Electronic supplementary material
Materials and methods:
Animals
Behavioral tests were conducted in wildtype (Tph2+/+), heterozygous (Tph2+/-), and homozygous (Tph2-/-) mutant mice
(Alenina et al., 2009) on a C57BL/6N background (Mosienko et al., 2012). In several studies comparing the social
behavior of various mouse strains (with up to >10 strains), the highly related mouse strain C57BL/6J was found to
display high levels of social approach behavior and a clear preference for social novelty in the Crawley sociability test,
with its sociability scores being in the typical range (Nadler et al., 2003; Moy et al., 2004; Moy et al., 2007; Moy et al.,
2008). This strain was further shown to emit high levels of ultrasonic vocalizations (USV) as pups, juveniles, and adults
(Scattoni et al., 2008; Scattoni et al., 2011; Scattoni et al., 2013; Wöhr et al., 2008; Wöhr et al., 2011). Importantly,
differences between C57BL/6N and C57BL/6J in the production of USV were found to be minor (Wöhr et al., 2008).
Juvenile behavior
Time and frequency of physical contacts during juvenile social interaction were manually scored blindly to genotype
and sex of male and female pairs of Tph2+/+ (n=10/9), Tph2+/- (n=9/10), and Tph2-/- (n=4/6).
For detecting only murine USV in the experimental room, all fresh testcages were isolated from external sounds by
Styrofoam walls, leaving only one side open for video and sound detection. For detection of USV an UltraSoundGate
Condenser Microphone CM 16 (Avisoft Bioacoustics, Berlin, Germany) was used. Due to technical problems, some
audio-files had to be excluded from the USV analysis and was performed with the remaining male and female pairs of
Tph2+/+ (n=9/8), Tph2+/- (n=9/8) and Tph2-/- (n=4/5) pairs.
USV analysis was performed for the juvenile interaction test and for both urine expositions of adult mice. The USV
microphone (sensitivity: 15-180 kHz; flat frequency response: ±6 dB between 25-140 kHz) was connected to an
UltraSoundGate 416 USGH audio device (Avisoft Bioacoustics). Audio-files were stored on a personal computer and
were recorded in 16 bit format with a sampling rate of 250,000 Hz (Avisoft RECORDER version 2.97). Files were
analyzed in Avisoft SASLab Pro (version 5.20) with following specifications: 512 Fast Fourier Transformation (FFT)
length, 100 % frame, Hamming window and 75 % time window overlap. Produced spectrograms had 488 Hz frequency
resolution, 0.512 ms time resolution.A cut off frequency of <30 and >500 kHz was used. Spectrograms were manually
analyzed and USV-shouts were labeled blind to the respective test-conditions.
Social behavior in adult males
During social interaction with male or female conspecifics different behavioral types of test-mice were scored manually.
(1) Digging behavior, i.e. the animal was moving the forepaws in a digging manner or pushes saw dust to a small hill;
(2) rearings, i.e. the animal lifted itself on the hindpaws, raised the upper body with either forepaws on the cage-walls
or freely; (3) aggressive attacks, i.e. the animal approaches the other conspecific and bite, kick or savage the animal
such that the conspecific jumps visibly away; (4) social contacts, i.e. the animal has physical contact to the other
animal with the nose, flank or backside and stays with this contact for some seconds; (5) genital sniffing, i.e. the animal
pushed the nose to the ano-genital region of the conspecific and may even lift the other animal up; (6) mountings, i.e.
the animal mounts the conspecific from behind, hold it with both forepaws laterally and shows several lumbar trusts.
Besides these behavioral types of the test-mouse, also the amount of counter-attacks of the smaller FVB/N interaction
partners was scored, but only one animal showed a positive attack once during the whole test.
Results
Correlation analysis between different social and sexual behaviors
To compare co-appearance of the respective behavioral types, all variables collected during the three test-phases
were z-transformed to adjust the scoring level and a Pearson’s correlation was performed separately for each
genotype. Tph2+/+ male mice had a highly significant positive correlations between attacks and mountings of female
partners (r2=.851, p<.001), number of USVs to male urine before and after the interaction (r 2=.623, p=.017) and
between the number of genital sniffings to males and USV frequency to female urine after interaction (r2=.732, p=.003).
High negative correlations were visible between the time spent proximal and distal to female urine before interaction
(r2=-.903, p<.001), the time spent proximal to male urine before interaction and time spent distal to male urine after
interaction (r2=-.815, p<.001), and the number of mountings on males and number of attacks to females (r2=-.789,
p=.001). Thus, in wildtype Tph2+/+ mice sexual activity (mounting) and aggression (attacks) are related, while many
behavioral responses have a sex-specific bias.
In contrast, Tph2-/- male mice had no correlation between mountings and attacks to females, but high negative
correlation between the duration spent proximal and distal to the female urine after interaction (r2=-.795, p=.001), as
well as a high positive correlation between genital sniffings and social contacts to male partners (r 2=.732, p=.003) (for
detailed overview see Supplementary Table 1).
An overview of the detailed statistical analysis of juvenile interaction and vocalization is presented in supplementary
Table 2.
References
Alenina N, Kikic D, Todiras M, Mosienko V, Qadri F, Plehm R, Boyé P, Vilianovitch L, Sohr R, Tenner K, Hörtnagl H, Bader M.
Growth retardation and altered autonomic control in mice lacking brain serotonin. Proc Natl Acad Sci U S A. 2009 Jun
23;106(25):10332-7.
Mosienko V, Bert B, Beis D, Matthes S, Fink H, Bader M, Alenina N. Exaggerated aggression and decreased anxiety in mice
deficient in brain serotonin. Transl Psychiatry. 2012 May 29;2:e122.
Moy SS, Nadler JJ, Perez A, Barbaro RP, Johns JM, Magnuson TR, Piven J, Crawley JN. Sociability and preference for social
novelty in five inbred strains: an approach to assess autistic-like behavior in mice. Genes Brain Behav. 2004 Oct;3(5):287-302.
Moy SS, Nadler JJ, Young NB, Perez A, Holloway LP, Barbaro RP, Barbaro JR, Wilson LM, Threadgill DW, Lauder JM, Magnuson
TR, Crawley JN. Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains. Behav Brain Res. 2007 Jan
10;176(1):4-20.
Moy SS, Nadler JJ, Young NB, Nonneman RJ, Segall SK, Andrade GM, Crawley JN, Magnuson TR. Social approach and repetitive
behavior in eleven inbred mouse strains. Behav Brain Res. 2008 Aug 5;191(1):118-29.
Nadler JJ, Moy SS, Dold G, Trang D, Simmons N, Perez A, Young NB, Barbaro RP, Piven J, Magnuson TR, Crawley JN.
Automated apparatus for quantitation of social approach behaviors in mice. Genes Brain Behav. 2004 Oct;3(5):303-14.
Scattoni ML, Gandhy SU, Ricceri L, Crawley JN. Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism.
PLoS One. 2008 Aug 27;3(8):e3067.
Scattoni ML, Ricceri L, Crawley JN. Unusual repertoire of vocalizations in adult BTBR T+tf/J mice during three types of social
encounters. Genes Brain Behav. 2011 Feb;10(1):44-56.
Scattoni ML, Martire A, Cartocci G, Ferrante A, Ricceri L. Reduced social interaction, behavioural flexibility and BDNF signalling in
the BTBR T+ tf/J strain, a mouse model of autism. Behav Brain Res. 2013 Aug 15;251:35-40.“
Wöhr M, Dahlhoff M, Wolf E, Holsboer F, Schwarting RK, Wotjak CT. Effects of genetic background, gender, and early
environmental factors on isolation-induced ultrasonic calling in mouse pups: an embryo-transfer study. Behav Genet. 2008
Nov;38(6):579-95.
Wöhr M, Roullet FI, Crawley JN. Reduced scent marking and ultrasonic vocalizations in the BTBR T+tf/J mouse model of autism.
Genes Brain Behav. 2011 Feb;10(1):35-43.
Supplementary Table 1
Pearson’s product moment correlation between z-transformed values of scored behaviors before, during or after social
interaction separately for Tph2+/+ and Tph2-/- male mice (each n=14). Highlighted are only correlations that reached
significant level of p<0.05 and r2-values of more than ±0.560 for at least one genotype-group. n.s. not significant,
SocInt
Variable
Test
between
Adjustment
Statistics of factors
P
LSD post-hoc
er
Numbers of
One Way
genotypes
--F(2)=5.660
p=0.006
Tph2+/+ vs. Tph2+/Social
contacts
ANOVA
n.s.
Interac
+/+
-/Tph2 vs. Tph2
tion
p=0.018
Tph2+/- vs.Tph2-/phase
p=0.002
during
sex
--F(1)=1.020
n.s.
the
Two Way
Genotype and
5 min as levels Time
n.s.+/+
Tph2
Tph2-/testRepeated
sex
Time x geno
Sex of stimulus measurement
Behavior 1
Behavior 2
r2= n.s.P=
r2=
P=
battery
Time x sex
n.s.
Female
Duration
Duration distal to urine before
-.903
<.001
-.795
.001
ANOVA,
minproximal to urine
(days
Time x geno x sex
n.s.
SocInter
1-5before SocInter
3 & 4).
Genotype
p=0.013
Tph2+/+ vs. Tph2+/Female
Female
Female
Female
Female
Duration of
contacts
Female
Female
Female
Female
Males
Male
Male
Male
Male
Average
Contact
Inter-sex
duration
min 1-5
Inter-sex
Inter-sex
Inter-sex
Inter-sex
Number of aggressive
attacks
Duration proximal to urine
after SocInter
Number of mountings
Number of attacks
Number
to urine
One
Way of USVs
genotypes
before SocInter
ANOVA
Number of mountings
Duration distal to urine
before SocInter
sex
Number of self-groomings
Two Way
Genotype and
sex
Repeated
Number of genital sniffings
measurement
ANOVA,
minproximal to urine
Duration
1-5before SocInter
Number of USVs before
SocInter
Number of social contacts
Duration proximal to urine
after SocInter
Number of USVs before
One
SocInter
Way
genotypes
ANOVA
Genital sniffings on females
Number of USVs to female
urine after social interaction
Mountings
on females
Two
Way
Genotype and
Repeated
sex
measurement
Attacks on females
ANOVA, min
1-5
Attacks on females
Number of mountings
F(2)=4.799
.851
<.001
Duration distal to urine after
SocInter
Number of social contacts
Sex
Number of social contacts
Sex x geno
Number
of USVs to
urine after
--F(2)=5.657
SocInter
Number of shouts to urine after
SocInter
Number of rearings
-.645
.013
--F(1)0.196
Number of shouts to urine after
5 min as levels Time
SocInter
F(3.116)=9.031
Number of social Time
contacts
x geno
Duration distal toTime
urinexafter
sex
Time x geno x sex
SocInter
Genotype
Number of USVs after
SocInter
F(2)=5.166
Number of genital sniffings
Duration distal to urine after
SocInter
Sex
Duration proximalSex
to urine
before
x geno
SocInter
--F(2)=8.715
Duration distal to male urine
after social interaction
Genital sniffings on males
.581
.029
n.s.
.394
n.s.
n.s.
-.118 p=0.006
n.s.
.333
n.s.
-.523
.055
.448
n.s.
n.s.
p<0.001
-.092 n.s.n.s.
-.815 n.s.<.001
-.566
.035
.896
+/+
Tph2 vs.<.001
Tph2+/n.s.
.654+/+ vs..011
Tph2
Tph2-/p=0.038
+/-/Tph2
.629 vs.Tph2
.016
p=0.002
.602
.023
.587
-.789
.027
.001
n.s.
.623 p=0.010
.017
.328
-.447
n.s.
n.s.
n.s.
-.105 n.s.n.s.
p=0.001
-.789
.001
.732
.003
Number
USVs to
male
urine
-.710 p<0.001
.004
5
min as of
levels
Time
F(2.741)=7.450
after SocInter
Time
x geno
Number of USVs to
male
urine
-.539 n.s..047
Time x sex
n.s.
after SocInter
Time
x
geno
x
sex
Mountings on males
-.789 n.s..001
Genotype
-.304
n.s.
n.s.
+/+
Tph2 vs. Tph2-/p=0.020
-.701
.005
Tph2+/- vs.Tph2-/p=0.002
.083
n.s.
p=0.001
Tph2
Tph2+/-.091+/+ vs.n.s.
n.s.
+/+
Tph2
Tph2-/.732 vs..003
p=0.045
-.710+/- vs.Tph2
.004 -/- p=002
Tph2
-.539
.047
Tph2+/+ vs. Tph2+/n.s.
.160
n.s.
Tph2+/- vs.Tph2-/p<0.001
-.306
n.s.
Tph2+/+ vs. Tph2-/p=0.002
-.136
n.s.
-.018
n.s.
-.190
n.s.
Tph2+/+ vs. Tph2+/-
Suppl
ement
ary
Table
2:
Sex
n.s.
Sex x geno
n.s.
detaile
d statistics for juvenile interaction, behavior.The table shows One-Way and Two-Way repetitive measurement
ANOVA analysis with related post-hoc tests of juvenile contact frequency and duration with “genotype” and “sex” as
independent variables. n.s. = not significant.
F(2)=7.733
n.s.
Tph2+/+ vs. Tph2-/p=0.003
Tph2+/- vs.Tph2-/p<0.001
Supplementary Table 3: detailed statistics for juvenile interaction, USV production.
The table shows One Way, Two Way, and repetitive measurement ANOVA analysis with related Post Hoc tests of
juvenile USV numbers and duration with “genotype” and “sex” as independent variables. n.s. = not significant.
Variable
USV
numbers min
1-5
Test
One Way
ANOVA
between
genotypes
Adjustment
---
Subgroup
P
n.s.
sex
genotypes
--Separated for
sex
Separated for
genotypes
p=0.004
n.s.
n.s.
n.s.
n.s.
p=0.036
5 min as levels
F(1)=9.401
Within males
Within females
Within Tph2+/+
Within Tph2+/Within Tph2-/F(1)=6.668
Time (F4)=10.476
n.s.
genotypes
---
Time X geno
F(8)=0.416
Time x sex
F(4)=2.935
Time x geno x sex
F(8)=0.443
Genotype
Sex F(1)=11.648
Sex X geno
F(2)=4.098
sex
genotypes
--Separated for
sex
F(1)=10.226
Within males
Within females
F(2)=4.411
p=0.003
n.s.
p=0.028
sex
Two-Way
Repeated
measurement
ANOVA
USV duration
min 1-5
One Way
ANOVA
Genotype
and sex
LSD PostHoc
p<0.001
p=0.023
n.s.
n.s.
p=0.002
p=n.s.
p=0.024
Tph2+/+ vs. Tph2-/p=0.032
Tph2+/+ vs. Tph2+/- n.s.
Tph2+/- vs. Tph2-/p=0.007
Tph2+/+ vs. Tph2+/- n.s.
Tph2+/+ vs. Tph2-/p=0.044
Tph2+/- vs. Tph2-/p=0.009
sex
Two-Way
Repeated
measurement
ANOVA
Genotype
and sex
Separated for
genotypes
5 min as levels
Within Tph2+/+
F(1)=4.353
Within Tph2+/Within Tph2-/F(1)=6.143
Time (F4)=8.290
p=0.054
Time X geno
Time x sex
F(4)=2.736
Time x geno X sex
Genotype
F(2)=4.414
n.s.
p=0.031
Sex F(1)=16.287
Sex X geno
F(2)=3.514
p<0.001
P=0.040
n.s.
p=0.042
p<0.001
n.s.
p=0.019
Tph2+/+ vs. Tph2-/p=0.013
Tph2+/+ vs. Tph2+/- n.s.
Tph2+/- vs. Tph2-/p=0.002