neonatal ultrasonic emissions may be related to the maturation of vocal production mechanisms or to the increased motor control, even though the association between changes in physiological mechanisms and USV acoustic features is not yet clear. Therefore, additional studies are needed to determine how these changes occur during early postnatal life.
VII STRAIN AND GENOTYPE DIFFERENCES IN MOUSE PUP USVs The production of isolation calls strongly varies with the mouse strain and its genotype (Bell, Nitschke, & Zachman, 1972; D’Udine, Robinson, & Oliverio, 1982; W€ ohr et al., 2008). The knowledge that USVs have a robust genetic basis has come from a large number of studies that consistently characterized ultrasonic patterns of approximately 20 different laboratory mouse strains. Interestingly, clear differences have been found even among closely related mouse strains (Hahn & Lavooy, 2005). The strain differences concern the call rate as well as other spectrographic USV characteristics (Hahn et al., 1998; Hahn, Hewitt, Adams, & Tully, 1987; Hahn, Hewitt, Schanz, Weinreb, & Henry, 1997; Hahn & Schanz, 2002; Nitschke et al., 1972; Thornton et al., 2005). For instance, NZB and C57BL/6J mouse strains emitted a very low number of USVs compared to other inbred mice, such as BALB/c, DBA, A/J, BTBR, and FVB/NJ. The analysis of duration, sound frequency, and bandwidth measured for each pup call showed that these parameters differ among three strains— 129SV, C57BL6, and BALB/c—with the highest fluctuation of values estimated during the second postnatal
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week (Wiaderkiewicz, Glowacka, Grabowska, & Jaroslaw-Jerzy, 2013). Some years ago, Scattoni and coworkers carried on a detailed spectrographic analysis, based on the abovedescribed ultrasonic categorization, of four different strains of mouse pups (BTBR, C57BL/6J, 129X1 and FVB/NJ), classifying every call emitted by 8-day-old pups. In particular, they found that pups belonging to the C57BL/6J, FVB/NJ, and 129X1 strains produced a wide repertoire of calls, which included high numbers of frequency steps and complex USVs. By contrast, pups belonging to the BTBR strain, a potential mouse model of ASD, emitted a narrower repertoire of calls that included high levels of harmonic and composite USVs but a minimal number of other shape USVs (Scattoni et al., 2008). Moreover, as the spectrographic characterization of the 8-day-old CD-1 outbred mice was compared to results obtained with C57BL/6J, 129X1, and FVB/NJ strains, CD-1 pups emitted a higher percentage of frequency steps and complex USVs but low numbers of flat, short, and complex calls. Finally, the ontogenetic profile of USV emission has also been found to be strain-dependent (Branchi, Santucci, & Alleva, 2006; Scattoni et al., 2008). For instance, C57BL/6 and BALB/c strains showed a peak in the calling rate early, around postnatal day 3 (D’Udine et al., 1982), while other inbred (BTBR, FVB/ NJ, 129X1, DBA, A/J, SEC, C3H) or outbred (CD-1) strains showed a peak around postnatal day 7 (Branchi et al., 1998; Elwood & Keeling, 1982; Hennessy et al., 1980). Over the last 15 years, the definitive role of genetic differences in USV emission among mice of different strains has been corroborated by a large number of studies carried out on knock-out and other genetically modified mice, showing that deletion or insertion of selected genes markedly modifies USV production. In particular, it has been found that genes affecting USVs encode a broad spectrum of molecular signals, acting in complex pathways involved in the correct maturation and function of the central nervous system. Their mutations could be responsible for the onset of such neurodevelopmental disorders as ASD (Ey et al., 2013; Romano, Michetti, Caruso, Laviola, & Scattoni, 2013; Roy, Watkins, & ohr, 2016; Young, Heck, 2012; Sungur, Schwarting, & W€ Schenk, Yang, Jan, & Jan, 2010). Both calling rate and spectrographic differences in the vocal repertoire of animal models of ASD have been interpreted as developmental alterations within the social/communication domain (see Chapter 42 in this book). In summary, data from several mouse strains and genetically modified lines show that genetic factors have great influence on USV emission, producing a multitude of effects on both quantitative and qualitative characteristics of calls.
VIII EFFECTS OF PHARMACOLOGICAL COMPOUNDS ON PRODUCTION OF MOUSE PUP USVs The effect of many compounds on pup USV emission has been thoroughly investigated (Cuomo, De Salvia, Maselli, Santo, & Cagiano, 1987; Laviola, Adriani, Gaudino, Marino, & Keller, 2006). In particular, it has been shown that pharmacological treatments associated with the modulation of the main neurotransmitter systems in the central nervous system clearly affected USV signaling (Benton & Nastiti, 1988; Dastur, McGregor, & Brown, 1999; Hodgson, Guthrie, & Varty, 2008; Insel, Hill, & Mayor, 1986; McGregor, Dastur, McLellan, & Brown, 1996; Takahashi et al., 2009; Varga, Fodor, Klausz, & Zelena, 2015; Vivian, Barros, Manitiu, & Miczek, 1997). Great attention has been paid to the GABAergic and glutamatergic systems that are strongly implicated in the modulating rate and features of USVs emitted by isolated mouse pups. For instance, benzodiazepines and other agonists of GABAA receptors reduced neonatal calls while inverse agonists enhance them (Fish, Sekinda, Ferrari, Dirks, & Miczek, 2000; Nastiti, Benton, & Brain, 1991; Rowlett, Tornatzky, Cook, Ma, & Miczek, 2001). Interestingly, ethanol exposure, having anxiolytic properties similar to benzodiazepines, also caused a reduction of the USV rate (Barron & Gilbertson, 2005). A detailed sonographic analysis has shown that ethanol produced a rather selective reduction in specific waveforms (rising frequency, U-shaped, and 3-sweep calls) (Wellmann, George, Brnouti, & Mooney, 2015), instead of affecting the overall production of USVs. Glutamatergic drugs, such as NMDA receptor antagonists, have a bidirectional effect on USV emission in a dose-dependent fashion, that is, the number of USVs was enhanced after moderate glutamate doses and reduced after high doses (Swanson et al., 2005; Takahashi et al., 2009). Similarly to the glutamatergic system, the serotonergic system also had a bidirectional effect on USV production dependent on the type of 5-HT receptor stimulated (Nastiti et al., 1991; W€ ohr et al., 2015). As to the dopaminergic modulation, agonists of D1, D2, and D3 receptors have an important role in reducing USV production (Dastur et al., 1999). Moreover, prenatal cocaine exposure, a potent dopamine reuptake inhibitor, decreases the number of USVs emitted as well as modifies the spectrotemporal USV parameters (Cox et al., 2012; McMurray et al., 2013). Because pharmacological studies have proven that the emission of USVs can be affected by anxiolytic and anxiogenic drugs, it is assumed that neonatal USV production in response to separation from the nest environment reflects a state of anxiety and thus is considered as a valid tool to test emotional states postnatally.
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REFERENCES
IX CONCLUSIONS AND POTENTIAL FUTURE DIRECTIONS Mice emit USVs throughout their lives, especially during early postnatal days upon separation from the nest. Interestingly, the analysis of this vocal response permits us to study emotional development and responsiveness to environmental signals in the newborn as well as the communication between the pup and mother. Recently, the growing literature on neonatal ultrasonic emission has suggested that the ontogenetic profile represents an interesting tool to phenotype mouse lines modeling neurodevelopmental disorders, in which socio-communicative deficits are one of the core symptoms. Although animal models cannot be compared to the complexity of human language, the study of vocal communication in mouse pups could provide valuable insight into the genetic basis of communication and speech disorders. The quantitative and qualitative approaches allow comparing vocal repertoires emitted by pups under different conditions over the course of development and by pups of different strains or genotypes. A USV profile strongly changes within the first two postnatal weeks and appears extremely different from the adult repertoire. This result opens the controversial question on the innate nature of USVs emitted by rodent pups and on the possibility of analyzing them according to the same approach exploited for birds, in which chicks learn vocalization patterns through experience (Arriaga & Jarvis, 2013; Hammerschmidt, Whelan, Eichele, & Fischer, 2015). Studies in which USVs are emitted by juvenile mice could help to understand the vocal changes during developmental stages, filling the gap between pup and adult repertoires. Moreover, it is interesting to speculate that the sets of USV categories generated by each strain may represent specific “dialects.” Further studies using a wider number of mice inbred and outbred strains or genetically modified lines are warranted to make definitive statements about strain or genotype variations in complex vocal production. There are still important limitations in the qualitative characteristics of USVs by means of the instruments for USV investigation currently available. Nowadays, the most used approach is based on observation of USV features by the experimenter in order to classify each call into the different categories. This approach is error-prone and leads to poor reproducibility. In order to circumvent these limitations, efforts are focused on developing sophisticated software based on automated algorithms, which permit a faster and proper identification of salient features of mouse USVs. These automatic recognition systems could be useful to the scientific community as they can standardize USV analysis across laboratories and can potentially allow a detailed analysis aimed at increasing the translational value of the study of USVs emitted by mouse pups.
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Hahn, M. E., Hewitt, J. K., Adams, M., & Tully, T. (1987). Genetic influences on ultrasonic vocalizations in young mice. Behavior Genetics, 17(2), 155–166. Hahn, M. E., Hewitt, J. K., Schanz, N., Weinreb, L., & Henry, A. (1997). Genetic and developmental influences on infant mouse ultrasonic calling. I. A diallel analysis of the calls of 3-day olds. Behavior Genetics, 27(2), 133–143. Hahn, M. E., Karkowski, L., Weinreb, L., Henry, A., Schanz, N., & Hahn, E. M. (1998). Genetic and developmental influences on infant mouse ultrasonic calling. II. Developmental patterns in the calls of mice 2–12 days of age. Behavior Genetics, 28(4), 315–325. Hahn, M. E., & Lavooy, M. J. (2005). A review of the methods of studies on infant ultrasound production and maternal retrieval in small rodents. Behavior Genetics, 35(1), 31–52. Hahn, M. E., & Schanz, N. (2002). The effects of cold, rotation, and genotype on the production of ultrasonic calls in infant mice. Behavior Genetics, 32(4), 267–273. Hammerschmidt, K., Whelan, G., Eichele, G., & Fischer, J. (2015). Mice lacking the cerebral cortex develop normal song: insights into the foundations of vocal learning. Scientific Reports, 5, 8808. Hennessy, M. B., Li, J., Lowe, E. L., & Levine, S. (1980). Maternal behavior, pup vocalizations, and pup temperature changes following handling in mice of 2 inbred strains. Developmental Psychobiology, 13(6), 573–584. Hodgson, R. A., Guthrie, D. H., & Varty, G. B. (2008). Duration of ultrasonic vocalizations in the isolated rat pup as a behavioral measure: sensitivity to anxiolytic and antidepressant drugs. Pharmacology Biochemistry and Behavior, 88(3), 341–348. Hofer, M. A., Brunelli, S. A., Masmela, J., & Shair, H. N. (1996). Maternal interactions prior to separation potentiate isolation-induced calling in rat pups. Behavioral Neuroscience, 110(5), 1158–1167. Hofer, M. A., Brunelli, S. A., & Shair, H. N. (1994). Potentiation of isolation-induced vocalization by brief exposure of rat pups to maternal cues. Developmental Psychobiology, 27(8), 503–517. Hofer, M. A., Masmela, J. R., Brunelli, S. A., & Shair, H. N. (1998). The ontogeny of maternal potentiation of the infant rats’ isolation call. Developmental Psychobiology, 33(3), 189–201. Insel, T. R., Hill, J. L., & Mayor, R. B. (1986). Rat pup ultrasonic isolation calls: possible mediation by the benzodiazepine receptor complex. Pharmacology Biochemistry and Behavior, 24(5), 1263–1267. Kapusta, J., & Szentgy€ orgyi, H. (2004). Ultrasonic response of CBA pups to the odour of genetically different mice. Behaviour, 141, 157–167. Koch, M., & Ehret, G. (1989). Estradiol and parental experience, but not prolactin are necessary for ultrasound recognition and pup-retrieving in the mouse. Physiology and Behavior, 45(4), 771–776. Laviola, G., Adriani, W., Gaudino, C., Marino, R., & Keller, F. (2006). Paradoxical effects of prenatal acetylcholinesterase blockade on neuro-behavioral development and drug-induced stereotypies in reeler mutant mice. Psychopharmacology (Berlin), 187(3), 331–344. Liu, R. C., Miller, K. D., Merzenich, M. M., & Schreiner, C. E. (2003). Acoustic variability and distinguishability among mouse ultrasound vocalizations. Journal of the Acoustical Society of America, 114(6 Pt 1), 3412–3422. Marchlewska-Koj, A., Kapusta, J., & Olejniczak, P. (1999). Ultrasonic response of CBA newborn mice to bedding odour. Behaviour, 136, 269–278. McGregor, I. S., Dastur, F. N., McLellan, R. A., & Brown, R. E. (1996). Cannabinoid modulation of rat pup ultrasonic vocalizations. European Journal of Pharmacology, 313(1-2), 43–49. McMurray, M. S., Zeskind, P. S., Meiners, S. M., Garber, K. A., Tien, H., & Johns, J. M. (2013). Effect of prenatal cocaine on early postnatal thermoregulation and ultrasonic vocalization production. Frontiers in Psychology, 4, 882. Motomura, N., Shimizu, K., Shimizu, M., Aoki-Komori, S., Taniguchi, K., Serizawa, I., et al. (2002). A comparative study of isolation-induced ultrasonic vocalization in rodent pups. Experimental Animals, 51(2), 187–190.
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Nastiti, K., Benton, D., & Brain, P. F. (1991). The effects of compounds acting at the benzodiazepine receptor complex on the ultrasonic calling of mouse pups. Behavioural Pharmacology, 2(2), 121–128. Nitschke, W., Bell, R. W., & Zachman, T. (1972). Distress vocalizations of young in three inbred strains of mice. Developmental Psychobiology, 5(4), 363–370. Noirot, E. (1966). Ultra-sounds in young rodents. I. Changes with age in albino mice. Animal Behaviour, 14(4), 459–462. Noirot, E. (1968). Ultrasounds in young rodents. II. Changes with age in albino rats. Animal Behaviour, 16(1), 129–134. Noirot, E. (1972). Ultrasounds and maternal behavior in small rodents. Developmental Psychobiology, 5(4), 371–387. Noirot, E., & Pye, D. (1969). Sound analysis of ultrasonic distress calls of mouse pups as a function of their age. Animal Behaviour, 17, 340–349. Okabe, S., Nagasawa, M., Kihara, T., Kato, M., Harada, T., Koshida, N., et al. (2013). Pup odor and ultrasonic vocalizations synergistically stimulate maternal attention in mice. Behavioral Neuroscience, 127(3), 432–438. Okon, E. E. (1970). The effect of environmental temperature on the production of ultra sounds by isolated non-handled albino mouse pups. Journal of Zoology (London), 162, 71–83. Oswalt, G. L., & Meier, G. W. (1975). Olfactory, thermal, and tactual influences on infantile ultrasonic vocalization in rats. Developmental Psychobiology, 8(2), 129–135. Panksepp, J. B., Jochman, K. A., Kim, J. U., Koy, J. J., Wilson, E. D., Chen, Q., et al. (2007). Affiliative behavior, ultrasonic communication and social reward are influenced by genetic variation in adolescent mice. PLoS One, 2(4)e351. Portfors, C. V. (2007). Types and functions of ultrasonic vocalizations in laboratory rats and mice. Journal of the American Association for Laboratory Animal Science, 46(1), 28–34. Romano, E., Michetti, C., Caruso, A., Laviola, G., & Scattoni, M. L. (2013). Characterization of neonatal vocal and motor repertoire of reelin mutant mice. PLoS One, 8(5), e64407. Rowlett, J. K., Tornatzky, W., Cook, J. M., Ma, C., & Miczek, K. A. (2001). Zolpidem, triazolam, and diazepam decrease distress vocalizations in mouse pups: differential antagonism by flumazenil and betaCarboline-3-carboxylate-t-butyl ester (beta-CCt). Journal of Pharmacology and Experimental Therapeutics, 297(1), 247–253. Roy, S., Watkins, N., & Heck, D. (2012). Comprehensive analysis of ultrasonic vocalizations in a mouse model of fragile X syndrome reveals limited, call type specific deficits. PLoS One, 7(9), e44816. Sales, G. D., & Pye, D. (1974). Ultrasonic Communication by Animals (1st ed.). London: Chapman and Hall Ltd. Sales, G. D., & Smith, J. C. (1978). Comparative studies of the ultrasonic calls of infant murid rodents. Developmental Psychobiology, 11, 595–619. Scattoni, M. L., Crawley, J., & Ricceri, L. (2009). Ultrasonic vocalizations: a tool for behavioural phenotyping of mouse models of neurodevelopmental disorders. Neuroscience and Biobehavioral Reviews, 33(4), 508–515. Scattoni, M. L., Gandhy, S. U., Ricceri, L., & Crawley, J. N. (2008). Unusual repertoire of vocalizations in the BTBR T + tf/J mouse model of autism. PLoS One, 3(8)e3067. Sewell, G. D. (1970). Ultrasonic communication in rodents. Nature, 227(5256), 410. Shair, H. N., & Jasper, A. (2003). Decreased venous return is neither sufficient nor necessary to elicit ultrasonic vocalizations of infant rat pups. Behavioral Neuroscience, 117(4), 840–853. Shair, H. N., Rupert, D. D., Rosko, L. M., Hofer, M. A., Myers, M. M., & Welch, M. G. (2015). Effects of maternal deprivation and the duration of reunion time on rat pup ultrasonic vocalization responses to isolation: possible implications for human infant studies. Developmental Psychobiology, 57(1), 63–72.
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Smith, J. C., & Sales, G. D. (1980). Ultrasonic behavior and mother-infant interactions in rodents. In R. W. Bell & W. P. Smotherman (Eds.), Maternal influences and early behavior (pp. 105–133). New York: Spectrum Press. Smotherman, W. P., Bell, R. W., Starzec, J., Elias, J., & Zachman, T. A. (1974). Maternal responses to infant vocalizations and olfactory cues in rats and mice. Behavioral Biology, 12(1), 55–66. Sungur, A. O., Schwarting, R. K., & W€ ohr, M. (2016). Early communication deficits in the Shank1 knockout mouse model for autism spectrum disorder: developmental aspects and effects of social context. Autism Research, 9(6), 696–709. Swanson, C. J., Bures, M., Johnson, M. P., Linden, A. M., Monn, J. A., & Schoepp, D. D. (2005). Metabotropic glutamate receptors as novel targets for anxiety and stress disorders. Nature Reviews Drug Discovery, 4(2), 131–144. Takahashi, A., Yap, J. J., Bohager, D. Z., Faccidomo, S., Clayton, T., Cook, J. M., et al. (2009). Glutamatergic and GABAergic modulations of ultrasonic vocalizations during maternal separation distress in mouse pups. Psychopharmacology (Berlin), 204(1), 61–71. Thornton, L. M., Hahn, M. E., & Schanz, N. (2005). Genetic and developmental influences on infant mouse ultrasonic calling. III. Patterns of inheritance in the calls of mice 3–9 days of age. Behavior Genetics, 35(1), 73–83. Uematsu, A., Kikusui, T., Kihara, T., Harada, T., Kato, M., Nakano, K., et al. (2007). Maternal approaches to pup ultrasonic vocalizations produced by a nanocrystalline silicon thermo-acoustic emitter. Brain Research, 1163, 91–99. Varga, J., Fodor, A., Klausz, B., & Zelena, D. (2015). Anxiogenic role of vasopressin during the early postnatal period: maternal separationinduced ultrasound vocalization in vasopressin-deficient Brattleboro rats. Amino Acids, 47(11), 2409–2418. Vivian, J. A., Barros, H. M., Manitiu, A., & Miczek, K. A. (1997). Ultrasonic vocalizations in rat pups: modulation at the gammaaminobutyric acidA receptor complex and the neurosteroid recognition site. Journal of Pharmacology and Experimental Therapeutics, 282(1), 318–325. Weller, A., Leguisamo, A. C., Towns, L., Ramboz, S., Bagiella, E., Hofer, M., et al. (2003). Maternal effects in infant and adult phenotypes of 5HT1A and 5HT1B receptor knockout mice. Developmental Psychobiology, 42(2), 194–205. Wellmann, K. A., George, F., Brnouti, F., & Mooney, S. M. (2015). Docosahexaenoic acid partially ameliorates deficits in social behavior and ultrasonic vocalizations caused by prenatal ethanol exposure. Behavioural Brain Research, 286, 201–211. Wiaderkiewicz, J., Glowacka, M., Grabowska, M., & Jaroslaw-Jerzy, B. (2013). Ultrasonic vocalizations (USV) in the three standard laboratory mouse strains: developmental analysis. Acta Neurobiologiae Experimentalis, 73(4), 557–563. W€ ohr, M., Dahlhoff, M., Wolf, E., Holsboer, F., Schwarting, R. K., & Wotjak, C. T. (2008). Effects of genetic background, gender, and early environmental factors on isolation-induced ultrasonic calling in mouse pups: an embryo-transfer study. Behavior Genetics, 38(6), 579–595. W€ ohr, M., van Gaalen, M. M., & Schwarting, R. K. (2015). Affective communication in rodents: serotonin and its modulating role in ultrasonic vocalizations. Behavioural Pharmacology, 26(6), 506–521. Young, D. M., Schenk, A. K., Yang, S. B., Jan, Y. N., & Jan, L. Y. (2010). Altered ultrasonic vocalizations in a tuberous sclerosis mouse model of autism. Proceedings of the National Academy of Sciences of the United States of America, 107(24), 11074–11079. Zippelius, H. M., & Schleidt, W. M. (1956). Ultraschall-Laute bei jungen M€ausen [Ultrasonic vocalization in infant mice]. Naturwissenschaften, 43, 502–503.
E. ULTRASONIC COMMUNICATION OF INFANT RODENTS
13 Quantitative and Qualitative Features of Neonatal Vocalizations in Mice Angela Caruso1,2, Mara Sabbioni1, Maria Luisa Scattoni1,*, Igor Branchi3,* 1
Research Coordination and Support Service, Istituto Superiore di Sanità, Rome, Italy 2 Sapienza University of Rome, Rome, Italy 3 Center for Behavioral Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy
I AUDIBLE AND ULTRASONIC VOCALIZATIONS EMITTED BY MOUSE PUPS
II FUNCTIONAL ROLE OF PUP ISOLATION-INDUCED USVs
Mus musculus pups are born blind and deaf with overall limited sensory-motor abilities; they are also incapable of feeding or thermoregulation. Therefore, during the first two postnatal weeks, their survival is strictly dependent on the mother. It is well known that at this earliest stage, mouse pups are highly vocal, emitting a variety of calls in different motivationally relevant contexts that elicit adequate maternal care (Branchi, Santucci, Vitale, & Alleva, 1998; Ehret & Bernecker, 1986). For instance, they can produce vocalizations in the audible range when cleaned by the mother, in response to pain or roughhandling, or when accidentally pinched. They can also emit low-frequency wriggling calls when they push for the mother’s nipples or when crawling over each other inside the nest (Hahn & Lavooy, 2005). Mouse pups also produce abundant ultrasonic vocalizations (USVs), whistle-like sounds within the 30–90 kHz frequency range above the human hearing threshold of 20kHz (Sales & Pye, 1974; Sales & Smith, 1978; Zippelius & Schleidt, 1956). A large amount of data indicate that, although both audible sounds and USVs are generated in the larynx by air passing over the vocal cords, the mechanisms underlying the production of these two types of sounds markedly differ (for further details of the mechanisms of USV production, see Chapter 5 in this volume). The focus of the present chapter is on the emission of USVs because these have been shown to play a highly relevant role in mother-pup communication, allowing a fine-tuning of pup behaviors with maternal responses. *Co-last senior authors Handbook of Ultrasonic Vocalization, Volume 25 ISSN: 1569-7339 https://doi.org/10.1016/B978-0-12-809600-0.00013-5
Neonatal USVs are emitted by mice as well as by other altricial rodent species. The USVs are produced by pups when they are outside the nest and far from the mother and littermates. Therefore, these calls are defined as “isolation-induced USVs” (Branchi, Santucci, & Alleva, 2001; Hofer, Brunelli, Masmela, & Shair, 1996; Sales & Pye, 1974; Sales & Smith, 1978; Sewell, 1970; Smith & Sales, 1980; Zippelius & Schleidt, 1956). Upon hearing the USVs from an isolated pup, the mother rapidly investigates the sound, identifies the pup’s location, and retrieves it to the nest (Ehret & Bernecker, 1992; Noirot, 1972; Fig. 13.1). Similar to other behavioral responses, USV features change with the age of the pups. Indeed, the calling rate emitted by mouse pups follows an ontogenetic profile: it increases during the first 6–7 days of life, reaches a peak around postnatal day 8, and then starts to decrease until the total disappearance of calls when pups are two weeks old (Hahn et al., 1998; Noirot, 1966; Thornton, Hahn, & Schanz, 2005). The reduction of isolation call emission parallels the emergence of pups’ ability to thermoregulate, move, and see (eye opening), and with the consequent capability to forage for themselves or search for their mother (Elwood & Keeling, 1982). The notion that USVs are emitted by pups when isolated from the nest to gain their mother’s attention suggests a communicative role for USVs. In fact, neonatal USVs emitted under social isolation are considered distress calls for help and therefore interpreted as essential communicative signals. However, several authors have challenged this interpretation, opening a controversial debate about the functional role of pup USVs.
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Frequency (kHz)
Maternal response
Isolation condition
120 90 60 30 300
0 Time (ms) Ultrasonic emission
FIG. 13.1 The communicative role of ultrasonic vocalizations. After social isolation, mouse pups emit USVs that elicit in the dam a prompt response concerning care-giving behaviors. The sonogram illustrates three two-component ultrasonic calls of approximately 50–70 ms in duration and 60–90 kHz in frequency.
Blumberg and Alberts (1990) supported the idea that the ultrasonic emission is an acoustic by-product of a respiratory maneuver that physiologically increases oxygen delivery to metabolically active tissues (Blumberg, 1992; Blumberg & Alberts, 1990). Later, Blumberg and Sokoloff (2001) affirmed that USV production is caused by an abdominal compression reaction that increases blood flow return to the heart in response to decreased temperature when the pup falls out of the nest trying to right themselves or is handled roughly by adult mice or human experimenters (Blumberg & Sokoloff, 2001). These hypotheses were, however, later disproved (Shair & Jasper, 2003). In order to elucidate the functional role of neonatal USVs, the emission pattern has been extensively studied by monitoring several crucial factors such as physiological or environmental parameters, and by characterizing maternal response to USV production (Blumberg & Sokoloff, 1998; Branchi et al., 1998; Farrell & Alberts, 2002; Noirot, 1972). Another approach that provided insight into the role of ultrasonic signals, but has been so far rarely investigated, is the accurate evaluation of motor behavioral response in conjunction with the ultrasonic emission. It was, however, clearly demonstrated that a connection between USV emission and pup motor activity exists. The high calling rate of USVs during locomotor behavior is an active response to isolation, helping the pups to rejoin the mother (Branchi, Santucci, Puopolo, & Alleva, 2004).
In conclusion, regardless of the origin of USVs, as a result of adaptive pressure for expression of an emotional state or in association with motor activity, pup USVs evolved as biologically meaningful signals communicating a state of negative arousal that results in promoting maternal care (Ehret, 2005; Portfors, 2007).
III FACTORS ELICITING MOUSE PUP USVs Although mouse pups are in a highly immature state at birth, they are already responsive to tactile and olfactory stimuli and able to process them. Mouse pups are indeed able to modulate their USV production according to several environmental conditions such as thermal and olfactory stimuli or as a result of a combination of these factors (Elwood & McCauley, 1983). A great deal of evidence suggests exposure to a cold environment as a potent eliciting stimulus for neonatal USVs because newborn mice depend upon the mother for keeping their body temperature constant. During this poikilothermic period, pups encourage maternal care by calling the dam through the USVs. Experiments that demonstrated a relationship between USVs and the development of homeothermy have been performed by Okon (1970). He reported a limited vocalizing behavior at birth, its increase during the transition to homeothermy, and finally its decrease after homeothermy has been fully achieved (Okon, 1970). A narrow
E. ULTRASONIC COMMUNICATION OF INFANT RODENTS
IV THE IMPORTANCE OF USVs IN THE MOUSE MOTHER-OFFSPRING RELATIONSHIP
correlation between decreased environmental temperature and increased USV calling rate has also been described in a large number of later studies (Blumberg, Efimova, & Alberts, 1992; Elwood & Keeling, 1982; Noirot, 1972). In addition to the thermal condition, another important factor affecting USV production is the presence of olfactory cues. Oswalt and Meier (1975) found that infant rats vocalized less when tested under soiled bedding conditions with home cage material as compared to clean bedding or no bedding conditions (Oswalt & Meier, 1975). A few years later, Conely and Bell (1978) reported that newborn rats produce more USVs in the presence of olfactory stimuli from adult females than in the presence of that from adult males (Conely & Bell, 1978). Similar findings in isolated mouse pups confirmed that the odors of littermates and mothers led to a calming response with a reduction in USV emission. By contrast, the exposure to a novel odor such as clean bedding induced a stress response that, in turn, increased USV production orgyi, (D’Amato & Cabib, 1987; Kapusta & Szentgy€ 2004; Marchlewska-Koj, Kapusta, & Olejniczak, 1999; W€ ohr, van Gaalen, & Schwarting, 2015). Overall, infant USVs are emitted in response to stressful conditions that generate aversive arousal in the pup. This observation is in agreement with the hypothesis that USVs represent communicatory signals.
IV THE IMPORTANCE OF USVs IN THE MOUSE MOTHER-OFFSPRING RELATIONSHIP USV emission plays an important communicative role in the adult-pup relationship, and is critical, in particular, for the well being and survival of the pups (Smith & Sales, 1980). A great number of studies have shown that USVs emitted by newborns affect various aspects of adult behavior. In particular, they are able to stop maternal ongoing activities and switch to specific responses such as retrieving a lost infant and nest building (Hennessy, Li, Lowe, & Levine, 1980; Nitschke, Bell, & Zachman, 1972; Sewell, 1970; Smotherman, Bell, Starzec, Elias, & Zachman, 1974). The first study in this field, demonstrating an early mother-pup relationship induced by USVs, has been performed by Zippelius and Schleidt (1956). They reported that USVs produced by scattered pups (1–12 days old) elicited maternal activities aimed at searching for the pups and guiding them to the nest. In addition, although they did not control for confounding factors, Zippelius and Schleidt found that pups unable to emit USVs (e.g., being dead or anesthetized) were not retrieved by the mother. Furthermore, the cessation of ultrasonic emission by older pups (two weeks old) coincided with a considerable decrease in the mother’s
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retrieving and nest-building behaviors and an increased probability of being attacked or killed by adults (Noirot, 1966; Noirot & Pye, 1969). Further evidence confirming the effects of isolation calls on the maternal response has been provided by Sewell (1970). Mouse females were exposed to a purely acoustic stimulus (recorded calls emitted by a 5-day-old isolated mouse litter) or control signals (recorded background noise or artificial 45-kHz pulses). The females responded by going toward the speaker replaying the infant calls. This and other playback studies have shown that vocalizing pups can be replaced by a loudspeaker emitting pup calls, confirming the idea that acoustic signals elicit maternal response and that visual and olfactory cues are not always necessary (Ehret & Haack, 1982; Okabe et al., 2013; Uematsu et al., 2007). Moreover, it has been demonstrated that a variety of ultrasonic sounds, even if they contain acoustic features never found in the pup USVs, can be attractive to the dams (Ehret, 2005; Ehret & Haack, 1981). Taken together, these findings suggest that ultrasonic emission could be considered as an arousal-producing stimulus for the mother, who has the natural tendency to approach any type of ultrasonic sound although they significantly prefer those with acoustic features similar to pup calls. In the literature, a considerable amount of data indicate that both the physiological and emotional states of the mother affect ultrasonic emission by the offspring (D’Amato & Populin, 1987; D’Amato, Scalera, Sarli, & Moles, 2005; Weller et al., 2003; W€ ohr et al., 2008). Female genotype or hormonal changes affect maternal response to pup USVs (Curry et al., 2013; Greene-Colozzi, Sadowski, Chadwick, Tsai, & Sahin, 2014). For instance, lactating mothers respond faster than nonlactating mothers (Ehret & Haack, 1984; Koch & Ehret, 1989). Moreover, D’Amato et al. (2005) found that mouse pups raised by mothers with higher maternal responsiveness emitted lower call rates than pups raised by mothers with low maternal responsiveness, suggesting these maternal states or behaviors are negatively correlated with the neonatal USV calling rate (D’Amato et al., 2005). A phenomenon called “maternal potentiation” confirmed the importance of maternal cues for the emission of pup USVs (Shair et al., 2015). In 1994, Hofer and colleagues showed that the calling rate of rat pups reisolated after a brief reunion with the mother increases approximately two- or three-fold over the rate shown during the first isolation. Interestingly, this USV potentiation does not develop until postnatal day 7–9, reaches a peak around day 13, and declines when all USV responses cease (Hofer, Brunelli, & Shair, 1994; Hofer, Masmela, Brunelli, & Shair, 1998; see also Chapter 15 in this volume). More recently, the maternal potentiation of USVs has been shown also in mouse pups. In addition, altered levels of maternal potentiation have been associated with
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social deficits in genetic mouse models of neurodevelopmental disorders, such as autism spectrum disorders (ASD) (Scattoni, Crawley, & Ricceri, 2009).
V RECORDING AND ANALYSIS OF MOUSE USVs While a great number of studies have been focused on the circumstances leading to pup USV production, fewer have explored the acoustic features of USVs. Given that USVs, by definition, are well beyond the human hearing threshold, their recording and analysis are dependent on computerized electronic equipment. The first studies aimed at investigating USVs used bat detectors, which transform sounds by lowering their frequency to the human hearing range and allowing the estimation of the vocalization rate (Brudzynski, 2009). Over the years, due to progress in equipment technology, advanced measurement systems have been developed providing the opportunity for detailed analyses of USV structure, thus adding an accurate qualitative insight to the common quantitative one (Branchi et al., 2001; Scattoni, Gandhy, Ricceri, & Crawley, 2008). USVs are characterized by acoustic properties of sound waves, which are sound frequency, wavelength, period, amplitude, intensity, and direction of propagation. Indeed, the parameters commonly considered for describing each ultrasonic call are peak frequency (in kHz), duration (in ms) and amplitude (expressed in dB as a relative unit). The digital analysis of the spectrogram has allowed us to visualize these sounds as sonograms that are threedimensional images in which the x-axis represents time, the y-axis represents frequency, and the color represents the amplitude. The spectrographic analyses performed in a large number of studies confirmed that USVs are whistle-like sounds emitted within the 30–90kHz frequency band, with a great variation in duration (approximately 5–200 ms per call). Also, they are separated by breaks (inter-call intervals) and emitted in bouts at rates of about 10 calls per second (Brudzynski, 2009; for further details, see Chapter 5 in this volume). Recently, Scattoni and colleagues classified mouse calls into 10 categories defined according to the internal frequency changes, duration, and spectrographic shape, in line with previously published categorizations (Branchi et al., 1998; Brudzynski, Kehoe, & Callahan, 1999; Panksepp et al., 2007; Sales & Smith, 1978; Scattoni et al., 2008). USVs are classified as complex calls if one call element contains two or more directional changes in pitch, each 6.25 kHz. Harmonic calls are characterized by one main call element, resembling the complex call, and additional call fragments of different frequencies surrounding the main call. A two-component call consists of two sound components: a main call
component (flat or downward) with an additional punctuated fragment toward the end. An upward call exhibits a continuous increase in pitch that is 12.5 kHz with a terminal dominant frequency at least 6.25 kHz higher than the frequency at the beginning of the vocalization. A downward call exhibits a continuous decrease in pitch that is 12.5 kHz with a terminal dominant frequency at least 6.25 kHz lower than the frequency at the beginning of the vocalization. A flat call displays a constant frequency from the beginning to the end of the call (with changes not more than 3 kHz). A chevron call resembles an inverted-U shape, which is identified by a continuous increase in pitch 12.5 kHz followed by a decrease that is 6.25 kHz. Short calls are punctuated and shorter than 5 ms in duration. Composite calls are formed by two harmonically independent components, emitted simultaneously. Frequency-step calls are characterized by instantaneous frequency changes appearing as a vertically discontinuous “step” on a spectrogram, but with no interruption in time (see Fig. 13.2). The 10 categories described above are used to analyze the call distribution (the relative number of calls emitted within each category), and their probability of occurrence. The experimenter determines the call category of each emitted USV based on the visual analysis of the spectrogram. The development of new techniques for automatic identification of the acoustic features of USVs is warranted for a faster and more detailed analysis of large data sets (Burkett, Day, Penagarikano, Geschwind, & White, 2015; Grimsley, Gadziola, & Wenstrup, 2012). Overall, a quantitative and qualitative characterization, rather than a quantitative count only, helps in better appreciating subtle differences in mouse USV emission. Further studies are needed to investigate the potential biological significance of the differences among the call categories.
VI DEVELOPMENTAL CHANGES OF MOUSE PUP USVs FEATURES The structural features of sound production that occur during the first two postnatal weeks have been extensively examined (Ehret, 1980; Grimsley, Monaghan, & Wenstrup, 2011; Noirot, 1966, 1968; Noirot & Pye, 1969). The increase in call rate during the first days of life is primarily due to a higher number of bouts produced, whereas its subsequent decline is due to a lower number of USVs within each bout. The reduction with age of the interval between calls (the inter-call interval) has been hypothesized to reflect an increased lung capacity allowing a higher emission rate (Elwood & Keeling, 1982). The study of call categories within a bout has shown that ultrasonic patterns vary extensively over development
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VII STRAIN AND GENOTYPE DIFFERENCES IN MOUSE PUP USVs
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FIG. 13.2 Typical sonograms of USVs of mouse pups (C57BL/6J strain) illustrating five distinct call categories. From left to right: USV representing the short, chevron, complex, two-components, and frequency-steps call categories. Time (in ms) is indicated on the x-axis, frequency (in kHz) is indicated on the y-axis, and relative intensity or loudness is indicated by color (see color-coded bar on the right side of the figure).
with old pups emitting a larger vocal repertoire compared to young ones. Usually all neonates start repeatedly emitting the same vocal component. However, when they are able to produce more complex vocal sequences, each pup will have a unique and individual ultrasonic profile different from those of littermates. An interesting example of developmental changes is the increased proportion of harmonic calls over the initial days (Grimsley et al., 2011). This phenomenon could be interpreted as extremely advantageous for pups under isolation from the evolutionary standpoint. First, harmonic calls, as broadband sounds, are easier to locate than tonal sounds. Second, because harmonic calls are characterized by nonlinear components, they may convey negative emotional states that are appropriate to isolation condition. Taken together, these considerations support the hypothesis that harmonic calls may enhance the probability of isolated pups to be found and retrieved by the mother. In addition, changes in the spectral and temporal parameters of the mouse pup USVs during the first postnatal weeks have been studied in detail. Even though sound frequency usually increases with age, resulting in a higher peak frequency that is above the dominant frequency of adult calls, and call duration declines, it has been reported that each call type can show a specific pattern of developmental changes (Ey et al., 2013; Hahn et al., 1998; Liu, Miller, Merzenich, & Schreiner, 2003; Motomura et al., 2002; Noirot & Pye, 1969; Scattoni et al., 2008). On one hand, accurate analysis suggests that developmental changes that are reflected in the acoustic features and vocal repertoire may have a highly communicative value because they may be used by the mother to recognize pups on the basis of their age. On the other, the age-
dependent changes in neonatal ultrasonic emissions may be related to the maturation of vocal production mechanisms or to the increased motor control, even though the association between changes in physiological mechanisms and USV acoustic features is not yet clear. Therefore, additional studies are needed to determine how these changes occur during early postnatal life.
VII STRAIN AND GENOTYPE DIFFERENCES IN MOUSE PUP USVs The production of isolation calls strongly varies with the mouse strain and its genotype (Bell, Nitschke, & Zachman, 1972; D’Udine, Robinson, & Oliverio, 1982; W€ ohr et al., 2008). The knowledge that USVs have a robust genetic basis has come from a large number of studies that consistently characterized ultrasonic patterns of approximately 20 different laboratory mouse strains. Interestingly, clear differences have been found even among closely related mouse strains (Hahn & Lavooy, 2005). The strain differences concern the call rate as well as other spectrographic USV characteristics (Hahn et al., 1998; Hahn, Hewitt, Adams, & Tully, 1987; Hahn, Hewitt, Schanz, Weinreb, & Henry, 1997; Hahn & Schanz, 2002; Nitschke et al., 1972; Thornton et al., 2005). For instance, NZB and C57BL/6J mouse strains emitted a very low number of USVs compared to other inbred mice, such as BALB/c, DBA, A/J, BTBR, and FVB/NJ. The analysis of duration, sound frequency, and bandwidth measured for each pup call showed that these parameters differ among three strains— 129SV, C57BL6, and BALB/c—with the highest fluctuation of values estimated during the second postnatal
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week (Wiaderkiewicz, Glowacka, Grabowska, & Jaroslaw-Jerzy, 2013). Some years ago, Scattoni and coworkers carried on a detailed spectrographic analysis, based on the abovedescribed ultrasonic categorization, of four different strains of mouse pups (BTBR, C57BL/6J, 129X1 and FVB/NJ), classifying every call emitted by 8-day-old pups. In particular, they found that pups belonging to the C57BL/6J, FVB/NJ, and 129X1 strains produced a wide repertoire of calls, which included high numbers of frequency steps and complex USVs. By contrast, pups belonging to the BTBR strain, a potential mouse model of ASD, emitted a narrower repertoire of calls that included high levels of harmonic and composite USVs but a minimal number of other shape USVs (Scattoni et al., 2008). Moreover, as the spectrographic characterization of the 8-day-old CD-1 outbred mice was compared to results obtained with C57BL/6J, 129X1, and FVB/NJ strains, CD-1 pups emitted a higher percentage of frequency steps and complex USVs but low numbers of flat, short, and complex calls. Finally, the ontogenetic profile of USV emission has also been found to be strain-dependent (Branchi, Santucci, & Alleva, 2006; Scattoni et al., 2008). For instance, C57BL/6 and BALB/c strains showed a peak in the calling rate early, around postnatal day 3 (D’Udine et al., 1982), while other inbred (BTBR, FVB/ NJ, 129X1, DBA, A/J, SEC, C3H) or outbred (CD-1) strains showed a peak around postnatal day 7 (Branchi et al., 1998; Elwood & Keeling, 1982; Hennessy et al., 1980). Over the last 15 years, the definitive role of genetic differences in USV emission among mice of different strains has been corroborated by a large number of studies carried out on knock-out and other genetically modified mice, showing that deletion or insertion of selected genes markedly modifies USV production. In particular, it has been found that genes affecting USVs encode a broad spectrum of molecular signals, acting in complex pathways involved in the correct maturation and function of the central nervous system. Their mutations could be responsible for the onset of such neurodevelopmental disorders as ASD (Ey et al., 2013; Romano, Michetti, Caruso, Laviola, & Scattoni, 2013; Roy, Watkins, & ohr, 2016; Young, Heck, 2012; Sungur, Schwarting, & W€ Schenk, Yang, Jan, & Jan, 2010). Both calling rate and spectrographic differences in the vocal repertoire of animal models of ASD have been interpreted as developmental alterations within the social/communication domain (see Chapter 42 in this book). In summary, data from several mouse strains and genetically modified lines show that genetic factors have great influence on USV emission, producing a multitude of effects on both quantitative and qualitative characteristics of calls.
VIII EFFECTS OF PHARMACOLOGICAL COMPOUNDS ON PRODUCTION OF MOUSE PUP USVs The effect of many compounds on pup USV emission has been thoroughly investigated (Cuomo, De Salvia, Maselli, Santo, & Cagiano, 1987; Laviola, Adriani, Gaudino, Marino, & Keller, 2006). In particular, it has been shown that pharmacological treatments associated with the modulation of the main neurotransmitter systems in the central nervous system clearly affected USV signaling (Benton & Nastiti, 1988; Dastur, McGregor, & Brown, 1999; Hodgson, Guthrie, & Varty, 2008; Insel, Hill, & Mayor, 1986; McGregor, Dastur, McLellan, & Brown, 1996; Takahashi et al., 2009; Varga, Fodor, Klausz, & Zelena, 2015; Vivian, Barros, Manitiu, & Miczek, 1997). Great attention has been paid to the GABAergic and glutamatergic systems that are strongly implicated in the modulating rate and features of USVs emitted by isolated mouse pups. For instance, benzodiazepines and other agonists of GABAA receptors reduced neonatal calls while inverse agonists enhance them (Fish, Sekinda, Ferrari, Dirks, & Miczek, 2000; Nastiti, Benton, & Brain, 1991; Rowlett, Tornatzky, Cook, Ma, & Miczek, 2001). Interestingly, ethanol exposure, having anxiolytic properties similar to benzodiazepines, also caused a reduction of the USV rate (Barron & Gilbertson, 2005). A detailed sonographic analysis has shown that ethanol produced a rather selective reduction in specific waveforms (rising frequency, U-shaped, and 3-sweep calls) (Wellmann, George, Brnouti, & Mooney, 2015), instead of affecting the overall production of USVs. Glutamatergic drugs, such as NMDA receptor antagonists, have a bidirectional effect on USV emission in a dose-dependent fashion, that is, the number of USVs was enhanced after moderate glutamate doses and reduced after high doses (Swanson et al., 2005; Takahashi et al., 2009). Similarly to the glutamatergic system, the serotonergic system also had a bidirectional effect on USV production dependent on the type of 5-HT receptor stimulated (Nastiti et al., 1991; W€ ohr et al., 2015). As to the dopaminergic modulation, agonists of D1, D2, and D3 receptors have an important role in reducing USV production (Dastur et al., 1999). Moreover, prenatal cocaine exposure, a potent dopamine reuptake inhibitor, decreases the number of USVs emitted as well as modifies the spectrotemporal USV parameters (Cox et al., 2012; McMurray et al., 2013). Because pharmacological studies have proven that the emission of USVs can be affected by anxiolytic and anxiogenic drugs, it is assumed that neonatal USV production in response to separation from the nest environment reflects a state of anxiety and thus is considered as a valid tool to test emotional states postnatally.
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REFERENCES
IX CONCLUSIONS AND POTENTIAL FUTURE DIRECTIONS Mice emit USVs throughout their lives, especially during early postnatal days upon separation from the nest. Interestingly, the analysis of this vocal response permits us to study emotional development and responsiveness to environmental signals in the newborn as well as the communication between the pup and mother. Recently, the growing literature on neonatal ultrasonic emission has suggested that the ontogenetic profile represents an interesting tool to phenotype mouse lines modeling neurodevelopmental disorders, in which socio-communicative deficits are one of the core symptoms. Although animal models cannot be compared to the complexity of human language, the study of vocal communication in mouse pups could provide valuable insight into the genetic basis of communication and speech disorders. The quantitative and qualitative approaches allow comparing vocal repertoires emitted by pups under different conditions over the course of development and by pups of different strains or genotypes. A USV profile strongly changes within the first two postnatal weeks and appears extremely different from the adult repertoire. This result opens the controversial question on the innate nature of USVs emitted by rodent pups and on the possibility of analyzing them according to the same approach exploited for birds, in which chicks learn vocalization patterns through experience (Arriaga & Jarvis, 2013; Hammerschmidt, Whelan, Eichele, & Fischer, 2015). Studies in which USVs are emitted by juvenile mice could help to understand the vocal changes during developmental stages, filling the gap between pup and adult repertoires. Moreover, it is interesting to speculate that the sets of USV categories generated by each strain may represent specific “dialects.” Further studies using a wider number of mice inbred and outbred strains or genetically modified lines are warranted to make definitive statements about strain or genotype variations in complex vocal production. There are still important limitations in the qualitative characteristics of USVs by means of the instruments for USV investigation currently available. Nowadays, the most used approach is based on observation of USV features by the experimenter in order to classify each call into the different categories. This approach is error-prone and leads to poor reproducibility. In order to circumvent these limitations, efforts are focused on developing sophisticated software based on automated algorithms, which permit a faster and proper identification of salient features of mouse USVs. These automatic recognition systems could be useful to the scientific community as they can standardize USV analysis across laboratories and can potentially allow a detailed analysis aimed at increasing the translational value of the study of USVs emitted by mouse pups.
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E. ULTRASONIC COMMUNICATION OF INFANT RODENTS