Current Biology, Volume 29

Supplemental Information

Oxytocin Signaling in the Central Amygdala

Modulates Emotion Discrimination in Mice
Valentina Ferretti, Federica Maltese, Gabriella Contarini, Marco Nigro, Alessandra
Bonavia, Huiping Huang, Valentina Gigliucci, Giovanni Morelli, Diego Scheggia, Francesca
Managò, Giulia Castellani, Arthur Lefevre, Laura Cancedda, Bice Chini, Valery
Grinevich, and Francesco Papaleo
 D e m o n s tra to rs (T e s t)

A S n if f in g G r o o m in g R e a r in g B itin g F r e e z in g

60 60 60 60 60

45 45 45 45 45
***
Seconds

30 30 30 30 30

15 15 15 15 15

0 0 0 * 0 0
2 4 6 2 4 6 2 4 6 2 4 6 2 4 6
T im e (m in ) T im e (m in ) T im e (m in ) T im e (m in ) T im e (m in )

B S n if f in g G r o o m in g R e a r in g B itin g

60 60 60 60 N e u tr a l

45 45 45 45
Seconds

30 30 30 30 Fear

15 15 15 15

0 0 0 0 R e lie f
2 4 6 2 4 6 2 4 6 2 4 6
T im e (m in ) T im e (m in ) T im e (m in ) T im e (m in )

C
U S V s fre q u e n c y (K H z )

U S V s a m p lit u d e ( D b )
U S V s d u r a t io n ( m s )

5 25 30 10
USVs num ber

4 20
20 0
3 15

2 10
10 -1 0
1 5

0 0 0 -2 0
2 4 6 2 4 6 2 4 6 2 4 6
T im e (m in ) T im e (m in ) T im e (m in ) T im e (m in )

Figure S1. Demonstrators behavior during emotion discrimination test, Related to Figure 1, 2 and 3.
(A) Behaviors displayed by neutral and fear demonstrator mice during the fear emotion discrimination
paradigm. No demonstrator defecated or urinated during the whole test session. Emotion-by-time statistical
interaction for sniffing (F2,36=2.72, p=0.08), grooming (F2,36=1.07, p=0.35), rearing (F2,36=5.09, p=0.01),
biting (F2,36=1.28, p=0.29), and freezing (F2,36=48.82, p<0.0001). *p<0.05, and ***p<0.0001 versus all other
points. n=10 demonstrators per group. (B) Behaviors displayed by neutral and relief demonstrator mice
during the relief emotion discrimination paradigm. No demonstrator defecated or urinated during the whole
test session. No significant emotion-by-time statistical interaction was evident for sniffing (F2,36=0.09,
p=0.92), grooming (F2,36=0.34, p=0.71), rearing (F2,36=0.31, p=0.73), and biting (F2,36=0.84, p=0.44). n=10
demonstrators per group. (C) Schematic drawing of the test setting to record USVs. Mean number and
duration, peak frequency and amplitude at the maximum of the spectrum of USVs emitted by single
demonstrator mice in neutral (grey bar), fear (red bar), or relief (yellow bar) emotional state are reported.
Two-Way ANOVAs showed no significant differences. n=6 demonstrators per group.
Error bars represent standard error of the mean.
A 60
B 60
N e u tra l

S n if f in g ( s e c )
S n if f in g ( s e c )
**
40
* 40
Fear

R e lie f

20 20

0 0
2 4 6 2 4 6
T im e (m in ) T im e (m in )
C 40 D 40
S n if f in g ( s e c )

S n if f in g ( s e c )
** **
* *
20 20

0 0
M a le F e m a le M a le F e m a le

E F G
Test 30 40 *
S n if f in g ( s e c )

S n if f in g ( s e c )
30
20 *
20
10
10

0 0
2 4 6 2 4 6
T im e (m in ) T im e (m in )
R e -te s t 30 40
S n if f in g ( s e c )

S n if f in g ( s e c )

* 30
20 **
20
10
10

0 0
2 4 6 2 4 6
T im e (m in ) T im e (m in )

H I N e u t r a l- N e u t r a l
C o r t ic o s t e r o n e
d is c r im in a t io n

( f o ld c h a n g e )

1 .6 1 .5 N e u t r a l - R e li e f
r= -0 ,0 7
E m o t io n

0 .8
1
0 .0
0 .5
- 0 .8

- 1 .6 0
0 25 50 75 100
S o c ia l in t e r a c t io n
Figure S2. Characterization of the mouse emotion discrimination test, Related to Figure 1 and 2. (A-B)
Time (in seconds) spent by each male and female observer mice sniffing the two demonstrators in the (A)
fear and (B) relief paradigms. Emotion-by-time interaction for fear (F2,108=5.00, p=0.008), or relief
(F2,116=6.71, p=0.002) paradigm. n=28/30 observers per group. *p<0.05 and **p<0.005 versus all other
points. (C-D) Direct comparison between male and female emotion discrimination abilities in the (C) fear
(last 2-min RM ANOVA Male: F1,15=6.51, p=0.022; Female: F1,11=10.98, p=0.006), or (D) relief (first 2-min
RM ANOVA Male: F1,14=15.07, p=0.001; Female: F1,14=14.60, p=0.001) paradigm. *p<0.05 and **p<0.005
versus the neutral demonstrator. n=12/16 observers per group. (E) Schematic drawing of the test-retest
reliability validation. (F-G) Time (in seconds) spent by observer mice sniffing the two demonstrators in the
(F) fear or (G) relief paradigms during their first and second exposure (one week later) to the emotion
discrimination test. RM ANOVA for the “fear” manipulation, last 2-minute session, Test: F1,13=6.10,
p=0.028; Re-Test: F1,13=8.59, p=0.012. RM ANOVA for the “relief” manipulation, first 2-minute session,
Test: F1,10=5.15, p=0.046; Re-Test: F1,10=22.88, p=0.0008. *p<0.05, and **p<0.005 versus the exploration of
the neutral demonstrator. n=11/14 observers per group. Time spent sniffing neutral, fear and relief
demonstrators is depicted in grey, red and yellow, respectively. (H) Correlation analyses between the emotion
discrimination index (in y axis) and social interaction (in x axis). No significant correlation was found (r=-
0.07). n=101 observers. (I) Blood corticosterone levels displayed by observer mice immediately after being
tested in the emotion discrimination test with two neutral demonstrators (grey bar), or one neutral and one
relief demonstrators (yellow bar). Data are expressed as fold changes compared to observers exposed to two
neutral demonstrators (T-test: df: 9; p=0.18). n=5/6 observers per group.
Error bars represent standard error of the mean.
A B
40

S n iffin g (s e c )
30 **
20

10

0
Veh CNO
N e u tr a l

e ffe c t o f C N O tre a tm e n t p e r s e Fear

C D R e lie f

20 40
* *
*
S n iffin g (s e c )

S n iffin g (s e c )

* 30

10 20

10

0 0
Veh CNO Veh CNO

Figure S3. Control experiments for DREADDs manipulations, Related to Figure 4 and 5. (A) Scheme
of the viral vector used to infect PVN OXT neurons. (B) Time (in seconds) spent sniffing the two
demonstrators during the first 2 minutes of the emotion discrimination test, displayed by the same virus-
injected observer female mice treated with vehicle or CNO (3mg/kg in a volume of 10 ml/kg, i.p., 30 minutes
before the test), and shown separately for each demonstrator’s emotion. Time spent sniffing neutral
demonstrators is represented by grey bars. Time spent sniffing demonstrators with water-induced relief states
(first 2-min RM ANOVA Veh: F1,9=15.61, p=0.0033; CNO: F1,9=1.04, p=0.33) is represented by yellow bars.
**p<0.005 versus the neutral demonstrator within the same observer treatment. n=10 observers per group.
(C-D) Assessment of CNO treatment per se in the emotion discrimination test. Time (in seconds) spent
sniffing two demonstrator mice during (C) the last 2 minutes of the fear paradigm (RM ANOVA Veh:
F1,5=8.55, p=0.032; CNO: F1,5=8.60, p=0.042) and (D) the first 2 minutes of the relief paradigm (RM
ANOVA Veh: F1,5=9.55, p=0.027; CNO: F1,5=10.56, p=0.022) displayed by the same observer mice treated
with vehicle or CNO (i.p., 3mg/kg in a volume of 10 ml/kg, 30 minutes before the test). Time spent sniffing
neutral demonstrators is represented by grey bars. Time spent sniffing demonstrators with tone-induced
fearful states or water-induced relief states is represented by red or yellow bars, respectively. *p<0.05 versus
the neutral demonstrator within the same observer treatment. n=6 observers per group.
Error bars represent standard error of the mean.
 N e u tra l N e u tra l

Fear R e lie f
A F K
40 40 150
*

S n if f in g ( s e c )

S n if f in g ( s e c )
S n if f in g ( s e c )
30 * 30
100
20 20
50
10 10

0 0 0
T im e (m in ): 2 4 6 2 4 6 2 4 6 2 4 6
Veh CNO Veh CNO Veh CNO

B G L
40 40 150
*

S n if f in g ( s e c )
S n if f in g ( s e c )

S n if f in g ( s e c )
30 30
100
20 * 20
50
10 10

0 0 0
T im e (m in ): 2 4 6 2 4 6 2 4 6 2 4 6
Veh CNO Veh CNO Veh CNO
C H M
40 40 150
S n if f in g ( s e c )

S n if f in g ( s e c )

S n if f in g ( s e c )
30 30 * *
100
20 * * 20
50
10 10

0 0 0
T im e (m in ): 2 4 6 2 4 6 2 4 6 2 4 6
Veh CNO Veh CNO Veh CNO

D I N
40
* 40 150
S n if f in g ( s e c )

S n if f in g ( s e c )

S n if f in g ( s e c )
30 * 30 * 100
20 20
*
50
10 10

0 0 0
T im e (m in ): 2 4 6 2 4 6 2 4 6 2 4 6
Veh CNO Veh CNO Veh CNO
E J O
40 40 150
S n if f in g ( s e c )

S n if f in g ( s e c )
S n if f in g ( s e c )

30 30 *
* 100
20 * * 20
50
10 10

0 0 0
T im e (m in ): 2 4 6 2 4 6 2 4 6 2 4 6
Veh CNO Veh CNO Veh CNO

Figure S4. Effect of selective inhibition of PVN OXT projections in the emotion discrimination test and
one-on-one social interaction, Related to Figure 4 and 5. (A-E) Scheme of the viral vector used to infect
the PVN OXT neurons and respective projecting area (CeA, NAcc, mPFC or CA2). (F-J) Time (in seconds)
spent sniffing the two demonstrators during the emotion discrimination test displayed by the same observer
mice treated with vehicle or CNO, and shown separately for each demonstrator’s emotion. Time spent
sniffing neutral demonstrators is represented by grey bars. Time spent sniffing demonstrators with tone-
induced fearful states, or water-induced relief states is represented by red or yellow bars, respectively.
*p<0.05 versus the exploration of the neutral demonstrator. n=7/11 observers per group. (K-O) Time (in
seconds) spent sniffing an unfamiliar age- and sex-matched conspecific during 4 minutes of free social
interaction displayed by the same mice treated with vehicle or CNO. PVN (one-way ANOVA F1,4=0.19,
p=0.68); PVN-CeA (one-way ANOVA F1,18=0.081, p=0.78); PVN-NAc (one-way ANOVA F1,16=0.006,
p=0.94); PVN-mPFC (one-way ANOVA F1,10=0.005, p=0.94). n=4/10 per group.
Error bars represent standard error of the mean.
 A B
IN LV

OXT promoter Venus BLA

MeA
ITR ITR

-1,06 mm -1,58 mm +0,86 mm

BLA MeA IN
PVN

-1,06 mm

C D
-64mV
-62mV

80pA 80pA

0pA 0pA
-60pA -60pA
-30pA -30pA

E F Pre-CNO
G Pre-CNO
hM4D 20 20
OXT promoter
ITR
CAV-Cre
ITR
15 15 **
# of spikes

# of spikes
CA2
10 10
Post-CNO Post-CNO
5 5
PVN
0 0

Pre-CNO Post-CNO Pre-CNO Post-CNO

H OTp-hM4D-mcherry

PVN
-0,83 -0,94 -1,06 -1,22

I OTp-DIO-hM4D-mcherry
CAV-Cre

CeA PVN

J OTp-DIO-hM4D-mcherry

CAV-Cre

PVN
NAcc

K OTp-DIO-hM4D-mcherry

CAV-Cre
mPFC

PVN

L OTp-DIO-hM4D-mcherry
CAV-Cre
CA2

PVN
Figure S5. Anatomy of PVN OXT projections, ex vivo electrophysiological validation and placement of
back-labeled PVN neurons, Related to Figure 4 and 5. (A) Scheme of the viral vector used to infect PVN
OXT neurons. (B) OXT projections from the PVN to the basolateral amygdala (BLA), medial amygdala
(MeA), and insular cortex (IN). Scale bar: 100 μm. (C-D) Representative IR-DIC image of back labeled (C)
parvocellular and (D) magnocellular neurons during patch-clamp recordings. Right: in current clamp mode in
response to depolarizing current pulses (IHold: 0 pA): (C) the parvocellular neuron lacks transient outward
rectification (black arrow in the zoomed trace), whilst (D) the magnocellular neuron displays an inward
rectification and a strong transient outward rectification (black arrow in zoomed trace). (E) Scheme of the
viral vector used to infect the PVN OXT neurons and respective projecting area (CA2). (F-G) Example traces
of membrane potential changes (left) and quantification (right) of the number of spikes evoked by a 1 sec of
20 pA current step (IHold: 0 pA) in (F) non infected neurons and in (G) PVN-CA2 neurons pre- and post- bath
application of CNO. Scale bars: 40 mV and 500 ms. Two-tailed Wilcoxon matched-pairs signed rank test: (F)
n=12 from 8 mice (p=0.25); (G) n=8 from 3 mice (p=0.0078). **p<0.01. (H) Placements of PVN injected
with hM4D(Gi) DREADD receptor under the control of the OXT promoter. (I-L) Placements of selective
PVN projecting neurons to the (I) CeA, (J) NAcc, (K) mPFC, and (L) CA2 obtained by bilateral injection in
these areas of CAV2-Cre virus combined with bilateral injection in the PVN of rAAV carrying a double-
floxed inverted open reading frame (ORF) (DIO) of the inhibitory hM4D(Gi) DREADD receptor and
mCherry under the control of the OXT promoter.
Figure S6. Dysbindin+/- show no deficits in sociability and social novelty in the 3-chamber task.
Validation of AAV-mediated rescue of CeA OXTR levels, Related to Figure 6. (A) Time (in seconds)
spent sniffing one unfamiliar mouse (novel mouse 1) and one novel object during the sociability phase of the
3-chamber task displayed by Dys+/+ and Dys+/- male and female littermates. RM ANOVA, Males Dys+/+:
F1,12=32.07, p=0.0001; Dys+/-: F1,12=26.16, p=0.0003; Females Dys+/+: F1,12=28.60, p=0.0002; Dys+/-:
F1,13=36.67, p=0.001. **p<0.005 and ***p<0.0005 versus the novel object within the same genotype. n=8/14
per group. (B) Time (in seconds) spent sniffing one unfamiliar mouse (novel mouse 2) and the now familiar
mouse (novel mouse 1) during the social novelty phase of the 3-chamber task displayed by Dys+/+ and
Dys+/- male and female littermates. RM ANOVA, Males Dys+/+: F1,12=42.41, p<0.0001; Dys+/-:
F1,12=13.44, p=0.0032; Females Dys+/+: F1,12=32.05, p=0.0001; Dys+/-: F1,13=29.59, p=0.0001. **p<0.005
and ***p<0.0005 versus the novel mouse within the same genotype. n=8/14 per group. (C) Scheme of the
viral vector used to unilaterally infect the CeA of OXTR-/- mice (left) and autoradiograms of OXT binding
sites (right) that show efficiency and spreading of virus expression and spreading. (D) Scheme of the viral
vectors used to bilaterally infect the CeA of Dys+/+ and Dys+/- littermates (left) and autoradiograms of OXT
binding sites (right). Expression of OXTR was assessed by using the ligand binding of 20 pmol/l I125-labeled
OVTA. (E) Quantification of the autoradiographic I125 receptors in Dys+/+ and Dys+/- is expressed as fold
changes from the Dys+/+ control group (Two-Way ANOVA genotype-by-treatment interaction F2,13=12.86;
p=0.0008). *p<0.05 and ***p<0.0005 versus Dys+/+ control virus group. n=3/6 per group. (F) Placements of
Dys +/- mice injected in the CeA with AVV expressing OXTR and EYFP under the control of the EF1a
promoter.
Error bars represent standard error of the mean.