Trac

ing

expe

rimen

t

a

BNST ILCx

CTb

cc

CTb

BNSTILCx

CTb

b cvSUB/CA1

CTb

Supplementary Figure 1

vSUB/CA1

Supplementary Figure 1: The amBNST receives strong innervations from both ILCx and vSUB/CA1. a. Experimental protocol. b. Injection site of retro-grade tracer CTb in the amBNST. Scale bar: 0.5 mm. c. Detection of the retrograde tracer CTb in the vSUB/CA1 and in the ILCx. Scale bar: 0.1 mm. LQVHW��VFDOH�EDU� ����ȝP�

In v

ivo

re

co

rdin

gs

Rec: BNST Stim: vSUB/CA1 Stim: ILCx

BNSTILCx vSUB/CA1

Stim. HFSvSUB/CA1

Rec.

d HFS stimulation protocol

10 pulses, 400 Hz

1 s

10 min; 500 pulses; 400 Hz

60 s

e

f

Supplementary Figure 2

a

vSUB/CA1

Rec.vSUB/CA1

1 s

0.5V

250s

0

125(Hz)

inst.

fre

qu

en

cy

Sp

ike

s

5ms

0.5V

bvSUB/CA1 neuron

200

150

100

50

0

n=

12

vSUB/CA1

ma

xim

um

in

st.

fire

qu

en

cy

(Hz)

c

Supplementary Figure 2: High frequency stimula-

tion protocol. a. Experimental protocol (top) and

example of histological of recording area (bottom).

Scale: 0.5 mm. b. Example trace of a vSUB/CA1

neuron recorded with the instantaneous frequency

(inst. Frequency) and an inset , scale bar 5 ms. c.

Histogram of the mean average of the maximum

instantaneous frequency of vSUB/CA1 neurons

expressed in Hz. d. High frequency stimulation

protocol. e-f. Experimental protocol and histological

control of recording and stimulating areas where

HFSvSUB/CA1

has been assessed. Scale bar: 0.5 mm.

In v

ivo

reco

rdin

gs

aStim. HFS

vILCxRec.

Supplementary Figure 3

b

150

100

50

0

ILCx

Rm

ag (

% b

asel

ine)

vSUB/CA1

Before HFSILCx

After HFSILCX

Time (min.)

150

100

50

0

200

10 20 30 400-10

Rm

ag (%

bas

elin

e)

HF

SIL

Cx

ILCx (n=5)

vSUB/CA1 (n=4)

c

Supplementary Figure 3: HFS applied in the ILCx

do not induce plasticity in the ILCx nor in the vSUB.

a. Experimental protocol. b. Kinetic (left) and quantifi-

cation (right) of the mean percentage change (± sem)

in ILCx evoked spike probability (green) and in

vSUB/CA1 evoked spike probability (purple), normal-

ized to the baseline, after HFSILCx

. Rmag, excitatory

response magnitude.

0

10

20

30

40

50

60

70

aCSF

AP5

SHAM

a

b

Circ

adia

n cy

cle

Be

am

bre

aks/

10

min

Beam

breaks/6

0m

in

200

100

400

300

200

22 0 2 4 6 8 10 12 14 16 18

aCSF-SHAM

AP5-SHAM

Time (h.)

20

Light on Light off

Supplementary Figure 4

Loco

mot

or a

ctiv

ity

aCSF-HFSvSUB/CA1

AP5-HFSvSUB/CA1

HFSvSUB/CA1

Anx

iety

test

s

SHAM HFSvSUB/CA1

0

5

10

15

20

aCSF

AP5

cbasal situation

d

0

5

10

15

20

SHAM HFSvSUB/CA1

SHAM HFSvSUB/CA1

e

0

5

10

15

20

25

Tra

nsitio

ns

Tra

nsitio

ns

Tra

nsitio

ns

anxiogenic situation anxiogenic situation

EPMLD box LD box

Supplementary Figure 4: Locomotor activity and circadian

rhythms of general activity in sham or HFSvSUB/CA1

treated animals

after ACSF or AP5 infusion in the amBNST. a. Locomotor activity

in a novel environment. b. circadian rhythms of general activity.

No between-group differences in novelty-induced locomotor

activity or circadian rhythms of general activity excluded

differences unrelated to anxiety. c. Histograms showing the

number of transitions in the light-dark box test after AP5 (grey) or

vehicle in the BNST (aCSF, white) followed by HFSvSUB/CA1

or

sham manipulation in basal situation. d-e. Histograms showing

the number of transitions in the light-dark box test (d) and in the

open arms in the EPM (e) in the SHAM group and in HFS vSUB/CA1

group in anxiogenic situation.

ILCx

BNST

ILCx

HFSvSUB/CA1

BNST neuron

LTDILCx

LTPvSUB/CA1

Supplementary Figure 5

vSUB/CA1

vSUB/CA1

NMDA non NMDA

anxiolytic effect

NMDA R- dependent

plasticity

Supplementary Figure 5: NMDA-R-dependent

plasticity in the amBNST induced by HFSvSUB/CA1

triggers anxiolytic effect.

A proposed diagram of the circuit’s mechanisms in

response to HFSvSUB/CA1

. amBNST neurons

integrate, at the single-cell level, inputs from both

ILCx and vSUB/CA1. HFSVSUB/CA1

induced a NMDA

dependent LTP vSUB/CA1

whereas it promoted an

NMDA independent LTDILCx

. In this model, in vivo

NMDA-R dependent plasticity in amBNST neurons

after HFSvSUB/CA1

induced anxiolytic effect in rats.