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Ultrasonic vocalizations emitted during dyadic interactions in female mice: A possible index of sociability?
Behavioural Brain Research 182 (2007) 223–230
Review
Ultrasonic vocalizations emitted during dyadic interactions in female mice: A possible index of sociability? Anna Moles ∗ , Fabrizio Costantini, Luciana Garbugino, Claudio Zanettini, Francesca R. D’Amato ∗ Institute of Neuroscience, CNR-CERC, Via del Fosso di Fiorano 64/65, I-00143 Rome, Italy Received 3 October 2006; received in revised form 16 January 2007; accepted 23 January 2007 Available online 31 January 2007
Abstract Despite the evidence that ultrasonic vocalizations are a consistent component of the behavioural repertoire of female mice, only few studies have investigated this phenomenon. In this paper, we reported new data about ultrasonic vocalisations emitted during female–female mice social encounters. In particular, we first showed that the resident female utters a considerable number of 70 kHz calls and that the number of calls seems to be modulated by the motivational state of the emitter during the estrous cycle: sexually receptive females emitted fewer ultrasonic vocalizations than non-receptive ones in the presence of a female intruder. A strong positive correlation linked the number of calls and the time spent by the resident sniffing the intruder female. Moreover, the number of calls uttered during interaction with an unknown female partner significantly decreased with pregnancy and ageing. Secondly we reported that 1-year-old female mice showed a reduction of ultrasonic calls in the presence of a partner they had been exposed to, only if the re-exposure (test) occurred 30 min after the previous presentation. If the test was performed with a delay of 60 min, the number of calls emitted did not decrease. These results confirm that ultrasonic vocalizations emitted during social interaction with a female conspecific can be used as an index of social recognition and can be useful to detect age-related disruption of social memory in female mice. © 2007 Elsevier B.V. All rights reserved. Keywords: Ultrasonic vocalizations; Social interactions; Estrous; Diestrous; Pregnancy ageing; Social recognition; Female mice
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Estrus cycle determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Mating procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Behavioral tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Social interaction test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Social recognition test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Behavioral measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.4. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. UVs during social interaction test and resident female estrous cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. UVs during social interaction test and resident female reproductive condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. UVs during social interaction test and resident female age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. UVs and social recognition test in elderly resident females . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding authors. Tel.: +39 06 501703275/6; fax: +39 06 501703331. E-mail addresses: [email protected] (A. Moles), [email protected] (F.R. D’Amato).
0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.01.020
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Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Ultrasonic vocalizations (UVs) emitted by adult mice for long time have been considered as a component of the sexual behavioral repertoire of males [42]. Sewell [43] was the first to detect UVs also during social interaction in pairs of female mice, especially when they were engaged in olfactory investigation of the other. Subsequently, sporadic studies reporting ultrasonic calls in female mice [15,16,45] were published until Maggio and Whitney [29] demonstrated that female mice of different genotypes emitted a large number of UVs during encounters with other females, at rates comparable to those of the males. Despite this convincing study, the opinion persisted that mouse UVs were a chiefly male trait [52]. In the last few years, we have started a line of research aimed at understanding the occurrence and function of UVs in female mice. In particular, using the resident/intruder experimental paradigm, we have demonstrated that the resident female emitted a great number of ultrasonic calls when a same sex partner was introduced in the cage [33]. The ultrasounds recorded were emitted by the resident animal, as demonstrated by several experiments performed with either anesthetized resident or intruder [12,29]. As recently confirmed in BALB/c mice, ultrasonic vocalizations recorded during female–female encounters ranged from 50 to 70 kHz [23] and are emitted during the first minutes of social interaction. This is in line with data previously reported in female [29], as well as in male mice [42] showing that the majority of vocalizations can be detected in concomitance with high levels of social investigation. Olfactory investigation is very important for the individual identification, allowing animals to gain information on the features (i.e. sex, rank and reproductive state) of a conspecific [7]. We have investigated the emission of UVs during social recognition. Individual recognition of conspecifics, is a crucial prerequisite for a wide range of social behaviors such as the development of bonds and the establishment of hierarchies that limit aggressive interactions and allow group living [24]. According to its original design, the social recognition/social discrimination test was based on the natural propensity of a mouse to investigate another mouse introduced into its home cage [14,17]. If a different, novel, conspecific was presented, the initial level of social investigation could be reinstated in the resident mouse (dishabituation). We explored the possibility that the observed decrease of ultrasonic vocalizations detected during the re-exposure to the same intruder could have been the consequence of the reduction in social interest of the resident towards the already familiar animal. Thus, we hypothesized that this reduction in UVs could be used as a measure of social recognition [12]. Indeed, when re-exposed to the same partner, there was a decline in the number of UVs emitted by resident mice when 15, 30, or 60 min passed elapsed between the two social
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sessions. After 24 h, this effect disappeared. In contrast, when the resident was exposed to a novel female partner, the number of UVs remained unchanged. Interestingly, female mice treated with the classic cholinergic antagonist scopolamine when reexposed to the familiar conspecific after 30 min from the first social exposure did not show the expected decrease in UVs. Scopolamine seemed specifically disrupting the social memory process and not the UVs emission per se. Indeed, at the same dose this drug did not affect the UVs of female mice exposed to an unfamiliar partner. We thus have provided behavioral and pharmacological evidences that ultrasonic calls could be used as an index of social memory in female mice. Moreover, we proved that this parameter was more sensitive than the measures of social investigation or social contacts as we did not find any significant decrease in these parameters. This test has been recently used in a neonatal neurotoxicological study in outbred CD-1 female mice allowing to detect treatment-induced differences in baseline levels of UVs, but not in the recognition capability, since all females showed the expected decrease in UVs when encountering the familiar partner [51]. In mice and rats, olfactory cues coming from conspecifics are also useful to acquire information about the food they have eaten [20,21,47]. This kind of phenomenon provides an experimental context suitable to study the ultrasonic vocalisation in parallel to the olfactory investigation. Interestingly, we found that the resident/observer female emitted a great number of ultrasonic calls during the first minute of social interaction, when the intruder had previously fed on a palatable food [33]. By contrast, fewer UVs were associated with partners fed on unpalatable food. These calls were associated with resident’s intense olfactory investigation of the partner. Data reviewed above strongly suggest that ultrasonic vocalizations are an important component of the female mice behavioural repertoire. Indeed, ultrasonic emission is a consistent and robust phenomenon during social interaction in females of this species that can be also “exploited” as an index of social interest in these animals. As already hypothesized for male calls during sexual interactions [38], these vocalizations seem to have an affiliative function facilitating proximity. This proximity is a pre-requisite for the acquisition of relevant information about the intruder by the resident female. To better characterize this aspect of the female behavioral repertoire neglected so far, in the first part of the present paper we will report a series of experiment showing variations of UVs in female mice according to estrous status, reproductive condition and age. As for aging, rodent models have been used successfully to study the behavioral and neurophysiological changes associated with cognitive changes [22], but the majority of studies have focused on males. However, as males tend to perform better than females in spatial performance tests in general [5], it is important to set up behavioural tests not involving spatial
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navigation in order to investigate aging cognitive decline in female mice. Being the social recognition test based on UVs specifically designed for females [12], in the second part of this paper, we will expand our previous report on UVs and memory, testing the hypothesis that ultrasonic vocalizations can be used as a useful parameter to investigate age associated deficits in memory in female mice. 2. Methods 2.1. Subjects Outbred albino NMRI mice were used in this study. They were purchased from Harlan, Italy, at about 45 days of age (weight 23–25 g) and housed in group of 4 in Plexiglas cages (40 cm × 23 cm × 15 cm) with sawdust on the floor and water and food available ad libitum. They were maintained on a 12 h light–dark cycle (light on at 7:00) at least for 2 weeks before animal testing. Room temperature was kept at 21 ± 1 ◦ C.
2.2. Estrus cycle determination At the end of the 3 min social interaction session, 40 resident females were checked for the vaginal estrous condition. The four stages (proestrous, estrous, metaestrous and diestrous) of estrous cycle were determined by examining the proportion and morphology of leukocytes and epithelial cells present in the smear under 400× magnification of a light microscope.
2.3. Mating procedure Pairs of females were moved in the cage of an adult male (day 0) and left there for 48 h. Thereafter the male was removed and the two females were left in this “pregnancy cage”. On pregnancy days 3 (±1), 10 (±1) and 17 (±1) females were isolated for 24 h and underwent the social interaction test. Unfamiliar female were used as intruders in order to never re-expose the resident to the same partner. To avoid social isolation, females were returned in their pregnancy cage with their female mate, after each test session. Ten females out of 36 were pregnant and successfully delivered their pups. The remaining 26 were used as non-pregnant controls.
2.4. Behavioral tests 2.4.1. Social interaction test Female subjects were individually housed in clean cages for 1–3 days before the experimental session and served as resident. Other females were left in social group of 4, individually marked, and served as intruders. Intruders were always 3–4 months old. On the day of test, residents and intruders were moved to a soundproof cabin provided with video and ultrasounds recording systems. A female intruder (partner) was introduced into the cage of the resident for three minutes. 2.4.2. Social recognition test Twelve-month-old female mice were individually housed in clean cages for 3 days and served as residents. The next day a female partner was introduced for 3 min into the home cage of the isolated resident (pretest). At the end of this session, the partner was returned into its home cage. After 30 or 60 min from the pretest, the resident was exposed to either the same or a different partner, for 3 min (test) (see Fig. 1). Same and different mice had already been exposed once to a conspecific so that they would represent comparable stimuli for the experimental residents. In the different condition, partners used for the pretest and test sessions were never drawn from the same social group. Because of the high interindividual variability in the ultrasound emission, the experimental groups were formed on the basis of the number of UVs emitted in the pretest UVs count. In this way, we obtained groups that were comparable with regard to the amount of UVs emitted in the pretest. Mice that did not vocalize in the pretest or test session (around 3%) were not included in the analysis.
Fig. 1. Schematic representation of the social recognition test in 1-year-old female mice. Ultrasonic vocalizations (UVs) emitted by the resident female are recorded during the three sessions (test, pretest same, pretest different). Modified from Ricceri et al. [39]. 2.4.3. Behavioral measures The number of UVs emitted during the 3 min social interaction was measured by using the Ultravox 2.0 (Noldus Information Technology, The Netherlands) set to a frequency of 70 kHz (±4) connected to a computer. The behavior of resident mice in their home cage during the 3 min of social interaction was videotaped. Later, an experimenter unaware of the experimental design recorded the behavioral parameters by the focal sample method [1], using a keyboard connected to a computer. The Observer software (Version 1.0, Noldus Information Technology, Wageningen, the Netherlands) was used for data acquisition. Time (s) spent in the following behavioral activities was used for the analysis of resident behavioral profile [49]: social investigation (sniffing any part of the partner’s body), social grooming (grooming the partner), cage exploration (sniffing the physical environment, rearing), self-grooming (lick and scratch own body fur), and mounts (all attempts to mount the partner). 2.4.4. Statistical analysis To meet the normality criteria, the number of ultrasounds recorded during the 3 min social interaction test was square-root transformed. Ultrasonic vocalizations and the time spent in each behavior by resident female mice during the 3 min social interaction were analyzed by ANOVA or t-test. Student’s t-test with the Bonferroni’s correction or Tukey HSD was used as post hoc as appropriate.
3. Results 3.1. UVs during social interaction test and resident female estrous cycles The correlation analysis indicated a significant positive relation between the number of UVs and the time spent by the resident in social investigation (r = 0.35, n = 39, p < 0.05). A negative correlation was found between UVs and cage exploration by the resident animal (r = −0.34, n = 39, p < 0.05) with no other significant correlations detected. The time spent in social investigation of the partner and in cage exploration were affected by the estrous phase (social investigation: F3,35 = 4.05, p = 0.01; cage exploration: F3,35 = 3.72, p < 0.05), whereas the number of ultrasounds recorded tended to differ among the four stages of the estrous cycle (UVs: F3,35 = 2.56, p = 0.07) (Fig. 2). Sexual receptivity in the mouse is primarily promoted by estradiol that peaks in proestrous, but progesterone, peaking late in proestrous, may enhance this effect, being important
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Fig. 2. Square root (SQRT) transformed number (mean ± S.E., upper panel) of ultrasonic vocalisations (UVs) emitted by proestrous (pro), estrous (est), metaestrous (met) and diestrous (di) resident female mice and time (mean ± S.E.) spent by these females investigating the same sex unknown intruder or exploring the cage (lower panel). * p < 0.01 Student’s t-test with Bonferroni correction: receptive (pro + est) vs. non-receptive (met + di).
in terminating receptivity [6]. Considering thus a behavioural receptive (proestrous + estrous) and non-receptive phase (diestrous + metaestrous), significant differences emerged for all behavioural parameters (Student’s t-tests with Bonferroni correction, p ≤ 0.01). These results suggest that sexually receptive resident females are less interested in same sex intruders, in comparison to non-receptive ones. 3.2. UVs during social interaction test and resident female reproductive condition Overall, pregnancy reduced social investigation of female mice (main effect: F1,34 = 7.10, p = 0.01). Fig. 3 showed that, while non-pregnant mice showed the same amount of social investigation toward the unfamiliar females across time, females on pregnancy days 10 and 17 spent less time in social investigation compared to the non-pregnant mice (p < 0.05, Tukey HSD, Fig. 3 lower panel) and concomitantly were more engaged in cage exploration (pregnancy × time F3,102 = 6.12; p < 0.001, data not shown) This reduction in the social interest was paralleled by a decrease in the amount of UVs emitted by pregnant residents (pregnancy × time F3,102 = 5.93; p < 0.0001: Fig. 3, upper panel) that was significantly lower in comparison to nonpregnant mice both on days 10 and 17 of pregnancy (p < 0.05, Tukey HSD). Before mating occurred (day 4) no behavioural differences were found between the two groups of animals (mice that became pregnant and animals that did not). No differences were found for the other behavioural parameters recorded.
3.3. UVs during social interaction test and resident female age Ageing was associated with a reduction in the social investigation of a female intruder (t67 = 4.19, p < 0.0001: Fig. 4, lower panel), as well as with a decrease in ultrasonic vocalisations (t67 = 3.23, p < 0.01: Fig. 4, upper panel). Even when the time residents engaged in social behaviors was reduced by ageing, a significant positive correlation was found between social investigation and the number of UVs emitted (r = 0.355, n = 34, p < 0.05). 3.4. UVs and social recognition test in elderly resident females When these females were exposed to the same or to a different partner (Fig. 5) there was a general decrease in the UVs emitted (F1,40 = 9.53; p < 0.01), regardless the time interval between exposures (30 or 60 min: F1,40 = 0.36, ns) and the partner they have been exposed to (same or different: F1,40 = 0.60, ns). However, post hoc analysis revealed that aged female mice vocalized significantly less if re-exposed to the same female only when the interval between the pre- and test session was 30 min (Tukey HSD, p < 0.05). By contrast, in this group no significant decrease in time spent in social investigation in the presence of the same or a different was observed (partner: F1,12 = 0.25, ns; pretest versus test: F1,12 = 1.73, ns; interaction effect: F1,12 = 0.10, ns; same female: pretest versus test, Tukey HSD, ns). These results
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Fig. 3. Square root (SQRT) transformed number (mean ± S.E., upper panel) of ultrasonic vocalisations (UVs) emitted by resident females and time (mean ± S.E.) spent investigating same sex unknown intruders (lower panel) before mating and at different pregnancy days (PD). * p < 0.05 same day; p < 0.05 Tukey HSD vs. pre-mating.
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Fig. 4. Square root (SQRT) transformed number (mean ± S.E., upper panel) of ultrasonic vocalisations (UVs) emitted by 3–6 months and 12-month-old resident females and time (mean ± S.E.) spent investigating same sex unknown intruders (lower panel). * p < 0.01, Student’s t-test.
suggest that the number of UVs emitted during re-exposure to a familiar same sex mouse can be used as an index of agedependent impairments in social memory. 4. Discussion In the present study we confirmed that during female–female mice social encounters the resident animal utters a considerable number of 70 kHz calls. Moreover, the number of calls seems to be modulated by the estrous phase of the emitter as non-sexually receptive females emit more UVs than receptive ones. The number of calls uttered during interaction with an unknown female partner significantly decreases with pregnancy and ageing. In addition we showed that ultrasounds could be used to assess social memory in ageing female mice. In spite of the consistency of reports documenting the occurrence of ultrasonic vocalizations in female pairs, the function of these calls in female mice is still poorly understood. Some authors have suggested that female calls can act to establish female dominance [29,54]; others proposed that the primary function of 70 kHz vocalizations is “to announce the presence of another mouse and to attract other mice” [3]. UVs play an important role in regulating reproductive behavior in adult mice. Males emit 70 kHz vocalizations almost continuously prior to first mount, while they actively sniff and investigate the female. In this context, UVs are though to be important to maintain the
Fig. 5. Square root (SQRT) transformed number (mean ± S.E.) of ultrasonic vocalisations (UVs) emitted by resident female mice when exposed to the same or a different partner for the 3 min pretest and test sessions, after 30 or 60 min. * p < 0.05 Tukey HSD: test vs. pretest session.
female close to the vocalizing male rather than to attract her from a distance [38]. We suggest that also in female mice these calls facilitate proximity between animals in order to help the resident to acquire relevant social information on the intruder. Both the infantile origin of this behavior and the lack of UVs during agonistic male–male interaction [42,53] support the view that UVs
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calls are an affiliative, rather than an agonistic signal, and these vocalizations seem compatible with the category of contact calls termed “approaching in a friendly manner” recorded in other mammalian species [35]. From the rat literature there is evidence that ultrasonic vocalizations can be associated with the animal’s motivational state. In particular it has been suggested that 50 and 22 kHz calls are associated to appetitive and aversive states in the rat, respectively [27]. In particular, 50 kHz ultrasounds have been postulated to reflect positive affective states in rats, since they correlate with approach behavior during courtship [32] and rough-and-tumble play [26] and social contact [8]. Whether or not 70 kHz have a similar motivational background in mice and can be a useful index of social motivation or sociability in adult female mice is a relevant question. To answer to this question, we used an experimental setting in our previous study [33] in which we made the “intruder” animal more or less attractive by changing the salience of the information it “carried”. The motivational state (food deprivation condition) strongly modulated the number of calls. In contrast, the quality of food eaten by the demonstrators affected both the behavior and the number of calls emitted by the observers. We concluded that the motivational state of the observer mouse affected the UVs release during the interaction with a demonstrator conspecific. In fact, nondeprived animals called more towards demonstrators fed on palatable food, whereas food-deprived subjects vocalized more to fed conspecifics, in comparison to non-fed ones, independently of the palatability of the food eaten. We suggest that the more the intruder was “attractive” the more UVs were uttered. Thus, the number of UVs reflects the “motivational properties” of the stimulus animal. Other data confirm the rewarding properties of social contacts in male and female mice [13,31]. For instance, in an operant task, socially isolated females responded faster for access to a female partner than the group-housed ones [31]. Moreover, the response rate seemed to be higher in female in diestrous in comparison to those with estradiol replacement. If the number of UVs is the reflection of the social motivation, this is in line with the decrease in UVs we found in sexually receptive females compared to nonreceptive ones. By contrast, our data are not in agreement with those reported by Tang et al. [46] showing that long-term estrogen replacement increased the interest in social interaction in C57BL/6 female mice. However, methodological differences such as the pharmacological induction of the estrous phase, test cage (home cage versus neutral cage) could explain this discrepancy. One further, albeit indirect, observation supporting the hypothesis that UVs can reflect the motivation to socially interact is the decrease in social investigation and UVs shown by female mice during the second half of pregnancy as compared to their non-pregnant controls. Indeed in non-human primates the late pregnancy/early lactation periods have been associated to a trend to withdraw from social life [2,28]. Likely, the biological basis of this behavior is the pregnancy-induced change in the steroids hormonal profile and peptide levels, including endogenous opioids peptides [40]. In a wide range of species it has been shown that opiates agonists disrupts different kinds of social behaviors ([4,36] for review). Moreover opioid ago-
nists administration decreases the emission of distress calls in rat pups [9] and knockout mice for the mu receptor genes emitted less UVs when separated from their mothers [34]. Due to the massive changes in the hormonal profile of pregnant animals, multiple factors are likely to be involved in the observed changes in social behavior. However, it is tempting to speculate that one of the factors contributing to the observed decrease in social interaction and the concomitant reduction in ultrasonic vocalizations is the drop of social motivation associated to the pregnancy-induced increase in beta-endorphin levels. In a wide variety of primate species, aging also leads to a withdrawal from social interactions and an increase in time spent alone; this social withdrawal is not a passive process, caused by a reduction in general activity, but rather an active avoidance of other group members [25,37,48,50]. Studies specifically exploring age-related changes in sociability of rodents are lacking. However, in a social interaction with age matched same sex conspecifics tested in a neutral arena, aged rats spent considerably less time in active social interaction than young rats [41]. Furthermore, in 13 months old male mice it has been shown a decrease in social investigation before agonistic interaction during male–male confrontation [18]. A drop in social motivation can account also for the decrease in the number of UVs and social investigation we found in 1-year-old female mice compared to 3-month-old ones. Mice, as well as rats, are macrosmates for which storage and recall of information acquired by olfaction is a prerequisite for a successful interaction with the living and nonliving world [45]. This implies that studies investigating the processing of such information provide a privileged access to learning and memory in these species. Recent studies have proposed that in female mice the process of social recognition begins with individual specific odor compounds that are detected respectively by the olfactory system. These, through synapses in the olfactory bulb, reach the amygdala where oxitocyin (OT) receptors mediate the facilitating OT effects on social recognition. In turn, OT mediation of social recognition itself is regulated by ovarian circulating estrogens [10,11]. Since age-related mnemonic decline in female rats [30] and mice [19] coincides with the loss of regular estrous cycling, this task seems particularly suitable to explore in animal models memory functions during the onset of reproductive senescence [44]. Ultrasounds uttered during the social recognition test represent an additional and more reliable behavioral parameter in adult [12] as well as in ageing female mice, as shown in this paper. The study of social behavior in laboratory animals is crucial for the understanding of biochemical, genetic and environmental factors underlying psychiatric disorders such as autism, schizophrenia and depression, characterized by deficits in social behavior and decrease of the desire to engage in social interaction [39]. However the translational value of animal models of human conditions is strictly correlated with the development of laboratory test focusing not only on the quantitative but also on the qualitative nature of social interaction. We think that the recording and possibly spectrographic analysis of the ultrasonic calls in mice can bring new information on motivational aspects underlying social behavior and subjective states related to social
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Review
Ultrasonic vocalizations emitted during dyadic interactions in female mice: A possible index of sociability? Anna Moles ∗ , Fabrizio Costantini, Luciana Garbugino, Claudio Zanettini, Francesca R. D’Amato ∗ Institute of Neuroscience, CNR-CERC, Via del Fosso di Fiorano 64/65, I-00143 Rome, Italy Received 3 October 2006; received in revised form 16 January 2007; accepted 23 January 2007 Available online 31 January 2007
Abstract Despite the evidence that ultrasonic vocalizations are a consistent component of the behavioural repertoire of female mice, only few studies have investigated this phenomenon. In this paper, we reported new data about ultrasonic vocalisations emitted during female–female mice social encounters. In particular, we first showed that the resident female utters a considerable number of 70 kHz calls and that the number of calls seems to be modulated by the motivational state of the emitter during the estrous cycle: sexually receptive females emitted fewer ultrasonic vocalizations than non-receptive ones in the presence of a female intruder. A strong positive correlation linked the number of calls and the time spent by the resident sniffing the intruder female. Moreover, the number of calls uttered during interaction with an unknown female partner significantly decreased with pregnancy and ageing. Secondly we reported that 1-year-old female mice showed a reduction of ultrasonic calls in the presence of a partner they had been exposed to, only if the re-exposure (test) occurred 30 min after the previous presentation. If the test was performed with a delay of 60 min, the number of calls emitted did not decrease. These results confirm that ultrasonic vocalizations emitted during social interaction with a female conspecific can be used as an index of social recognition and can be useful to detect age-related disruption of social memory in female mice. © 2007 Elsevier B.V. All rights reserved. Keywords: Ultrasonic vocalizations; Social interactions; Estrous; Diestrous; Pregnancy ageing; Social recognition; Female mice
Contents 1. 2.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Estrus cycle determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Mating procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Behavioral tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Social interaction test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Social recognition test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Behavioral measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.4. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. UVs during social interaction test and resident female estrous cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. UVs during social interaction test and resident female reproductive condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. UVs during social interaction test and resident female age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. UVs and social recognition test in elderly resident females . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding authors. Tel.: +39 06 501703275/6; fax: +39 06 501703331. E-mail addresses: [email protected] (A. Moles), [email protected] (F.R. D’Amato).
0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.01.020
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Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Ultrasonic vocalizations (UVs) emitted by adult mice for long time have been considered as a component of the sexual behavioral repertoire of males [42]. Sewell [43] was the first to detect UVs also during social interaction in pairs of female mice, especially when they were engaged in olfactory investigation of the other. Subsequently, sporadic studies reporting ultrasonic calls in female mice [15,16,45] were published until Maggio and Whitney [29] demonstrated that female mice of different genotypes emitted a large number of UVs during encounters with other females, at rates comparable to those of the males. Despite this convincing study, the opinion persisted that mouse UVs were a chiefly male trait [52]. In the last few years, we have started a line of research aimed at understanding the occurrence and function of UVs in female mice. In particular, using the resident/intruder experimental paradigm, we have demonstrated that the resident female emitted a great number of ultrasonic calls when a same sex partner was introduced in the cage [33]. The ultrasounds recorded were emitted by the resident animal, as demonstrated by several experiments performed with either anesthetized resident or intruder [12,29]. As recently confirmed in BALB/c mice, ultrasonic vocalizations recorded during female–female encounters ranged from 50 to 70 kHz [23] and are emitted during the first minutes of social interaction. This is in line with data previously reported in female [29], as well as in male mice [42] showing that the majority of vocalizations can be detected in concomitance with high levels of social investigation. Olfactory investigation is very important for the individual identification, allowing animals to gain information on the features (i.e. sex, rank and reproductive state) of a conspecific [7]. We have investigated the emission of UVs during social recognition. Individual recognition of conspecifics, is a crucial prerequisite for a wide range of social behaviors such as the development of bonds and the establishment of hierarchies that limit aggressive interactions and allow group living [24]. According to its original design, the social recognition/social discrimination test was based on the natural propensity of a mouse to investigate another mouse introduced into its home cage [14,17]. If a different, novel, conspecific was presented, the initial level of social investigation could be reinstated in the resident mouse (dishabituation). We explored the possibility that the observed decrease of ultrasonic vocalizations detected during the re-exposure to the same intruder could have been the consequence of the reduction in social interest of the resident towards the already familiar animal. Thus, we hypothesized that this reduction in UVs could be used as a measure of social recognition [12]. Indeed, when re-exposed to the same partner, there was a decline in the number of UVs emitted by resident mice when 15, 30, or 60 min passed elapsed between the two social
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sessions. After 24 h, this effect disappeared. In contrast, when the resident was exposed to a novel female partner, the number of UVs remained unchanged. Interestingly, female mice treated with the classic cholinergic antagonist scopolamine when reexposed to the familiar conspecific after 30 min from the first social exposure did not show the expected decrease in UVs. Scopolamine seemed specifically disrupting the social memory process and not the UVs emission per se. Indeed, at the same dose this drug did not affect the UVs of female mice exposed to an unfamiliar partner. We thus have provided behavioral and pharmacological evidences that ultrasonic calls could be used as an index of social memory in female mice. Moreover, we proved that this parameter was more sensitive than the measures of social investigation or social contacts as we did not find any significant decrease in these parameters. This test has been recently used in a neonatal neurotoxicological study in outbred CD-1 female mice allowing to detect treatment-induced differences in baseline levels of UVs, but not in the recognition capability, since all females showed the expected decrease in UVs when encountering the familiar partner [51]. In mice and rats, olfactory cues coming from conspecifics are also useful to acquire information about the food they have eaten [20,21,47]. This kind of phenomenon provides an experimental context suitable to study the ultrasonic vocalisation in parallel to the olfactory investigation. Interestingly, we found that the resident/observer female emitted a great number of ultrasonic calls during the first minute of social interaction, when the intruder had previously fed on a palatable food [33]. By contrast, fewer UVs were associated with partners fed on unpalatable food. These calls were associated with resident’s intense olfactory investigation of the partner. Data reviewed above strongly suggest that ultrasonic vocalizations are an important component of the female mice behavioural repertoire. Indeed, ultrasonic emission is a consistent and robust phenomenon during social interaction in females of this species that can be also “exploited” as an index of social interest in these animals. As already hypothesized for male calls during sexual interactions [38], these vocalizations seem to have an affiliative function facilitating proximity. This proximity is a pre-requisite for the acquisition of relevant information about the intruder by the resident female. To better characterize this aspect of the female behavioral repertoire neglected so far, in the first part of the present paper we will report a series of experiment showing variations of UVs in female mice according to estrous status, reproductive condition and age. As for aging, rodent models have been used successfully to study the behavioral and neurophysiological changes associated with cognitive changes [22], but the majority of studies have focused on males. However, as males tend to perform better than females in spatial performance tests in general [5], it is important to set up behavioural tests not involving spatial
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navigation in order to investigate aging cognitive decline in female mice. Being the social recognition test based on UVs specifically designed for females [12], in the second part of this paper, we will expand our previous report on UVs and memory, testing the hypothesis that ultrasonic vocalizations can be used as a useful parameter to investigate age associated deficits in memory in female mice. 2. Methods 2.1. Subjects Outbred albino NMRI mice were used in this study. They were purchased from Harlan, Italy, at about 45 days of age (weight 23–25 g) and housed in group of 4 in Plexiglas cages (40 cm × 23 cm × 15 cm) with sawdust on the floor and water and food available ad libitum. They were maintained on a 12 h light–dark cycle (light on at 7:00) at least for 2 weeks before animal testing. Room temperature was kept at 21 ± 1 ◦ C.
2.2. Estrus cycle determination At the end of the 3 min social interaction session, 40 resident females were checked for the vaginal estrous condition. The four stages (proestrous, estrous, metaestrous and diestrous) of estrous cycle were determined by examining the proportion and morphology of leukocytes and epithelial cells present in the smear under 400× magnification of a light microscope.
2.3. Mating procedure Pairs of females were moved in the cage of an adult male (day 0) and left there for 48 h. Thereafter the male was removed and the two females were left in this “pregnancy cage”. On pregnancy days 3 (±1), 10 (±1) and 17 (±1) females were isolated for 24 h and underwent the social interaction test. Unfamiliar female were used as intruders in order to never re-expose the resident to the same partner. To avoid social isolation, females were returned in their pregnancy cage with their female mate, after each test session. Ten females out of 36 were pregnant and successfully delivered their pups. The remaining 26 were used as non-pregnant controls.
2.4. Behavioral tests 2.4.1. Social interaction test Female subjects were individually housed in clean cages for 1–3 days before the experimental session and served as resident. Other females were left in social group of 4, individually marked, and served as intruders. Intruders were always 3–4 months old. On the day of test, residents and intruders were moved to a soundproof cabin provided with video and ultrasounds recording systems. A female intruder (partner) was introduced into the cage of the resident for three minutes. 2.4.2. Social recognition test Twelve-month-old female mice were individually housed in clean cages for 3 days and served as residents. The next day a female partner was introduced for 3 min into the home cage of the isolated resident (pretest). At the end of this session, the partner was returned into its home cage. After 30 or 60 min from the pretest, the resident was exposed to either the same or a different partner, for 3 min (test) (see Fig. 1). Same and different mice had already been exposed once to a conspecific so that they would represent comparable stimuli for the experimental residents. In the different condition, partners used for the pretest and test sessions were never drawn from the same social group. Because of the high interindividual variability in the ultrasound emission, the experimental groups were formed on the basis of the number of UVs emitted in the pretest UVs count. In this way, we obtained groups that were comparable with regard to the amount of UVs emitted in the pretest. Mice that did not vocalize in the pretest or test session (around 3%) were not included in the analysis.
Fig. 1. Schematic representation of the social recognition test in 1-year-old female mice. Ultrasonic vocalizations (UVs) emitted by the resident female are recorded during the three sessions (test, pretest same, pretest different). Modified from Ricceri et al. [39]. 2.4.3. Behavioral measures The number of UVs emitted during the 3 min social interaction was measured by using the Ultravox 2.0 (Noldus Information Technology, The Netherlands) set to a frequency of 70 kHz (±4) connected to a computer. The behavior of resident mice in their home cage during the 3 min of social interaction was videotaped. Later, an experimenter unaware of the experimental design recorded the behavioral parameters by the focal sample method [1], using a keyboard connected to a computer. The Observer software (Version 1.0, Noldus Information Technology, Wageningen, the Netherlands) was used for data acquisition. Time (s) spent in the following behavioral activities was used for the analysis of resident behavioral profile [49]: social investigation (sniffing any part of the partner’s body), social grooming (grooming the partner), cage exploration (sniffing the physical environment, rearing), self-grooming (lick and scratch own body fur), and mounts (all attempts to mount the partner). 2.4.4. Statistical analysis To meet the normality criteria, the number of ultrasounds recorded during the 3 min social interaction test was square-root transformed. Ultrasonic vocalizations and the time spent in each behavior by resident female mice during the 3 min social interaction were analyzed by ANOVA or t-test. Student’s t-test with the Bonferroni’s correction or Tukey HSD was used as post hoc as appropriate.
3. Results 3.1. UVs during social interaction test and resident female estrous cycles The correlation analysis indicated a significant positive relation between the number of UVs and the time spent by the resident in social investigation (r = 0.35, n = 39, p < 0.05). A negative correlation was found between UVs and cage exploration by the resident animal (r = −0.34, n = 39, p < 0.05) with no other significant correlations detected. The time spent in social investigation of the partner and in cage exploration were affected by the estrous phase (social investigation: F3,35 = 4.05, p = 0.01; cage exploration: F3,35 = 3.72, p < 0.05), whereas the number of ultrasounds recorded tended to differ among the four stages of the estrous cycle (UVs: F3,35 = 2.56, p = 0.07) (Fig. 2). Sexual receptivity in the mouse is primarily promoted by estradiol that peaks in proestrous, but progesterone, peaking late in proestrous, may enhance this effect, being important
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Fig. 2. Square root (SQRT) transformed number (mean ± S.E., upper panel) of ultrasonic vocalisations (UVs) emitted by proestrous (pro), estrous (est), metaestrous (met) and diestrous (di) resident female mice and time (mean ± S.E.) spent by these females investigating the same sex unknown intruder or exploring the cage (lower panel). * p < 0.01 Student’s t-test with Bonferroni correction: receptive (pro + est) vs. non-receptive (met + di).
in terminating receptivity [6]. Considering thus a behavioural receptive (proestrous + estrous) and non-receptive phase (diestrous + metaestrous), significant differences emerged for all behavioural parameters (Student’s t-tests with Bonferroni correction, p ≤ 0.01). These results suggest that sexually receptive resident females are less interested in same sex intruders, in comparison to non-receptive ones. 3.2. UVs during social interaction test and resident female reproductive condition Overall, pregnancy reduced social investigation of female mice (main effect: F1,34 = 7.10, p = 0.01). Fig. 3 showed that, while non-pregnant mice showed the same amount of social investigation toward the unfamiliar females across time, females on pregnancy days 10 and 17 spent less time in social investigation compared to the non-pregnant mice (p < 0.05, Tukey HSD, Fig. 3 lower panel) and concomitantly were more engaged in cage exploration (pregnancy × time F3,102 = 6.12; p < 0.001, data not shown) This reduction in the social interest was paralleled by a decrease in the amount of UVs emitted by pregnant residents (pregnancy × time F3,102 = 5.93; p < 0.0001: Fig. 3, upper panel) that was significantly lower in comparison to nonpregnant mice both on days 10 and 17 of pregnancy (p < 0.05, Tukey HSD). Before mating occurred (day 4) no behavioural differences were found between the two groups of animals (mice that became pregnant and animals that did not). No differences were found for the other behavioural parameters recorded.
3.3. UVs during social interaction test and resident female age Ageing was associated with a reduction in the social investigation of a female intruder (t67 = 4.19, p < 0.0001: Fig. 4, lower panel), as well as with a decrease in ultrasonic vocalisations (t67 = 3.23, p < 0.01: Fig. 4, upper panel). Even when the time residents engaged in social behaviors was reduced by ageing, a significant positive correlation was found between social investigation and the number of UVs emitted (r = 0.355, n = 34, p < 0.05). 3.4. UVs and social recognition test in elderly resident females When these females were exposed to the same or to a different partner (Fig. 5) there was a general decrease in the UVs emitted (F1,40 = 9.53; p < 0.01), regardless the time interval between exposures (30 or 60 min: F1,40 = 0.36, ns) and the partner they have been exposed to (same or different: F1,40 = 0.60, ns). However, post hoc analysis revealed that aged female mice vocalized significantly less if re-exposed to the same female only when the interval between the pre- and test session was 30 min (Tukey HSD, p < 0.05). By contrast, in this group no significant decrease in time spent in social investigation in the presence of the same or a different was observed (partner: F1,12 = 0.25, ns; pretest versus test: F1,12 = 1.73, ns; interaction effect: F1,12 = 0.10, ns; same female: pretest versus test, Tukey HSD, ns). These results
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Fig. 3. Square root (SQRT) transformed number (mean ± S.E., upper panel) of ultrasonic vocalisations (UVs) emitted by resident females and time (mean ± S.E.) spent investigating same sex unknown intruders (lower panel) before mating and at different pregnancy days (PD). * p < 0.05 same day; p < 0.05 Tukey HSD vs. pre-mating.
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Fig. 4. Square root (SQRT) transformed number (mean ± S.E., upper panel) of ultrasonic vocalisations (UVs) emitted by 3–6 months and 12-month-old resident females and time (mean ± S.E.) spent investigating same sex unknown intruders (lower panel). * p < 0.01, Student’s t-test.
suggest that the number of UVs emitted during re-exposure to a familiar same sex mouse can be used as an index of agedependent impairments in social memory. 4. Discussion In the present study we confirmed that during female–female mice social encounters the resident animal utters a considerable number of 70 kHz calls. Moreover, the number of calls seems to be modulated by the estrous phase of the emitter as non-sexually receptive females emit more UVs than receptive ones. The number of calls uttered during interaction with an unknown female partner significantly decreases with pregnancy and ageing. In addition we showed that ultrasounds could be used to assess social memory in ageing female mice. In spite of the consistency of reports documenting the occurrence of ultrasonic vocalizations in female pairs, the function of these calls in female mice is still poorly understood. Some authors have suggested that female calls can act to establish female dominance [29,54]; others proposed that the primary function of 70 kHz vocalizations is “to announce the presence of another mouse and to attract other mice” [3]. UVs play an important role in regulating reproductive behavior in adult mice. Males emit 70 kHz vocalizations almost continuously prior to first mount, while they actively sniff and investigate the female. In this context, UVs are though to be important to maintain the
Fig. 5. Square root (SQRT) transformed number (mean ± S.E.) of ultrasonic vocalisations (UVs) emitted by resident female mice when exposed to the same or a different partner for the 3 min pretest and test sessions, after 30 or 60 min. * p < 0.05 Tukey HSD: test vs. pretest session.
female close to the vocalizing male rather than to attract her from a distance [38]. We suggest that also in female mice these calls facilitate proximity between animals in order to help the resident to acquire relevant social information on the intruder. Both the infantile origin of this behavior and the lack of UVs during agonistic male–male interaction [42,53] support the view that UVs
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calls are an affiliative, rather than an agonistic signal, and these vocalizations seem compatible with the category of contact calls termed “approaching in a friendly manner” recorded in other mammalian species [35]. From the rat literature there is evidence that ultrasonic vocalizations can be associated with the animal’s motivational state. In particular it has been suggested that 50 and 22 kHz calls are associated to appetitive and aversive states in the rat, respectively [27]. In particular, 50 kHz ultrasounds have been postulated to reflect positive affective states in rats, since they correlate with approach behavior during courtship [32] and rough-and-tumble play [26] and social contact [8]. Whether or not 70 kHz have a similar motivational background in mice and can be a useful index of social motivation or sociability in adult female mice is a relevant question. To answer to this question, we used an experimental setting in our previous study [33] in which we made the “intruder” animal more or less attractive by changing the salience of the information it “carried”. The motivational state (food deprivation condition) strongly modulated the number of calls. In contrast, the quality of food eaten by the demonstrators affected both the behavior and the number of calls emitted by the observers. We concluded that the motivational state of the observer mouse affected the UVs release during the interaction with a demonstrator conspecific. In fact, nondeprived animals called more towards demonstrators fed on palatable food, whereas food-deprived subjects vocalized more to fed conspecifics, in comparison to non-fed ones, independently of the palatability of the food eaten. We suggest that the more the intruder was “attractive” the more UVs were uttered. Thus, the number of UVs reflects the “motivational properties” of the stimulus animal. Other data confirm the rewarding properties of social contacts in male and female mice [13,31]. For instance, in an operant task, socially isolated females responded faster for access to a female partner than the group-housed ones [31]. Moreover, the response rate seemed to be higher in female in diestrous in comparison to those with estradiol replacement. If the number of UVs is the reflection of the social motivation, this is in line with the decrease in UVs we found in sexually receptive females compared to nonreceptive ones. By contrast, our data are not in agreement with those reported by Tang et al. [46] showing that long-term estrogen replacement increased the interest in social interaction in C57BL/6 female mice. However, methodological differences such as the pharmacological induction of the estrous phase, test cage (home cage versus neutral cage) could explain this discrepancy. One further, albeit indirect, observation supporting the hypothesis that UVs can reflect the motivation to socially interact is the decrease in social investigation and UVs shown by female mice during the second half of pregnancy as compared to their non-pregnant controls. Indeed in non-human primates the late pregnancy/early lactation periods have been associated to a trend to withdraw from social life [2,28]. Likely, the biological basis of this behavior is the pregnancy-induced change in the steroids hormonal profile and peptide levels, including endogenous opioids peptides [40]. In a wide range of species it has been shown that opiates agonists disrupts different kinds of social behaviors ([4,36] for review). Moreover opioid ago-
nists administration decreases the emission of distress calls in rat pups [9] and knockout mice for the mu receptor genes emitted less UVs when separated from their mothers [34]. Due to the massive changes in the hormonal profile of pregnant animals, multiple factors are likely to be involved in the observed changes in social behavior. However, it is tempting to speculate that one of the factors contributing to the observed decrease in social interaction and the concomitant reduction in ultrasonic vocalizations is the drop of social motivation associated to the pregnancy-induced increase in beta-endorphin levels. In a wide variety of primate species, aging also leads to a withdrawal from social interactions and an increase in time spent alone; this social withdrawal is not a passive process, caused by a reduction in general activity, but rather an active avoidance of other group members [25,37,48,50]. Studies specifically exploring age-related changes in sociability of rodents are lacking. However, in a social interaction with age matched same sex conspecifics tested in a neutral arena, aged rats spent considerably less time in active social interaction than young rats [41]. Furthermore, in 13 months old male mice it has been shown a decrease in social investigation before agonistic interaction during male–male confrontation [18]. A drop in social motivation can account also for the decrease in the number of UVs and social investigation we found in 1-year-old female mice compared to 3-month-old ones. Mice, as well as rats, are macrosmates for which storage and recall of information acquired by olfaction is a prerequisite for a successful interaction with the living and nonliving world [45]. This implies that studies investigating the processing of such information provide a privileged access to learning and memory in these species. Recent studies have proposed that in female mice the process of social recognition begins with individual specific odor compounds that are detected respectively by the olfactory system. These, through synapses in the olfactory bulb, reach the amygdala where oxitocyin (OT) receptors mediate the facilitating OT effects on social recognition. In turn, OT mediation of social recognition itself is regulated by ovarian circulating estrogens [10,11]. Since age-related mnemonic decline in female rats [30] and mice [19] coincides with the loss of regular estrous cycling, this task seems particularly suitable to explore in animal models memory functions during the onset of reproductive senescence [44]. Ultrasounds uttered during the social recognition test represent an additional and more reliable behavioral parameter in adult [12] as well as in ageing female mice, as shown in this paper. The study of social behavior in laboratory animals is crucial for the understanding of biochemical, genetic and environmental factors underlying psychiatric disorders such as autism, schizophrenia and depression, characterized by deficits in social behavior and decrease of the desire to engage in social interaction [39]. However the translational value of animal models of human conditions is strictly correlated with the development of laboratory test focusing not only on the quantitative but also on the qualitative nature of social interaction. We think that the recording and possibly spectrographic analysis of the ultrasonic calls in mice can bring new information on motivational aspects underlying social behavior and subjective states related to social
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