Affiliative behavior, ultrasonic communication and social reward are influenced by genetic variation in adolescent mice.

Abstract

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.

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Affiliative Behavior, Ultrasonic Communication and
Social Reward Are Influenced by Genetic Variation in
Adolescent Mice
Jules B. Panksepp1*, Kimberly A. Jochman2¤, Joseph U. Kim2, Jamie J. Koy3, Ellie D. Wilson4, QiLiang Chen4, Clarinda R. Wilson4,
Garet P. Lahvis2,5*
1Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin, United States of America, 2Department of Surgery, University of
Wisconsin, Madison, Wisconsin, United States of America, 3Undergraduate Program in Religious Studies, University of Wisconsin, Madison, Wisconsin,
United States of America, 4Undergraduate Program in Biological Sciences, University of Wisconsin, Madison, Wisconsin, United States of America,
5Waisman Center for Developmental Disabilities, University of Wisconsin, Madison, Wisconsin, United States of America
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.
Citation: Panksepp JB, Jochman KA, Kim JU, Koy JJ, Wilson ED, et al (2007) Affiliative Behavior, Ultrasonic Communication and Social Reward Are
Influenced by Genetic Variation in Adolescent Mice. PLoS ONE 2(4): e351. doi:10.1371/journal.pone.0000351
INTRODUCTION
Social interactions constitute a broad range of forms that are
responsive to the particular environmental or social context where
they occur. Although food availability [1–3] disease prevalence [4–6]
and seasonal changes [7,8] can influence the level of sociability
among conspecifics, perhaps more familiar are the effects of group
structure on social interaction [9–15]. For example, an adult female
will perform a remarkable range of social behaviors throughout her
life, ranging from courtship to territorial defense to maternal care, all
ofwhicharecontingentuponhersocialcontext,individualstatusand
age. It is axiomatic that social interactions among adults are closely
tied to reproductive opportunities [16]. However, juvenile animals
lack reproductive competence [17–19], so it is conceivable that some
aspects of the juvenile social repertoire are sensitive to motivational
variables distinct from those that operate during adulthood.
Social interaction has been extensively described among
juvenile animals in the laboratory [20–24], as well as in more
naturalistic contexts [8,25–30]. Compared to adults, social inter-
actions between juveniles usually have a more playful quality [31–
33]. During pubertal and adolescent development [18,34,35],
these patterns of social behavior transition into forms that are
more typical of adults [35–37], yet very little is known about the
interplay between motivational variables and the expression of
approach behavior as it changes throughout adolescence. For
example, strong parent-offspring bonding and gregariousness
among kin, which are common features of sociality among
juvenile mammals, can dissipate during adolescent development.
As adolescent animals near adulthood, they begin to express
motivations to disperse and integrate into new social groups, to
establish a territory and interact with potential mates, and
ultimately to reproduce and care for offspring [28,38,39]. This
Academic Editor: Wim Crusio, Centre National de la Recherche Scientifique,
France
Received February 19, 2007; Accepted March 13, 2007; Published April 4, 2007
Copyright: ? 2007 Panksepp et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This project was funded by NIH research grants R03 HD046716 and R01
DA022543, a program-project grant (P30 HD03352) to the Waisman Center and
an institutional grant (U01 MH61971) to the Tennessee Genome Consortium. JBP
was supported by NIH training grants to the Neuroscience Training Program (T32
GM07507) and the Training Program in Emotion Research (T32 MH018931) at the
University of Wisconsin - Madison.
Competing Interests: The authors have declared that no competing interests
exist.
* To whom correspondence should be addressed. E-mail: jbpanksepp@wisc.edu
(JP); gplahvis@wisc.edu (GL)
¤ Current address: Department of Psychology, University of Texas, Austin, Texas,
United States of America
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reallocation of social interests is the cumulative product of a variety
of factors, including interactions between endocrine physiology
[17,40,41], social experience [42,43] and brain anatomy [44,45].
Adolescence is thus a period of dynamic social transformation in
which juvenile behavior is organized to face new challenges that
accompany being an adult.
A recent series of studies [46–48] demonstrated that pre-
pubescent mice from the B6 strain are particularly pro-social,
whereas age-matched BALB mice are much less responsive to
social opportunities. Specifically, these studies illustrated that
adolescent B6 mice approach and investigate social stimuli to
a greater extent than BALB mice [46,47]. Interestingly, adult mice
from these two genetic backgrounds appear to be much less
distinct than juvenile mice in terms of their motivation to
approach and investigate conspecifics [49,50], although such
a comparison has not been thoroughly tested. In the present study,
we evaluated patterns of social interaction within pairs of BALB
and B6 mice throughout adolescent development, in conjunction
with measurements of mounting behavior, investigation of a novel
olfactory stimulus, food consumption, ultrasonic communication
and social conditioned place preference during early adolescence.
Overall, our results demonstrate that genetic variation has a direct
impact on the expression of social approach and the production of
ultrasonic vocalizations by early-adolescent mice, as well as on the
underlying reward value they assign to the opportunity for social
contact. Importantly, the strain-dependent influence on social
approach was most pronounced during early adolescence and
diminished with the appearance of gender-specific social interests
during later stages of adolescent development. We thus propose
that specific alleles interact with a social reward process in the early-
adolescent mouse, modulating the value of social interaction, and
thereby influencing the degree to which conspecifics are motivated
to approach each other.
MATERIALS AND METHODS
Mouse husbandry
Mice from the BALB/cJ (BALB) and C57BL/6J (B6) strains were
purchased from Jackson Laboratories (Bar Harbor, ME, USA)
and subsequently bred at the University of Wisconsin, Biotron
(Madison, WI, USA) under tightly controlled temperature
(2161uC), humidity (range, 50–60%) and light (14:10 hr light/
dark, ‘lights off’ from 1130–2130) conditions. To reduce potential
influences from genetic drift, new mice were routinely introduced
to the breeding colony and brother-sister matings were avoided.
Mice were maintained under specific-pathogen free conditions and
housed in standard polyurethane cages (29061806130 mm) that
contained 1/8’’ grain-size corncob bedding (The Andersons,
Maumee, OH, USA) and nesting material (Ancare Corporation,
Bellmore, NY, USA), with ad libitum access to food (Teklad Rodent
Diet, Harlan, Madison, WI, USA) and water. Pregnant females
were isolated and pups were weaned on postnatal day (PD) 20–21
(day of birth=PD 0). Animal care and experimental protocols
were conducted in accordance with the regulations of the institu-
tional care and use committee at the University of Wisconsin -
Madison and the NIH Guide for the Care and Use of Laboratory Animals.
Our own laboratory personnel carried out all aspects of the mouse
husbandry under strict guidelines to insure gentle and consistent
handling of the mice.
Social interaction tests: general methodology and
measurements
At weaning, mice were formed into a social group (2 mice from
each sex) that served as the general housing arrangement for all
subsequent tests, with the exception of Experiment 4 (see below).
Sibling number, sex bias and maternal experience (primiparous vs.
multiparous) were recorded for each litter and a series of statistical
tests for correlation indicated that these variables were not related
to the social responsiveness of adolescent mice (data not shown),
and thus were not considered further. Twenty-four hours prior to
testing, each mouse from a social group was isolated within
a clean cage that contained fresh corncob bedding without nesting
material. After 24 hrs of social isolation, two mice from each
group were randomly designated as ‘test’ mice, while the remain-
ing individuals served as ‘stimulus’ mice. Testing entailed placing
a stimulus mouse into the cage where an isolated test mouse had
resided for the previous 24 hrs and then monitoring the amount of
social investigation (SI) that test mice directed towards the stimulus
mouse during a 5-min period (see Figures 1a & 1b). Behavioral
variables included: [i] sniffing or snout contact with the head/
neck/mouth area, [ii] sniffing or snout contact with the flank area,
[iii] direct contact with the anogenital area, [iv] social pursuit
within one body-length as the stimulus mouse moved continuously
throughout the cage and [v] social grooming. These variables were
highly correlated and combined into a composite measure of SI.
We also recorded additional features of social interaction, such as
social proximity (i.e., mice within one-body length of each other
without movement or direct contact) and ‘jerk-and-run’, a play-
like behavior (see ref. [30]), but these behaviors were infrequent
and highly variable among pairs of mice, and therefore not
considered in the comparisons of SI. Mounting behaviors were
also observed during some of the SI tests and recorded as
Figure 1. Social approach among adolescent mice. (a) The SI responses
of test mice were quantified during 5-min tests throughout adolescent
development. Weaning age and the average age of reproductive
maturity in females is illustrated for mice from both strains. (b)
Photograph of a B6 mouse investigating a former cage mate after
24 hrs of social isolation.
doi:10.1371/journal.pone.0000351.g001
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a categorical variable. These behavioral sequences were very brief,
usually entailing physical contact that was not conducive for
copulation, and were not included in the composite measure of SI.
In a set of preliminary experiments, where the testing period lasted
10 min, observations of tail rattling and wrestling behavior were
included in the ethogram. The social behaviors of stimulus mice
were also noted, but not included in the behavioral analyses.
SI testing was conducted during the dark phase (1300–1900),
under dim red illumination, in a sound-dampened room that was
structurally identical to the mouse colony. Mouse cages were
transported approximately 5 meters from the colony, through
a dimly-lit intervening room, to the procedure room .30 min
prior to testing. The top from the cage containing a test mouse was
replaced with a 1/8’’-thick piece of transparent PlexiglasH 5–
10 min before testing began. Following this habituation period,
a stimulus mouse was introduced to the side of the cage opposite to
the test mouse. Behaviors were videotaped (Sony, DCR-VX2100,
Japan) and stored on a Dell Pentium IV PC for additional analysis.
All behaviors were scored in duplicate with the aid of computer-
assisted analysis software (ButtonBox v.5.0, Behavioral Research
Solutions, Madison, WI, USA), once during the experiment and
once during a subsequent ‘off-line’ analysis session by a different
observer that was blind to the age and gender of the interacting
mice. The presentation of all SI data and statistical outcomes in
this study are based on an average of these two independent
measurements (inter-rater reliability,
r=0.93, d.f.=639).
Pearson’scorrelation,
Social interaction tests: experimental design
Experiment 1–Effects of genotype and sex composition
within a dyad on SI throughout adolescent develop-
ment
Mice were weaned on PD 20/21 and housed in a mixed-
sex social cohort containing 4 siblings. The identities of test and
stimulus mice within a social group were determined randomly on
the first day of SI testing and remained constant for all subsequent
tests. SI testing was conducted at 5-day intervals on PD 25/26, PD
30/31, PD 35/36, PD 40/41 and PD 45/46 (Fig. 1a). Stimulus
mice were identified by a small mark placed at the base of the tail
with a permanent marker. On average, SI tests were carried out at
4 of the 5 developmental time points for each pair of mice.
Following each testing period, all mice were weighed, placed into
a clean cage with fresh bedding and nesting material, returned to
the colony room, and left undisturbed until the next 24-hr isolation
period. The SI responses of male and female test mice were
measured in response to stimulus mice from both sexes. For tests
that involved a male and female, sample sizes were 12–19 test mice
per sex combination at each age (mice were derived from 18 litters
per strain). For tests that involved same-sex pairings, sample sizes
were 10–11 test mice per sex combination at each age (mice were
derived from 10 litters per strain).
Experiment 2–Influence of maternal care on strain differ-
ences in SI for early-adolescent male mice investigating
female mice
BALB (n=4) and B6 (n=3) litters were cross-
fostered to a postpartum dam from the alternate strain within
12 hrs of birth. Subsequent care for the mice was consistent with
Experiment 1. At weaning, four siblings (2 per sex) were formed
into a social group and left undisturbed until social isolation on PD
29/30, as previously described. On PD 30/31, male test mice were
tested for SI with a familiar stimulus female of the same strain.
N’s=10 test mice per strain.
Experiment 3–Early-adolescent BALB and B6 male mice
investigating female mice from the opposite strain
weaning, two males from one strain were housed with 2 females
from the alternate strain. Mouse husbandry and SI testing was
At
performed as described for Experiment 2. Following 8–10 days of
continuous housing in these social groups, all mice were socially
isolated for 24 hrs and then tested for SI. A 5-min social
interaction test was performed with male test mice (PD 30/31)
approaching a familiar, age-matched female from the alternate
strain. N’s=16 BALB and 14 B6 test mice from 5–6 litters per
strain.
Experiment 4–Early-adolescent BALB and B6 male mice
investigating females after long-term social isolation
weaning, individual male mice were placed in isolate housing and
left undisturbed for 8–10 days until SI testing. All mice were
socially isolated into a clean cage 24 hrs before SI testing. A 5-min
social interaction test was performed with male test mice (PD
30/31) approaching an unfamiliar female from the same strain.
N’s=8 BALB and 9 B6 test mice from 4 litters per strain.
Experiment5–Early-adolescent
investigating a novel olfactory stimulus
were formed into mixed-gender social groups and maintained as
previously described. Mice were socially isolated into a clean cage
24 hrs before testing. On PD 30–36, mice were presented with
a cotton ball scented with lemon extract (500 ml) and olfactory
investigation was measured for 5 min. Olfactory investigation
included contacting, actively manipulating or sniffing (,10 mm)
the cotton ball. Mice were re-grouped into a clean cage following
testing. N’s=13–15 mice per sex for each strain.
Experiment6–Early-adolescent
approaching and consuming a familiar food source after
food deprivation
Forty-eight hours after testing for investiga-
tion of a novel olfactory stimulus, mice from Experiment 5 were
re-isolated into a clean cage and food-deprived during a 24-hr
social isolation period. On PD 32–38, each mouse was provided
with a single pellet of standard lab chow on the floor of its cage
and the total amount consumed within a 10-min period was
measured. N’s=13–15 mice per sex for each strain.
At
BALB and
At weaning, mice
B6mice
BALBand B6 mice
Measurement and characterization of ultrasonic
vocalizations during social interaction
Ultrasonic vocalizations (USVs) were recorded for all SI tests
conducted during Experiment 1. An ultrasound microphone
(UltraSoundGate model CM16, Avisoft Bioacoustics, Berlin,
Germany) with a 10–180 kHz flat-frequency range was lowered
to the plane of the cage top, where there was a small opening (30-
mm diameter) centrally located within the PlexiglasH cage cover.
USVs were collected with an UltraSoundGate 116 acquisition
system and the Avisoft-Recorder v.2.97 (Avisoft Bioacoustics), and
stored as wav files for subsequent analysis. For practical purposes,
quantitative analyses of USV emission were focused on SI tests
conducted on PD 30/31, to compare with our previous work [51]
that was conducted during this particular period of adolescent
development.
Five sonograms, corresponding to the first 15-sec interval of
each minute during a SI test, were generated for all pairs of mice
tested on PD 30/31 and subjected to an extensive quantitative
analysis (SASLab Pro v.4.39, Avisoft Bioacoustics). A 40-kHz
band-pass filter was used to minimize background noise during
recordings; however, most wav files still contained a considerable
amount of ‘non-USV’ signal that compromised the accuracy of the
automated parameter-measurement functions available within the
SASLab Pro software format. Thus, extraneous noise was identi-
fied and removed from all of the sonograms. When a rater found
an ultrasound signal that was difficult to interpret, the call was
evaluated by a minimum of one additional, trained observer and
identification required a consensus by all raters. Each sonogram
was then evaluated with a series of automated parameter
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measurements that tallied the total number of USVs produced,
USV duration, the average dominant frequency of a USV and the
inter-vocalization interval.
Since a considerable amount of frequency modulation existed
within individual USVs, a quantitative framework was devised to
reliably identify USV subtypes based on their distinct sonographic
characteristics (see Results). Two blind observers categorized each
USV into a particular subtype, and the presentation of all data and
statistical outcomes in the present study are based on average of
these duplicate measurements (inter-rater reliabilities, Pearson’s
correlations, r=0.90–0.98 for each USV subtype). Additionally,
random samples of each USV subtype were subjected to a more
detailed quantitative analysis. USVs from each subtype were
sampled from a set of 15-sec sonogram files that represented $5
mouse pairs per sex combination for each strain. Sample sizes
ranged from 32–156 syllables per USV category for each strain. In
addition to measuring the duration and average pitch of these
calls, the pitch of each syllable was sampled at 1-msec intervals,
which permitted a higher temporal resolution for analyzing
internal frequency changes within individual USVs.
Social conditioned place preference
Experiment 7–Social reward in early-adolescent BALB and
B6 mice
Mice were weaned on PD 20/21, maintained in social
groups as previously described and left undisturbed until the
conditioning phase of the experiment. On PD 25–30, the day prior
to conditioning, mice were socially isolated at 1400–1600 into
a clean cage that contained corncob bedding and nesting material.
Social conditioning took place over the next 3 days, which
included 2 conditioning sessions per day (conditioning sessions
occurred at 0900–1100 and 1900–2100 each day). During
a conditioning session, mice from each social group (2 same-
strain mice per sex) were either reunited or socially isolated for
30 min in one of two distinct environments (aspen or paper
bedding) situated within a single compartment of a 3-chambered
testing arena (for additional details see ref. [51]). Unconditioned
mice from the control groups were socially isolated during all 30-
min conditioning sessions. To ensure that mice in the control
groups received an equivalent amount of experience with the
unconditioned stimulus (i.e., social interaction), they were reunited
with their respective social group once per day for 30 min in
a novel, plastic enclosure (38062006155 mm) that was lined with
corncob bedding and situated within a procedure room distinct
from where the conditioning sessions were conducted. All variables
associated with the conditioning procedure were randomized and
counter-balanced across the unconditioned and conditioned
groups of mice.
On the final day of conditioning, individual mice were allowed
to freely explore a 3-compartment testing arena (30061506
150 mm per compartment) for a 15-min habituation period with
no conditioning cues present (habituations took place at 1400–
1600 under dim red light). On the test day (PD 30–35), an
individual mouse was placed in the central compartment of the
testing arena (where no conditioning cues were present) and its
movement throughout the arena was videotaped for a 30-min
period. The time spent in each compartment (peripheral compart-
ments contained the socially-paired and isolation-paired beddings,
respectively) and the number of transitions made between each
compartment was quantified during a subsequent off-line analysis.
Preference scores were calculated as the duration a mouse spent in
the aspen bedding-lined compartment minus the duration spent in
paper bedding-lined compartment. Ten BALB mice (4 and 6
individuals from the conditioned and unconditioned groups,
respectively) were not included in the statistical comparisons due
a greatly reduced level of exploratory activity, as detailed in ref.
[51]. N’s=28–32 mice per strain for each of the unconditioned
and conditioned groups.
Statistical analyses
A 26465 analysis of variance (ANOVA), with the strain and sex
combination of mouse pairs as between-group factors and age as
a repeated measure, was used to analyze the amount of SI
expressed by test mice during Experiment 1. Orthogonal contrasts
(JMP v.6.0, SAS Institute Inc., Cary, NC; also see ref. [52]) were
used to evaluate the presence of strain differences at each sampling
point during adolescent development and within-strain differences
for PD 25/26 vs. PD 45/46 mice. For Experiments 2–4, one-
factor ANOVAs were used to assess the effect of genotype on SI.
For Experiments 5 and 6, two-factor ANOVAs (with strain and
sex as between-groups factors) were used to evaluate olfactory
investigation and food consumption, respectively. For Experiment
7, a 266 ANOVA (with sex and conditioning group as between-
groups factors) was used to assess the place-preference scores.
Tukey’s honestly significant different (HSD) tests and orthogonal
contrasts were employed to conduct post-hoc comparisons for
Experiments 6 and 7, respectively. A majority of the USV data
was assessed with 264 ANOVAs (with the strain and sex
combination of mice within an interacting pair as between-group
factors) and nested orthogonal contrasts when appropriate. For
variables that were not normally distributed, non-parametric x2-
approximations were used to evaluate differences in the median
value between strains. Linear regressions, Pearson’s correlations
and contingency-table analyses were conducted as necessary (tests
involving correlation matrices were corrected for multiple
comparisons). For all statistical tests, P,0.05 was considered
significant.
RESULTS
Strain-dependent variation in social investigation
during adolescent development:
We quantified the amount of SI that test mice direct towards
familiar conspecifics placed within their home cage during a 5-min
period at multiple points throughout adolescent development (see
Figure 1a & and Experiment 1 in Materials and methods).
Following 24 hrs of isolation, social reunion with a stimulus mouse
resulted in a larger SI response (Fig. 1b) by test mice from the B6
strain compared to age-matched BALB mice (Figs. 2a–d; main
effect of genotype, F [1,260]=61.1, P,0.0001). On PD 25/26,
which was the first day of SI testing, this strain-dependent
difference was evident for all test-by-stimulus mouse combinations;
i.e., males approaching females (M-F pairs; Fig. 2a), females
approaching males (F-M pairs; Fig. 2b), males approaching males
(M-M pairs; Fig. 2c) and females approaching females (F-F pairs;
Fig. 2d).
The social responsiveness of adolescent mice was noticeably
variable across adolescent development (main effect of age, F
[4,260]=2.3, P=0.06). Moreover, strain- and sex-dependent
effects on SI also changed during adolescence (P,0.01 for both
interaction effects, F-statistics not shown) and interacted with each
other as a function of age (genotype6sex6age interaction, F
[12,260]=2.6, P=0.003). This three-way statistical interaction
corresponded to distinct effects of the sex of interacting mice on SI
responses, which gradually came to overshadow the strain-
dependent influence during late adolescence. For example, BALB
test mice expressed larger SI responses towards a female stimulus
mouse on PD 45/46 compared to mice on PD 25/26 (Figs. 2a &
2d), corresponding to a decline in the strain difference that
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occurred during early adolescence. Older test mice (PD 45/46)
from the B6 strain expressed diminished SI responses, relative to
early-adolescent mice (PD 25/26), when they were reunited with
a male stimulus mouse (Figs. 2c & 2b). On PD 45/46, strain-
dependent variation in SI was evident only for female test mice
that had been paired with another female and this strain difference
was considerably smaller than the difference that was found for the
same pairs on PD 25/26 (see Figure 2d).
As adolescent test mice matured, we noted several instances of
social approach that were distinct from those included in the
composite measurement of SI (Table 1). For example, a pro-
portion of B6 males (PD 30/31) began to direct mounting
behaviors towards female stimulus mice approximately 5 days
earlier than male BALB mice (PD 35/36). These behavior patterns
usually encompassed 2–3 successive mounting attempts (,5 sec)
that occurred during the final 60 sec of the testing period.
Mounting was disproportionately expressed by male test mice in
response to the introduction of a female, accounting for 81% of all
occurrences, and was targeted towards both the posterior and
anterior portion of the stimulus mouse. The likelihood that mounts
occurred during M-F interactions increased as a function of age
(x2=32.5, d.f.=4, P,0.0001) and there was a general trend for
male test mice from the B6 strain to engage in more mounting
behaviors than age-matched BALB males at all sampling points
other than PD 25/26 (see Table 1; x2=8.3, d.f.=1, P,0.01).
We did not observe any instances of aggressive behavior
between adolescent mice during the 5-min SI tests. However,
during a preliminary set of experiments, which entailed 10-min
testing sessions, approximately 35% of BALB test males (PD 39–
42) engaged in wrestling or tail rattling when they were paired with
another male, whereas age-matched M-M pairs from the B6 strain
never expressed such patterns of behavior (J.B.P & G.P.L.,
personal observations). Importantly, all forms of ‘adult-like’
behavior were observed before females had exhibited patent
vaginal swelling, lordosis or estrus (see www.jax.org/phenome).
Furthermore, the development of mounting and aggressive
behaviors by male test mice occurred on a similar timeframe as
the appearance of gender-effects on SI. When male test mice that
had exhibited mounting attempts were excluded from the
statistical analysis (data not shown), differences between the
average SI responses of mice for each strain-by-age comparison
were not distinguishable from those presented in Figure 2a.
Figure 2. Differences in social investigation between adolescent mice as a function of genetic background, age and sex. SI responses of BALB and B6
test mice during adolescent development for (a) males approaching females, (b) females approaching males, (c) males approaching males and (d)
females approaching females. All data are presented as the mean6SEM (* P,0.05, ** P,0.01, *** P,0.001 for BALB vs. B6 mice;#P,0.05,##P,0.01
for PD 25/26 vs. PD 45/46 mice).
doi:10.1371/journal.pone.0000351.g002
Table 1. Proportion of male test mice that displayed
mounting behavior towards a female stimulus mouse.
......................................................................
Age BALB/cJC57BL/6J
PD 25/26 n=16 (n/a) n=18 (n/a)
PD 30/31 n=16 (n/a) 5 of 19 (26%)
PD 35/363 of 12 (25%)9 of 16 (56%)
PD 40/416 of 17 (35%) 9 of 13 (69%)
PD 45/465 of 18 (28%) 9 of 18 (50%)
doi:10.1371/journal.pone.0000351.t001
......................................
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To determine whether the strain difference in SI between early-
adolescent mice was attributable to the genetic background of the
test mouse, we conducted a series of control experiments in which
PD 30/31 males were given an opportunity to investigate age-
matched females after 24 hrs of social isolation. The strain-
dependent difference in SI (data for the PD 30/31 mice plotted in
Figure 2a are presented again in Figure 3a) was not altered
when BALB and B6 mice pups had been raised by a mother from
the opposite strain and then housed with siblings from weaning
until testing (see Experiment 2; Fig. 3b; F [1,18]=39.3, P,0.0001).
Furthermore, the strain difference was also present for mice that
were maintained in mixed-strain social housing for 8–10 days
prior to testing (see Experiment 3; Fig. 3c; F [1,28]=21.3,
P,0.0001), suggesting that differences between the specific
contexts associated with BALB and B6 housing could not account
for strain-dependent variability in SI. In another experiment, we
found that a longer period of social isolation (8–10 days) did not
affect the strain-dependent pattern in SI between early-adolescent
mice from the BALB and B6 strains (see Experiment 4; Fig. 3d; F
[1,14]=28.7, P,0.0001).
We also assessed the possibility that strain-dependent variation
in SI was due to a general difference in the capacity of adolescent
mice to express appetitive behavior. Male and female mice (PD
30–36) from the BALB and B6 strains exhibited an equivalent
amount of olfactory investigation towards a novel, lemon-scented
cotton ball following 24 hrs of social isolation (see Experiment 5;
Fig. 4a; F [1,52]=0.02, P=0.90). Two days later, following
24 hrs of food deprivation and social isolation, the mice used
during Experiment 5 were tested for consumption of standard
laboratory chow (see Experiment 6). All mice began to consume the
food source within 45 sec and BALB males (PD 32–38) consumed
more chow during the 10-min period than mice from all of the
other groups (Fig. 4b; genotype x gender interaction, F
[3,52]=6.3, P=0.02).
Ultrasonic vocalizations during social interactions
between early-adolescent mice:
On PD 30/31 (Experiment 1), we recorded ultrasonic vocaliza-
tions (USVs) that were produced by early-adolescent mice during
SI testing (see Materials and methods). One feature of USV
emission, which was common for mice from both strains, entailed
a positive association between the total number of vocalizations
produced (i.e., ultrasonic ‘syllable’ production, as defined in ref.
[53]) and the magnitude of the test mouse SI response (Figs. 5a &
5b). Furthermore, for both strains, USV production was more
frequent during mixed-sex (mean6standard error of the mean
Figure 3. Strain-dependent differences in social investigation as
a function of maternal care, stimulus mouse characteristics and length
of social isolation. (a) Following 24 hours of social isolation, on PD 30/
31, male B6 mice investigated a familiar female stimulus mouse for
a greater duration then age-matched BALB males. This strain-
dependent pattern was also expressed by (b) male mice that had
been raised by a mother of the alternate strain, (c) male mice
approaching a female from the alternate strain and (d) male mice
approaching a same-strain female after 8–10 days of social isolation. All
data are presented as the mean6SEM (** P,0.01, *** P,0.001).
doi:10.1371/journal.pone.0000351.g003
Figure 4. Approach behaviors of adolescent mice towards a novel
olfactory stimulus and a food source. After 24 hrs of social isolation, (a)
BALB and B6 mice investigated a lemon-scented cotton ball for a similar
amount of time. Following complete food deprivation during a 24-hr
social isolation period, (b) BALB males consumed more food (standard
lab chow) than mice from the other groups during a 10-min period. All
data are presented as the mean6SEM (* P,0.05 compared to BALB
females and B6 mice of both sexes).
doi:10.1371/journal.pone.0000351.g004
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[SEM]; 237610 USVs) versus same-sex social interactions
(149617 USVs; orthogonal contrast for M-F & F-M vs. M-M &
F-F pairs, F [1,103]=29.7, P,0.0001). Although we did not find
an overall difference in USV emission between BALB and B6 mice
(main effect of genotype, F [1,103]=1.5, P=0.23), there was
a strain-dependent sensitivity in USV production that was specific
to the sex of mice within an interacting pair (Fig. 5c; strain x sex
interaction, F [7,103]=5.4, P=0.002). For example, compared to
M-F pairs from the B6 strain, there were more USVs produced
when a female stimulus mouse was introduced to the cage of
a BALB male (orthogonal contrast for BALB vs. B6 M-F pairs, F
[1,103]=5.7, P=0.02). By contrast, USV emission was less
frequent for M-M pairs from the BALB strain relative to similar
pairs from the B6 strain (see Figure 5c; orthogonal contrast for
BALB vs. B6 M-M pairs, F [1,103]=10.4, P=0.002).
A detailed examination of the sonograms generated for each SI
test revealed several, additional strain-dependent differences in the
USVs of interacting mice. For example, while the timing of
individual syllables within each vocalization-sequence was similar
for mice from both strains (Figs. 5d & 5d9; x2=0.005, d.f.=1,
P=0.95), the average pitch and duration of individual USVs
segregated with the genetic background of mice. USVs produced
by B6 mice had a higher frequency (Figs. 5e & 5e9; x2=2674.4,
d.f.=1, P,0.0001) and shorter duration (Figs. 5f & 5f9;
x2=1401.8, d.f.=1, P,0.0001) than those of BALB mice. These
two particular features of USV emission were also influenced by
the sex composition of mice within a pair and its statistical
interaction with their collective genetic background (data and
statistics not shown).
Although it was not possible to categorize all of the USVs that
were present in the sonograms, we did classify 55620.1% (BALB)
and 32614.5% (B6) of these vocalizations (reported as mean6-
standard deviation), based on internal pitch-changes, into 5
distinct categories with high reliability between observers (see
Figure 5. Production and sonographic characteristics of ultrasonic vocalizations during the social interactions of early-adolescent mice. USV emission
was positively associated with the SI responses of early-adolescent (a) B6 and (b) BALB mice. (c) USV production was selectively modulated during
social interactions that involved a male test mouse from the BALB strain. (d–d9) Emission rates were similar for mice from both strains when USVs
were detected. However, compared to BALB mice, the USVs of B6 mice occurred at (e–e9) higher average frequencies and lasted for (f–f9) shorter
durations. Data in Figures d–f and d9–f9 are presented as frequency distributions of the raw acoustic signal that was collected during SI tests on PD
30/31. A portion of the data (,0.5% of the sample from each strain) is not illustrated to keep the abscissa of each distribution within a reasonable size
for presentation. Data in Figure 5c are presented as the mean6SEM (* P,0.05, ** P,0.01 for BALB vs. B6 mice).
doi:10.1371/journal.pone.0000351.g005
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Materials and methods). USVs were not classified when identi-
fication of a clear sonographic pattern was difficult (e.g., extremely
short calls were ,3 msec) or when the degree of frequency
modulation within a call did not fit the criteria outlined below (e.g.,
some ‘unmodulated’ calls contained single frequency sweeps that
were ,12.5 kHz). Our scheme for the classification of USVs was as
follows: Upwards-modulated calls exhibited a continuous increase
in pitch that was $12.5 kHz, with a final dominant frequency at
least 6.25 kHz greater than the initial pitch (see Figures 6a &
6a9). Downwards-modulated calls exhibited a continuous decrease
in pitch that was $12.5 kHz, with a terminal dominant frequency
at least 6.25 kHz less than the pitch at the beginning of the
vocalization (Figs. 6b & 6b9). The general sonographic profile of
chevron-classified calls resembled an ‘inverted-U’, which was
identified by a continuous increase in pitch $12.5 kHz followed
by a decrease that was $6.25 kHz (Figs. 6c & 6c9). USVs were
classified as complex if one syllable contained two or more direc-
tional changes in pitch, each $6.25 kHz (Figs. 6d & 6d9). A fifth
USV category, punctuated calls, was characterized by discontinuous
jumps in frequency that were $7.5 kHz. We identified these pitch-
jumps as ‘punctuations’ because they occurred rapidly (#1 msec),
dividing each ultrasonic syllable into two or more distinct elements
(see Figures 7b1–7b4).
We detected strain-dependent effects on the probability of
producing calls from each of the five classes of USV. For example,
early-adolescent BALB mice emitted more upwards-modulated
USVs than B6 mice (Fig. 6a0; main effect of genotype, F
[1,103]=125.5, P,0.0001), whereas age-matched B6 mice
expressed downwards-modulated USVs more frequently than
BALB mice (Fig. 6b0; main effect of genotype, F [1,103]=109.8,
P,0.0001). Chevron-call production was relatively common
during the social interactions of BALB mice, but not B6 mice
(Fig. 6c0; main effect of genotype, F [1,103]=142.4, P,0.0001).
Furthermore, although the production of complex calls was less
common than vocalizations from other USV subtypes, B6 mice
emitted more of these calls than BALB mice (Fig. 6d0; main effect
of genotype, F [1,103]=6.1, P=0.02).
The production of upwards-, downwards- and chevron-
classified USVs also varied as a function of the sex of mice within
a pair and its interaction with their genetic background (P,0.05
for the sex and genotype x sex interaction factors, data and F-
statistics not shown). Interestingly, genetic background was the
only variable that influenced complex call production during social
interactions between early-adolescent mice (see Figure 6d0;
P.0.05 for the sex and genotype6sex interaction factors, data
and F-statistics not shown). Similarly, punctuated calls were
influenced by the genetic background of mice (Fig. 7a; main effect
of genotype, F [1,103]=48.6, P,0.0001), but not by the sex of
mice or its interaction with genetic background (P.0.05 for both
factors, as reported above).
Figure 6. Classification of ultrasonic vocalizations into distinct categories. Representative sonograms and descriptive statistics for different types of
USV that were emitted by (a–d) BALB and (a9–d9) B6 mice (see Results for classification criteria). Descriptive statistics (mean6std. dev.) are given for
the duration of each call type, as well as the beginning and ending dominant frequency. The USV traces are arbitrarily aligned to the 100-msec
demarcation on the abscissa of each sonogram. Intensity changes within each representative vocalization are color-coded. (a0–d0) The production of
each USV subtype was strain-dependent (* P,0.05, *** P,0.001 for BALB vs. B6 mice).
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A strain difference in the sonographic pattern of punctuated
USVs was evident when the dominant frequencies of selected calls
were plotted at t (the x-axis in Figures 7c & 7d) and contrasted to
changes in pitch within the same call that occurred at t+1 msec
(the y-axis in Figures 7c & 7d). As illustrated in Figures 7c and
7d, data points near the diagonals have a paired-pitch ratio
(pitch[t]/pitch[t+1]) approximating 1.0 and thus represent contin-
uous changes in the dominant frequency (or pitch) of a call.
Paired-pitch ratios deviating from 1.0 aligned off the diagonals
and represent discontinuous breaks in the dominant frequency of
a call (these punctuations were tallied as ‘upwards’ [pitch[t+1]$
(pitch[t]+7.5 kHz)] or ‘downwards’ [pitch[t+1]#(pitch[t]+7.5 kHz)]
oriented pitch-jumps, as outlined above). The punctuated calls of
BALB mice were dominated by three distinct, paired-pitch ratios
(note the three data ‘clouds’ lying off the diagonal in Figure 7c).
By contrast, a much less stereotyped pattern of pitch-jumps was
evident for the punctuated calls of B6 mice (see Figure 7d). Since
the emission of punctuated USVs was less common during social
interactions involving BALB mice (see Figure 7a), greater
sampling of the BALB sonograms was required to attain a similar
number of paired-pitch ratios for each strain (<59% of BALB
[n=141] and <10% of B6 [n=140] punctuated USVs are plotted
in Figures 7c & 7d, respectively). Therefore, to examine whether
this apparent strain-dependent pattern had resulted from a sam-
pling bias, we sampled additional punctuated USVs from select
pairs of mice. Pitch-jumps from random samples (Figs. 7f & 7f9
re-illustrate data that are presented in Figures 7c & 7d,
respectively) were contrasted to pitch-jumps from SI tests that
we matched for the production of punctuated USVs a posteriori
(arrow g and arrowheads g19–g49 in Figure 7e denote pairs of mice
that emitted a comparable number of punctuated calls). The
strain-dependent pattern in paired-pitch ratios was still readily
apparent under conditions in which the pitch-jumps of one
particularly extreme BALB pair (Fig. 7g) were removed from the
plot (Fig. 7h) and when all of the pitch-jumps produced by 4
comparable B6 pairs (Figs. 7g19–7g49) were included (Fig. 7h9).
Additionally, although a majority of the punctuated USVs emitted
by early-adolescent mice contained 1–2 pitch-jumps, which
corresponded to 2–3 distinct elements per syllable, approximately
15% of the B6 punctuated calls included 3–4 pitch-jumps, as
illustrated in Figure 7i.
A set of tests for correlation revealed several additional relation-
ships between USV emission and the SI responses of early-
adolescent test mice (Table 2). While the total number of USVs
produced was positively associated with SI responses of test mice
from both strains (see Figures 5a & 5b), other patterns among
USV characteristics and SI were strain-specific. Interestingly,
longer calls predicted larger SI responses by BALB test mice (but
not B6 mice), whereas higher-pitched calls predicted greater social
responsiveness for B6 test mice (but not BALB mice).
Figure 7. Sonographic characteristics of punctuated ultrasonic vocalizations in relation to the genetic background of mice. (a) B6 mice emitted more
punctuated USVs than BALB mice. Four sonographic traces illustrate the varied form of punctuated USVs emitted by early-adolescent mice: (b1)
a BALB punctuated USV with 2 pitch-jumps, (b2) a B6 punctuated USV with 2 pitch-jumps and 2 harmonics corresponding to the 1stand 3rdelements
of the vocalization, (b3) a B6 punctuated USV with 1 pitch-jump and 1 harmonic corresponding to the 1stelement of the vocalization, and (b4) a B6
punctuated USV with 1 pitch-jump. (c) 141 BALB and (d) 140 B6 punctuated USVs were graphed as scatter-plots to illustrate the degree of internal
frequency modulation present within individual ultrasonic syllables. Data points lying off the diagonal of each plot represent pitch-jumps. (e) Line
graph depicting the number of punctuated USVs emitted during a 5-min interaction between mice that expressed the most punctuated USVs of each
strain. The arrow and arrowheads denote pairs of mice that emitted a similar number of punctuated USVs. (f–f9) Data re-plotted from Figures 7c and
7d, respectively. (g) The pitch-jumps of punctuated USVs from one extreme BALB pair. (g19–g49) The pitch-jumps of four B6 pairs that produced
punctuated USVs at a rate comparable to the BALB pair illustrated in Figure 7g. (h–h9) Data from Figures 7f and 7f9 with pitch-jumps from Figure 7g9
subtracted and the pitch-jumps from Figures 7g19–g49 added, respectively. (i) The total number of pitch-jumps within individual punctuated USVs for
each strain graphed as a relative-density histogram.
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Strain-dependent variation in the rewarding aspects
of social interaction:
To assess whether the strain-dependent difference in SI between
early-adolescent mice was related to the rewarding nature of social
interaction, we conducted an experiment using a social condi-
tioned place preference (SCPP) procedure (see Experiment 7).
Consistent with SCPP responses of early-adolescent B6 mice that
were exposed to a protocol which entailed 24-hr home cage
conditioning sessions (see ref. [51]), in the present study, PD 30–35
mice from the B6 strain expressed a robust place-preference
response for an aspen bedding-lined environment after it had been
associated with social interaction during three daily 30-min
conditioning sessions (Fig. 8; main effect of conditioning group,
F [5,82]=7.5, P,0.001). By contrast, age- and experience-
matched BALB mice were much less responsive to the same
conditioning contingency (between-strain orthogonal contrast, F
[1,82]=3.5, P,0.001). The sex of mice (F [1,82]=2.1, P=0.15),
nor its interaction with the particular conditioning-group (F
[5,82]=0.3, P=0.89), had an influence on the place preference
responses of adolescent mice.
Of note, repeated exposure of early-adolescent mice to each
environment (aspen and paper bedding), in the absence of social con-
ditioning, revealed a natural preference for paper bedding (t=4.8,
d.f.=39, P,0.0001 compared to HØ=0) for mice from both
strains (between-strain orthogonal contrast, F [1,82]=1.6, P=
0.12). Within the context of social conditioning, therefore, a strain-
dependent difference in SCPP was undetectable when paper
bedding had been associated with social enrichment (between-
strain orthogonal contrast, F [1,82]=0.9, P=0.37). However, as
mentioned above, early-adolescent B6 mice—unlike age-matched
BALB mice—spent substantially more time within an otherwise
negative environment (aspen bedding) once it had been associated
with opportunity for social interaction (see Figure 8).
DISCUSSION
Social approach
Adult female mice can give birth to a new litter at approximately
three-week intervals under favorable conditions. With the
emergence of new offspring, maternal behaviors towards the older
litter declines and juveniles are faced with the challenge of living
more independently as adolescents. This period of early adolescence is
a developmental stage when juveniles shift their social attention
away from maternal care and towards peers [19]. In feral
populations, young animals, including mice, experience a partic-
ularly high level of mortality after weaning [29,39,54,55], possibly
due to the inherent risks of traveling further from the nest.
Trapping studies have also demonstrated that adolescent mice are
captured in pairs more frequently than adults [56], which suggests
that young mice may travel in social groups while dispersing. A
heightened level of gregariousness among peers is in fact one of the
most consistent findings regarding studies of adolescent behavior
in mammals [8,20,22,24,32,33,57]. That robust forms of social
interaction are a regular occurrence among prepubescent animals
implies that some factor other than reproductive interests is the
primary motivation to engage in such aspects of sociality. While
adolescence includes the pubertal stages of development, it also
encompasses the more gradual acquisition of psychosocial skills
that precede, accompany and follow the capacity to produce
offspring [18,34,36]. Moreover, later stages of adolescence are
distinguishable from early adolescence and adulthood by differ-
ences in social behavior [20,22,24,58], responsiveness to environ-
mental or pharmacological manipulations [37,59–62] and hor-
mone sensitivity [41,63,64].
Consistent with this view of adolescent development, we found
that the motivation of early-adolescent B6 mice to investigate
a conspecific was unresponsive to the particular sex of the
individual they were paired with (i.e., the SI responses of B6 test
mice from all test-by-stimulus combinations were similar on PD
25/26). As mice matured, however, their social interests became
more sensitive to the sexual identity of the respective stimulus
mouse. Male test mice from the B6 strain, for example, expressed
larger SI responses towards females on PD 45/46, while age-
matched B6 females investigated other females more than males.
By contrast, sex-selective patterns of social approach were evident
for BALB mice on PD 25/26, as male test mice displayed a higher
level of social interest in females relative to males. Moreover, as
Table 2. Pearson’s correlation coefficients relating SI
responses of test mice to variables associated with USV
emission (* P,0.05, ** P,0.01, *** P,0.001).
......................................................................
USV variable BALB/cJ SIC57BL/6J SI
N[USV]
0.43** 0.40**
Pitch (kHz) 0.150.53***
Duration (msec) 0.58***0.10
N[upwards USV]
0.07 0.04
N[downwards USV]
0.58*** 0.52***
N[chevron USV]
0.53*** 0.08
N[complex USV]
0.45**0.03
N[punctuated USV]
0.30* 0.22
doi:10.1371/journal.pone.0000351.t002
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Figure 8. Strain-dependent variation in the social conditioned place
preference responses of early-adolescent mice. Unconditioned mice
from both strains expressed a preference for the paper bedding (control
bars) and this natural bias obscured any conditioning effect that might
have resulted from pairing social interaction with paper bedding (social
plus paper bars). However, there was a robust, strain-dependent SCPP
response for B6 mice when the less-preferred aspen bedding was
paired with social enrichment (social plus aspen bars). Preference scores
were calculated as the time each mouse spent in the aspen bedding-
lined environment minus the duration in the paper bedding-lined
compartment of the place-preference arena. All data are presented as
the mean6SEM (*** P,0.001 for BALB vs. B6 mice).
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BALB males matured, an additional increase in the bias towards
investigating females was evident. Overall, our results thus demon-
strate a strain-dependent influence on the social approach behaviors
of early-adolescent mice, which becomes increasingly obscured by
the emergence of sex-selective social interests in older mice.
Previously reported differences in the social tendencies of adult
BALB and B6 mice include a low level of parental care by
maternal BALB females, and both decreased sexual motivation
and increased aggressive tendencies among adult BALB males (see
ref. [49] for a review). Our preliminary studies indicated that,
when given a sufficient amount of time to interact (i.e., 10 min
instead of the 5-min duration that was used for Experiment 1), tail
rattling and wrestling behaviors occurred in approximately 35% of
M-M interactions that involved BALB mice (PD 39–42), but never
during the social interactions of age-matched pairs of B6 males.
Furthermore, five days before it became evident for similarly
paired BALB males, mounting behaviors occurred during
approximately 25% of the social interactions in which B6 test
males had been paired with a female stimulus mouse. These
patterns of social behavior lacked the overall intensity that is
typical of sexual and aggressive behaviors between adult mice. For
example, injuries were never observed on males that had been
tested and housed together throughout adolescence, while
mounting attempts were directed at the sides and front of a female
as often as they were targeted to the flank, and lordosis never
occurred. These findings are consistent with a view of adolescent
development in which a relatively strong and generalized form of
social motivation is gradually supplanted by behaviors that may
take on a function once puberty occurs. Importantly, we found
that the strain-dependent difference in SI was in fact smaller (F-F
pairings) or undetectable (M-F, F-M & M-M pairings) for mice
that were tested on PD 45/46. Together with our control
experiments (see Experiments 2–6), the data strongly suggest that
strain-dependent variation in the social approach phenotypes of
early-adolescent mice is attributable to a genetic influence on
a specific, social-motivational process.
Ultrasonic Vocalizations
Measurements of USV production among early-adolescent mice
complemented the strain difference that was found for SI. For
example, when the test mouse was a BALB male, the number of
USVs emitted during social interaction was particularly sensitive
to the sexual identity of the stimulus mouse. Despite this strain-
dependent difference, the quantity of USVs during social inter-
action was a strong, positive predictor of the extent that test mice
from both strains would investigate the stimulus mouse. This
finding is consistent with studies in rats, which suggest that USVs
reflect social affect [65]. The rate of USV emission by early-
adolescent mice in the present study (2.7 calls/sec collapsed across
strain, gender and time-into-trial) was very similar to what has
been previously reported for mice during contexts such as mating
[66,67] or infant social-isolations [68]. Interestingly, infant mice
from the B6 strain [69,70] and the related C57BL/10J strain [68]
tend to emit fewer USVs than BALB mice upon maternal separa-
tion, although infant B6 mice still appear to produce USVs of
a shorter duration and higher frequency [68]. To our knowledge,
the present study provides the first demonstration of USV emission
by mice outside of social contexts that are related to aggression,
mating or mother-infant attachments.
Aside from the twenty-two- and 50-kHz USVs produced by
young rats [71] and the lower-frequency distress calls of mouse
pups [72] few studies have evaluated the sonographic features of
rodent vocalizations within a behavioral context (also see refs.
[53,66,68,73]). Consistent with a recent study of song production
by adult mice [53], we found many USVs that were frequency-
modulated, occurring in repetitive bouts separated by periods of
silence. These vocalizations were remarkably complex, with
a significant effect of genotype on each distinct USV category that
was classified. Interestingly, the genetic background of mice within
an interacting pair was the sole influence on the production of
complex and punctuated USVs, which is topically similar to our
behavioral findings for early-adolescent B6 mice. Furthermore, the
pitch-jumps which characterized the sonographic profile of
punctuated USVs were much more variable for vocalizing B6 mice
relative to BALB mice. It is thus intriguing to consider whether
specific categories of vocalization may be functionally related to the
strain-dependent difference in SI among early-adolescent mice;
however, an assessment of social approach in the context of USV
play-backs will be necessary to fully evaluate this possibility.
Social Reward
We also found a strain difference in the reward value that early-
adolescent mice assigned to the opportunity for social interaction.
That young B6 mice found social contact rewarding is consistent
with our previous work [51]; however, it extends those findings by
demonstrating that a much shorter period of social interaction (i.e.,
30 min during the present study vs. 24 hrs during our previous
study) can also serve as an effective conditioning stimulus. Our
results thus indicate that some aspect of social contact, which
occurs within the first 30 min of reunion with conspecifics, can
serve as a powerful reward for early-adolescent B6 mice. By
contrast, age-matched BALB mice in the present study did not
express a SCPP response after a series of three daily 30-min
conditioning sessions nor do they following ten contiguous
conditioning sessions that alternate between 24-hr periods of
social enrichment and social isolation [51]. It is important to
mention one notable difference between the present findings and
our previous SCPP study. Unlike our previous experiments, we
found that unconditioned mice from both the BALB and B6
strains expressed a natural preference for the paper bedding.
Although this difference will require additional attention in future
experiments, one possible contributory factor is the amount of
experience that mice had with the aspen and paper beddings (i.e.,
mice had spent a total of 1.5 hrs with each bedding prior to testing
vs. 120 hrs with each bedding in our previous study).
Classical theories of motivation [74–76] propose that approach
behaviors are indicative of a state of reward and that neutral
stimuli can gain value through association with these rewarding
experiences [77], thereby extending the range of reward-related
opportunities that are available to an individual. In the present
study, we have shown that early-adolescent B6 mice are
particularly motivated to approach conspecifics, and while doing
so, produce USVs at a high rate. The B6 social phenotype is most
distinct from that of BALB mice during early adolescence, as the
strain difference gradually diminishes with the concurrent
emergence of sex-specific social approach by mice that are
approaching puberty. These findings are consistent with previous
studies that have underscored the fundamental importance of
‘timing’ for identifying the physiological [37,78] and gene-
regulatory [79,80] processes that underlie changes in behavior
(also see refs. [81,82]).
Well-defined genetic influences, which are operational during
a specific stage of behavioral development, provide a straightfor-
ward mechanism through which phenotypic variation can respond
to selection pressures that arise predictably during an animal’s
lifetime. The nature of social approach among early-adolescent B6
mice is consistent with observations that demonstrate social inter-
actions between young mammals can be amicable [8,22,33],
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involving unrelated individuals [83–85], and possibly functioning
to mitigate the risks associated with dispersal [86]. We propose
that the affiliative social interactions of young mice are driven by
a state of reward and that differences in the nature of these
interactions can be moderated by genetic factors during a relatively
narrow window of adolescent development.
ACKNOWLEDGMENTS
We thank Dr. Dan Goldowitz (University of Tennessee Health Sciences
Center, Nashville, TN, USA) and the Tennessee Genome Consortium for
their generous purchase of the ultrasound recording equipment. We also
thank EVP (Madison, WI, USA) for their stimulating contributions to
discussions regarding the design and analysis of the experiments presented
in this paper.
Author Contributions
Conceived and designed the experiments: JP GL. Performed the
experiments: JP JK. Analyzed the data: JP KJ JK JK EW QC CW GL.
Contributed reagents/materials/analysis tools: GL. Wrote the paper: JP
GL.
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