Affiliative Behavior, Ultrasonic Communication and Social Reward Are Influenced by Genetic Variation in Adolescent Mice

Social approach is crucial for establishing relationships among individuals. In rodents, social approach has been studied primarily within the context of behavioral phenomena related to sexual reproduction, such as mating, territory defense and parental care. However, many forms of social interaction occur before the onset of reproductive maturity, which suggests that some processes underlying social approach among juvenile animals are probably distinct from those in adults. We conducted a longitudinal study of social investigation (SI) in mice from two inbred strains to assess the extent to which genetic factors influence the motivation for young mice to approach one another. Early-adolescent C57BL/6J (B6) mice, tested 4–6 days after weaning, investigated former cage mates to a greater degree than BALB/cJ (BALB) mice, irrespective of the sex composition within an interacting pair. This strain difference was not due to variation in maternal care, the phenotypic characteristics of stimulus mice or sensitivity to the length of isolation prior to testing, nor was it attributable to a general difference in appetitive motivation. Ultrasonic vocalization (USV) production was positively correlated with the SI responses of mice from both strains. Interestingly, several USV characteristics segregated with the genetic background of young mice, including a higher average frequency and shorter duration for the USVs emitted by B6 mice. An assessment of conditioned place preference responses indicated that there was a strain-dependent difference in the rewarding nature of social contact. As adolescent mice aged, SI responses gradually became less sensitive to genetic background and more responsive to the particular sex of individuals within an interacting pair. We have thus identified a specific, genetic influence on the motivation of early-adolescent mice to approach one another. Consistent with classical theories of motivation, which propose a functional relationship between behavioral approach and reward, our findings indicate that reward is a proximal mechanism through which genetic factors affect social motivation during early adolescence

Empathy is moderated by genetic background in mice.

Empathy, as originally defined, refers to an emotional experience that is shared among individuals. When discomfort or alarm is detected in another, a variety of behavioral responses can follow, including greater levels of nurturing, consolation or increased vigilance towards a threat. Moreover, changes in systemic physiology often accompany the recognition of distressed states in others. Employing a mouse model of cue-conditioned fear, we asked whether exposure to conspecific distress influences how a mouse subsequently responds to environmental cues that predict this distress. We found that mice are responsive to environmental cues that predict social distress, that their heart rate changes when distress vocalizations are emitted from conspecifics, and that genetic background substantially influences the magnitude of these responses. Specifically, during a series of pre-exposure sessions, repeated experiences of object mice that were exposed to a tone-shock (CS-UCS) contingency resulted in heart rate deceleration in subjects from the gregarious C57BL/6J (B6) strain, but not in subjects from the less social BALB/cJ (BALB) strain. Following the pre-exposure sessions, subjects were individually presented with the CS-only for 5 consecutive trials followed by 5 consecutive pairings of the CS with the UCS. Pre-exposure to object distress increased the freezing responses of B6 mice, but not BALB mice, on both the CS-only and the CS-UCS trials. These physiological and behavioral responses of B6 mice to social distress parallel features of human empathy. Our paradigm thus has construct and face validity with contemporary views of empathy, and provides unequivocal evidence for a genetic contribution to the expression of empathic behavior

The fear conditioning apparatus and conditioning schedules.

<p>(a) Subject mice from the BALB (<i>white mouse</i>) and B6 (<i>dark grey mouse</i>) strains were separated from 2 novel, F1 object mice (<i>brown mice</i>) that received a 2-s electrical shock (UCS _objects) and/or a 30-s tone (CS) under different conditioning schedules (pre-exposure sessions). Both subjects and objects were exposed to the CS, but only objects directly experienced the UCS. Subjects had access to object distress cues (UCS _subjects) that were emitted as a consequence of receiving the UCS. (b) Photograph of the fear conditioning apparatus from an overhead perspective. (c–f) Each conditioning schedule is representative of one 120-s trial.</p

Baseline heart rates of test/subject mice during different phases of the conditioning protocol.

<p>Baseline heart rates of test/subject mice were calculated from a 90-s period that occurred immediately before the beginning of each habituation, conditioning/pre-exposure and testing period. Each of these periods began 20 min after a mouse was placed in the conditioning apparatus. There were no within-strain differences in baseline heart rate across different phases of the conditioning protocol. A previously described strain difference in baseline heart rate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004387#pone.0004387-Depino1" target="_blank">[42]</a> was found during all phases of the experiments (P<0.01). Data are presented as the mean±s.e.m.</p

Heart rate changes of subjects during pre-exposure to objects that were conditioned with the CS-UCS and during testing with the CS-only.

<p>HR was measured during (a) the first and second conditioning session in subjects that experienced objects receiving the paired CS-UCS, and during (b) testing, which entailed 5 repeated presentations of the CS-only. HR changes in subjects from both strains were similar across the 2 conditioning sessions and therefore pooled for graphical presentation. Each data point represents a 10-s bin. HR is presented as a change (Δ) from the baseline HR of each mouse (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004387#pone-0004387-t002" target="_blank">Table 2</a>). (c) Conditioned freezing responses of subjects during presentation of the CS-only (N = 15 subjects/genotype; age = 7–9 wks). Asterisks represent significant (P<0.05) HR differences between BALB and B6 subjects. Numeric symbols represent significant (P<0.05) differences in freezing between BALB and B6 subjects. Behavioral data were scored in duplicate by 2 independent raters and all data are presented as the mean±s.e.m.. Inter-rater reliability, r_p = 0.94, d.f. = 143.</p

Freezing responses of subjects after pre-exposure to the CS paired with playback of object distress vocalizations.

<p>(a) Subjects were exposed to objects during conditioning, but the CS-UCS was not presented to objects (N = 10 subjects/genotype; age = 5–7 wks). (b) Subjects were exposed to objects receiving the paired CS-UCS during conditioning (N = 8 subjects/genotype; age = 5–6 wks). (c) Subjects were not exposed to objects, but received the CS forward paired with the playback of object distress vocalizations during conditioning (N = 16 subjects/genotype; age = 5–7 wks). Asterisks represent a significant (P<0.05) difference between subjects from the BALB and B6 strains. All data were scored in duplicate by 2 independent raters and are presented as the mean±s.e.m. Inter-rater reliability, r_p = 0.96, d.f. = 659.</p

Distress vocalization of an object during UCS presentation.

<p>(a) Representative sonogram of an object distress vocalization recorded in real-time without the demonstration compartment enclosed by Plexiglas®. (b) Sonogram of the same vocalization played back through a speaker without the demonstration compartment enclosed. (c) Sonogram of the same vocalization played back through a speaker with the demonstration compartment enclosed. Color-coding indicates the relative decibel level for the call, with blues representing low-intensity energy and red/yellow representing high-intensity energy.</p

Descriptive statistics for object distress vocalizations.

<p>Vocalizations were recorded from 20 different pre-exposure sessions and sonograms were generated for each respective UCS presentation (10/session). Data are presented as the mean±std. dev.</p

Freezing responses of socially and isolate-housed subjects pre-exposed to objects receiving the CS-UCS contingency.

<p>Subjects were either isolated or remained in a social housing context outside of their observations of objects receiving the paired CS-UCS during the pre-exposure session. Since freezing responses of socially housed subjects in this experiment (N = 10 subjects/genotype; age = 6–8 wks) did not differ (P = 0.20) from responses of mice that were evaluated in the experiment presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004387#pone-0004387-g003" target="_blank">Figure 3a</a>, the data were pooled for graphical presentation and statistical analysis. Social isolation did not alter the freezing responses of BALB subjects, but it depressed fear acquisition to presentation of the paired CS-UCS in B6 subjects (N = 20 subjects/genotype; age = 7–8 wks). Socially housed B6 subjects expressed longer freezing responses than all other groups on trials 7-10 (P = 0.006). Asterisks represent significant differences between socially housed B6 subjects and all other groups as assessed by a Bonferroni step-down procedure on a trial-by-trial basis (<i>∝</i> = 0.013 for trials 7-10). All data were scored in duplicate by 2 independent raters and are presented as the mean±s.e.m. Inter-rater reliability (not including pooled data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004387#pone-0004387-g003" target="_blank">Fig. 3a</a>), r_p = 0.94, d.f. = 547.</p