1

Supplementary Information for

Basolateral amygdala input to the medial prefrontal cortex controls obsessive-compulsive disorder-like checking behavior

Tingting Sun1, Zihua Song1, Yanghua Tian1, Wenbo Tian, Chunyan Zhu, Gongjun Ji, Yudan Luo, Shi

Chen, Likui Wang, Yu Mao, Wen Xie, Hui Zhong, Fei Zhao, Min-Hua Luo, Wenjuan Tao, Haitao Wang, Jie Li, Juan Li, Jiangning Zhou, Kai Wang* and Zhi Zhang*

1These authors contributed equally to this work.

*Corresponding authors:

Zhi Zhang, Ph.D. Key Laboratory of Brain Function and Disease

Department of Biophysics and Neurobiology University of Science and Technology of China Hefei, Anhui 230027, China Tel.: (+86) 551-63602715 Fax: (+86) 551-63602715 E-mail: zhizhang@ustc.edu.cn Kai Wang, MD., Ph.D.

Department of Neurology The First Affiliated Hospital of Anhui Medical University Hefei, Anhui 230022, China Tel.: (+86) 551-62923704 Fax: (+86) 551-62923704 E-mail: wangkai1964@126.com

This PDF file includes:

Material and Methods

Supplementary Figures 1 to 12

References

www.pnas.org/cgi/doi/10.1073/pnas.1814292116

2

Materials and Methods

Animals. In all experiments, C57BL/6J (purchased from Beijing Vital River Laboratory Animal

Technology Co., Ltd., China), GAD2-Cre, CaMKII-Cre, Ai14 (RCL-tdT) male mice (purchased from

Charles River or Jackson Laboratories, USA) at 8-10 weeks of age were used. Until the cannula surgery,

the mice were housed five per cage in a colony with ad libitum access to water and food (standard mouse

chow). They were maintained under a 12-hour light/dark cycle (lights on from 7:00 a.m. to 7:00 p.m.) at a

stable temperature (23-25 ºC). All animal protocols were approved by the Animal Care and Use Committee

of the University of Science and Technology of China.

Animal model of compulsive sucrose drinking behavior. Chronic injection of dopamine D2 receptor

agonist quinpirole hydrochloride (0.75 mg/kg) was used to construct an OCD-like behavioral mouse model.

The behavioral field was a mouse cage (25 × 16 × 19 cm) containing a 7 × 7 × 8.5 cm opaque home base

for the mouse to rest, with an opening diameter of 4.5 cm. Two bottles containing water and 10% (w/v)

sucrose solution, respectively, were placed on the other side of the cage (Fig. 1A). A stopwatch was used to

record the mouse behavior.

In the training paradigm, mice were adapted in cages once every two days a total of three times (150

min per session), and then quinpirole was subcutaneously injected under the nape of the neck (once every

two days). Equivalent volumes (70 μl) of saline were used for control injections. On the first day, quinpirole

significantly suppressed the amount of locomotion and movement, which progressively increased in the

subsequent sessions. The quinpirole mice revisited the home base and 10% sucrose solution container in the

environment more excessively than any other place in the first 60 to 120 min after 30 days of training,

compared with saline controls. The frequency and duration of drinking of the solution from the 60th min to

the 120th min of the 150-min training period were analyzed (Fig. 1B). The consumption of sucrose and

water in this period was calculated (SI Appendix, Fig. S1A).

3

Virus injections. Prior to surgery, the mice were fixed in a stereotactic frame (RWD, Shenzhen, China)

under a combination of xylazine (10 mg/kg) anesthesia and ketamine (100 mg/kg) analgesia. A heating pad

was used to maintain the core body temperature of the animals at 36°C. A volume of 100-300 nl virus

(depending on the expression strength and viral titer) was injected using calibrated glass microelectrodes

connected to an infusion pump (micro 4, WPI, USA) at a rate of 30 nl/min. The coordinates were defined

as dorso-ventral (DV) from the brain surface, anterior-posterior (AP) from bregma, and medio-lateral (ML)

from the midline (in mm).

For retrograde monosynaptic tracing, helper viruses that contained rAAV-Ef1α-DIO-RVG-WPRE-pA

(AAV-DIO-RVG, AAV2/9, 2 × 1012 vg/ml) and rAAV-Ef1α-DIO-EGFP-2a-TVA-WPRE-pA (AAV-DIO-

TVA-GFP, AAV2/9, 2 × 1012 vg/ml; 1:2, 200 nl) were co-injected into the mPFC of Cre transgenic mice

(AP, +1.77 mm from bregma; ML, -0.3 mm; DV, -1.5 mm). After three weeks, the rabies virus RV-ENVA-

ΔG-DsRed (2 × 108 IFU/ml, 300 nl) was injected into the same site in the mPFC (1). Mice that had been

anesthetized with pentobarbital (20 mg kg-1, i.p.) were transcardially perfused 7 days after the last injection,

and brain slices were prepared (40 µm) for DsRed tracing or co-staining with glutamate antibody. In some

experiments, retrogradely transported Fluoro-Gold was injected into the mPFC (80 nl) for visualized

electrophysiological recordings in slices at 7 days after injection (2).

For anterograde tracing, Cre-dependent virus rAAV-Ef1α-DIO-hChR2 (H134R)-mCherry-WPRE-pA

(AAV-DIO-ChR2-mCherry, AAV2/9, 1.63 × 1013 vg/ml, 200 nl) was delivered into the BLA of CaMKII-

Cre mice (AP, -1.25 mm from bregma; ML, -3.0 mm; DV, -4.15 mm). After four weeks, the expression of

mCherry was detected in the whole brain. In some experiments, rAAV-Ef1α-DIO-eNpHR3.0-eYFP-

WPRE-pA (AAV-DIO-eNpHR3.0-eYFP, AAV2/9, 1.18 × 1013 vg/ml) was used for optogenetic

manipulation (3, 4). The rAAV-Ef1α-DIO-hM3D(Gq)-mCherry-WPRE-pA (AAV-DIO-hM3Dq-mCherry,

AV2/8, 2.69 × 1013 vg/ml) and rAAV-Ef1α-DIO-hM4D(Gi)-mCherry-WPRE-pA (AAV-DIO-hM4Di-

mCherry, AAV2/9, 3.69 × 1013 vg/ml) viruses were used for chemogenetic manipulations three weeks after

injection. CNO (3 mg/kg, i.p.) was injected and behavioral tests were performed 30 min after injection (5).

The rAAV-Ef1α-DIO-mCherry-WPRE-pA (AAV-DIO-mCherry, AAV2/8, 8.93 × 1012 vg/ml) and rAAV-

4

DIO-eYFP-WPRE-pA (AAV-DIO-eYFP, AAV2/9, 1.95 × 1012 vg/ml) viruses were used as the controls.

Unless otherwise stated, all viruses were packaged by BrainVTA (Wuhan, China). All mice were

transcardially perfused with 0.9% saline followed by ice-cold phosphate buffer (0.1 M) that contained 4%

paraformaldehyde. Images of the signal expression were acquired with a confocal microscope (LSM 710,

ZEISS, Germany). Animals with missed injections were excluded.

Optogenetic manipulations in vivo. An optical fiber was implanted into the mPFC, in the brain of an

anesthetized mouse that had been immobilized in a stereotaxic apparatus. The implant was secured to the

animal’s skull with dental cement. Chronically implantable fibers (diameter, 200 μm, Newdoon, Hangzhou)

were connected to a laser generator using optic fiber sleeves. The delivery of a 5-min pulse of blue light

(473 nm, 2-5 mW, 10-ms pulses, 20 Hz) or yellow light (594 nm, 5-8 mW, constant) was controlled by a

Master-8 pulse stimulator (A.M.P.I., Jerusalem, Israel). The same stimulus protocol was applied to the mice

in the control group. The location of the fibers was examined in all mice at the conclusion of the experiments,

and data obtained from mice in which the fibers were located outside of the desired brain region were

discarded. Behavioral assays were performed immediately after light stimulation.

Open field test. Mice were placed in one corner of an open field apparatus that consisted of a square area

(25 cm × 25 cm) and a marginal area (50 cm × 50 cm × 60 cm); the mice were allowed to freely explore

their surroundings. The animals’ movement trajectories were recorded for 5 min using EthoVision XT

software, which records the number of entries into and the amount of time spent in the central area. The area

was cleaned with 75% ethanol after each test to remove olfactory cues from the apparatus.

In vivo microdialysis-HPLC. Electrochemical detection in combination with HPLC (Antec, Netherlands)

was used to measure the glutamate concentration in the mPFC of freely moving mice (6). A microdialysis

probe (CMA7, CMA, USA) connected to a syringe infusion pump (CMA402, CMA, USA) via polyethylene

tubing was initially implanted into the mPFC of deeply anesthetized mice. The tissue was perfused with

normal Ringer’s solution (3 mM KCl, 145 mM NaCl, and 1.3 mM CaCl2) via the pump at a rate of 1 μL/min,

5

and the dialysate was collected in a 20-μL quantitative loop through the probe in freely moving mice. The

dialysate was reacted with 5 μL O-phthalaldehyde (OPA) for 3 min and then was loaded to the mobile phase

(90% 0.1 M NaH2PO4 and 10% methanol) and separated on a 1 mm × 50 mm column (ALF-105, Antec)

with a 3 μm particle size at a rate of 0.35 mL/min. Detection was conducted using an Alexys online analysis

system (Antec Leyden) that consisted of a DECADE II electrochemical detector and VT-3 electrochemical

flow cells. The data were analyzed using Clarity software (Antec, Netherlands) based on standard samples.

Brain slice preparation. Acute brain slices were prepared as previously described (7). Mice were deeply

anesthetized with pentobarbital sodium (2% w/v, i.p.) and intracardially perfused with ~20 ml ice-cold

oxygenated modified NMDG artificial cerebrospinal fluid (NMDG ACSF) that contained (in mM) 93 N-

methyl-D-glucamine (NMDG), 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5

Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2, 10 MgSO4, and 3 glutathione (GSH) (osmolarity: 300-310

mOsm/kg). The pH of NMDG ACSF was titrated to 7.3-7.4 with concentrated HCl. The pH of the ACSF

was 7.3-7.4, and its osmolarity was 300-305 mOsm/kg. Coronal slices (300 µm) that contained the mPFC,

or BLA were sectioned at 0.18 mm/s on a vibrating microtome (VT1200s, Leica, Germany). The brain slices

were initially incubated in NMDG ACSF for 10-12 min at 33°C, followed by N-2-hydroxyethylpiperazine-

N-2-ethanesulfonic acid (HEPES) ACSF that contained (in mM) 129 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgSO4,

1.2 KH2PO4, 20 NaHCO3, 3 HEPES and 10 glucose (pH: 7.4, osmolarity: 300-310 mOsm/kg) for at least 1

hour at 25°C. The brain slices were transferred to a slice chamber (Warner Instruments, USA) for

electrophysiological recording and were continuously perfused with ACSF at 2.5-3 ml/min at 28°C. The

temperature of the ACSF was maintained by an in-line solution heater (TC-344B, Warner Instruments,

USA).

Whole-cell patch-clamp recordings. Neurons in the slice were visualized using a 40 × water-immersion

objective on an upright microscope (BX51WI, Olympus, Japan) equipped with interference contrast

(IR/DIC) and an infrared camera connected to the video monitor. Whole-cell patch-clamp recordings were

obtained from visually identified BLA or mPFC cells. Patch pipettes (5-8 MΩ) were pulled from borosilicate

6

glass capillaries (VitalSense Scientific Instruments Co., Ltd., Wuhan, China) with an outer diameter of 1.5

mm on a four-stage horizontal puller (P-1000, Sutter Instruments, USA). The signals were acquired via a

MultiClamp 700B amplifier, low-pass-filtered at 2.8 kHz, digitized at 10 kHz and analyzed with Clampfit

10.7 software (Molecular Devices, Sunnyvale, CA, USA). If the series resistance changed by more than 20%

during the recording, the experimental recording was immediately terminated.

Synaptic transmission. Neurons were held at -60 mV using the voltage clamp mode for recording mIPSCs,

as performed in our previous study (7). The pipettes were filled with intracellular solution that contained (in

mM) 145 CsCl, 10 EGTA, 10 HEPES, 2 MgCl2, 2 CaCl2, 2 Mg-ATP, and 5 QX-314. The osmolarity of the

solution was adjusted to 285-290 mOsm/kg, and the pH was adjusted to 7.2 with KOH. 6,7-

dinitroquinoxaline-2,3 (1H,4H)-dione (DNQX, 10 μM) was added to eliminate excitatory components, and

1 μM tetrodotoxin (TTX) was added to the bath solution to eliminate spontaneous action potentials. The

current-evoked firing was recorded in current-clamp mode (I = 0 pA).

Light-evoked responses. Optical stimulation was delivered using a laser (Shanghai Fiblaser Technology

Co., Ltd., China) through an optical fiber 200 μm in diameter positioned 0.2 mm from the surface of the

brain slice. To test the functional characteristics of AAV-ChR2, fluorescently labeled neurons that expressed

ChR2 in GAD2-Cre or CaMKII-Cre mice 3-4 weeks after virus injection were visualized and stimulated

with a blue (473 nm, 5-10 mV) laser light using 5-Hz or 20-Hz stimulation protocols with a pulse width of

10 ms. In some experiments, the function of eNpH3.0 was assessed by applying sustained yellow (594 nm,

5-10 mV, 100 ms) laser light stimulation. IPSCs were recorded at 0 mV, and EPSCs were recorded at -70

mV after photostimulation of ChR2-expressing BLAGlu fibers in the mPFC slices.

Immunohistochemistry. The mice were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.)

and sequentially perfused with saline and 4% (w/v) paraformaldehyde (PFA). The brains were subsequently

removed and post-fixed in 4% PFA at 4°C overnight. After cryoprotection of the brains with 30% (w/v)

sucrose, coronal sections (40 µm) were cut on a cryostat (Leica CM1860) and used for immunofluorescence.

7

The sections were incubated in 0.3% (v/v) Triton X-100 for 0.5 h, blocked with 10% donkey serum for 1 h

at room temperature, and incubated with primary antibodies, including anti-c-Fos (1:500, rabbit, Santa Cruz

Biotechnology) and anti-glutamate (1:200, rabbit, Sigma Aldrich), at 4°C for 24 h, followed by the

corresponding fluorophore-conjugated secondary antibodies (Thermo Fisher) for 2 h at room temperature.

Functional Magnetic Resonance Imaging (fMRI)

Magnetic resonance image acquisition. MRI data were acquired by using a Biospec 94/30 preclinical system

(Bruker) operating at 400 MHz (9.4 T) equipped with a gradient coil of 12 cm inner diameter, a maximum

gradient strength of 660 mT/m, and mouse head orthogonal coil.

Before MRI scanning, the mice were initially anesthetized using the dexmedetomidine (50 μg/kg)

followed by 3% isoflurane in oxygen enriched air. During scanning, 0.5% isoflurane was delivered. Animals

were placed prone with their head placed firmly in a tooth bar and ear bar. The physiological conditions,

including body temperature and respiration rate, were monitored (SA Instruments, Stony Brook, NY, USA).

The core body temperature was maintained at 37 using a controlled warm water system (Thermo Fisher

Scientific SC100, Waltham, MA, USA).

A T2-weighted anatomical scan was acquired using a 2D TurboRARE (Turbo Rapid Acquisition with

Relaxation Enhancement) sequence with the following parameters: field of view (FOV) = 20 × 9 mm2;

matrix = 125 × 56; slice thickness = 0.16 mm; repetition time (TR) = 16,900 ms; echo time (TE) = 34 ms;

bandwidth = 34.7 kHz; number of averages (NEX) = 6.

fMRI was done using a 2D single-shot echo-planar imaging (EPI) pulse sequence with the following

parameters: FOV = 25.8 × 9 mm2; matrix = 87 × 77; slice thickness = 0.5 mm; TR = 1,500 ms; TE = 11 ms;

bandwidth = 340 kHz; NEX = 1. The reconstructed images have an isotropic voxel size of 300 μm.

Preprocessing. All resting-state fMRI data were preprocessed in a standard pipeline as follows: (1) deletion

of the first 10 time points and slice timing correction; (2) realignment to the first volume; (3) spatial

normalization to the Allen Mouse Reference Atlas (P56-Atlas) space with a 0.05 mm3 cubic size and

resampled to 0.15 mm isotropic voxel size; (4) regression of nuisance signals, including the linear trend, six

8

motion parameters, and their first-order differences; (5) bandpass temporal filtering (0.01-0.08 Hz) to reduce

high-frequency noise; (6) smoothing with a 0.5 mm kernel.

Functional connectivity. The bilateral BLA were selected from the P56-Atlas, as was the medial prefrontal

cortex. Functional connectivity analyses were performed for the left and right BLA. For each seed region,

a voxel-wise functional connectivity analysis was performed separately for each region of interest (ROI).

The mean time series from all of the voxels within the ROI were used as the seed reference time series, and

the Pearson’s correlation coefficient (between the average time series for that seed and each voxel in the

brain) was computed as the strength of functional connectivity. For further statistical analysis, the

correlation coefficients were transformed to z-values using the Fisher r-to-z transformation to improve the

normality of the correlation coefficients. Thus, a map that represented the functional connectivity strength

and the seed region (in terms of the z-values for each subject) was obtained. Pairwise functional

connectivities between the bilateral BLA and mPFC were calculated.

Statistics. Within each group, the individual z-values were entered into a one-sample t-test in a voxel-wise

manner to determine the brain regions that showed significant functional connectivity with the left BLA. A

combined threshold of contrast maps was set using clusters with a minimum volume of 20 voxels set at an

uncorrected individual voxel height threshold of P < 0.05. We then obtained the significant connectivity

map of the left BLA within each group. These same steps were performed for the right BLA.

A two-sample two-tailed t-test was performed between the saline mice and quinpirole mice to identify

the abnormality map after controlling for age and gender using P < 0.05 [T = 1.96, df = (1, 14)] for each

voxel and a cluster size of at least 10 voxels. A two-sample two-tailed t-test (P < 0.05) was performed to

identify the significant differences in the pairwise connectivities between BLA and mPFC.

Statistical analysis and drugs. Animals were randomly or pseudo-randomly assigned to experimental

groups, which minimized the influence of other variables such as weight or age on the experimental outcome.

We conducted simple statistical comparisons using two-tailed paired or two-tailed unpaired Student’s t test,

ANOVA (one-way and two-way repeated-measures), and Bonferroni post hoc analyses were used to

9

statistically analyze the data from the experimental groups with multiple comparisons. All data are

expressed as the mean ± SEM, and significance levels are indicated as *P < 0.05, **P < 0.01, and ***P <

0.001. OriginPro 2017 software (OriginLab Corporation, USA) and GraphPad Prism 5 (GraphPad Software,

Inc., USA) were used for the statistical analyses and graphing. Offline analysis of the data obtained from

electrophysiological recordings was conducted using Clampfit software version 10.7 (Axon Instruments,

Inc., USA) and MiniAnalysis software version 6.03 (Synaptosoft Inc., USA). Unless otherwise stated, all

drugs were purchased from Sigma-Aldrich (USA). TTX was obtained from Hebei Aquatic Science and

Technology Development Company, China and CNO was obtained from Tocris (UK).

10

Supplementary Figures

Fig. S1. Behavioral characteristics of OCD-like mice. (A) Quinpirole administration did not alter sucrose

consumption with the increase in the number of days (n = 7-9 mice/group, F1,14 = 0.63, P = 0.441). (B)

Quinpirole altered behaviors in the open field test, including the frequency into the center, but did not alter

the total distance traversed in the open field compared with saline mice (n = 12-15 mice/group, frequency

into center: t25 = 2.15, P = 0.042; total distance: t25 = 0.90, P = 0.377). (C and D) After quinpirole withdrawal,

the behavioral characteristics of OCD-like mice were maintained for about 21 days [n = 6-9 mice/group,

(saline, sucrose) vs. (quinpirole, sucrose), frequency: F1,13 = 51.18, P < 0.001; drink time: F1,13 = 46.86, P <

A B

C

*

Quinpirole, sucrose Quinpirole, waterSaline, sucrose Saline, water

0 0

5

10

15

20

25

1000

2000

3000

Saline Quinpirole

Freq

uenc

y in

toce

nter

Tota

l dis

tanc

e (c

m)

Cons

umpt

ion

(g)

0.2

0.4

0.6

0

N.S

Quinpirole, sucrose Quinpirole, water Saline, sucrose Saline, water

D

BLDay

2 6 10 14 18 22 26 30 32 34 36 38

******

*** ******

0

20

40

60

***

Day1 3 5 7 14 21 28

Withdrawal

Freq

uenc

y (t

imes

/h)

****

*********

0

Drin

k tim

e (s

/h)

50

100

150

200

***

Day1 3 5 7 14 21 28

Withdrawal

E F

Saline Quinpirole Saline Quinpirole

Freq

uenc

y (t

imes

/h)

Drin

k tim

e (s

/h)

0

50

150

200

0

10

20

30

40 ET-ET+ *** **

100

ET-ET+

11

0.001]. (E and F) Frequency and duration of sucrose drinking in mice without or with 30-day environmental

training (ET) (n = 6-7 mice/group, frequency: F1,11 = 23.43, P < 0.001; drink time: F1,11 = 11.21, P = 0.007).

Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way repeated-measures ANOVA with

Bonferroni post hoc analysis for (A), (C), and (D); unpaired t test for (B); one-way ANOVA with Bonferroni

post hoc analysis for (E) and (F).

12

Fig. S2. Mapping the c-Fos expression pattern in mouse brain provoked by OCD. (A) Distribution of c-Fos-

positive neurons in the primary somatosensory cortex barrel field (S1BF), thalamic nucleus, ventral

tegmental area (VTA), caudate putamen (striatum) (CPu), and accumbens nucleus core (AcbC) of mice

treated with quinpirole (bottom) or saline (up) for 30 days. Scale bars = 50 μm. (B) Average c-Fos-positive

neurons per 0.04 mm² imaging area (n = 5-7 slices from 3 mice/group, S1BF: t9 = -3.04, P = 0.01; thalamus:

t9 = 0.46, P = 0.655; VTA: t11 = -1.77, P = 0.104; CPu: t8 = -0.36, P = 0.728; AcbC: t8 = 1.15, P = 0.282).

Data are means ± SEM. *P < 0.05. Unpaired t test for (B).

AQ

uinp

irole

Salin

eQ

uinp

irole

Salin

e

S1BF

S1BF

MDC

MDL

PV

MDM

CM

IAM

LHb

MDC

MDL

PV

CM

IAM

MDM

LHb

CPu

CPu

CPu

CPu

aca

aca

AcbC

AcbC

RPC

RPC

ml

ml

VTA

VTA

IPR

IPR

c-Fo

s po

sitiv

e ne

uron

num

ber

0

20

40

80

60

SalineQuinpirole*

S1BF

ThalamusVTA CPu

AcbC

B

13

Fig. S3. c-Fos expression in mice treated with quinpirole or saline 3, 5, and 7 times. (A-C) Distribution of

c-Fos-positive neurons in the mPFC and BLA in mice treated with quinpirole or saline 3 times (n = 8-12

slices from 4 mice/group, mPFC: t20 = -1.85, P = 0.078; BLA: t14 = -1.15, P = 0.268). (D-F) Distribution of

c-Fos-positive neurons in the mPFC and BLA in mice treated with quinpirole or saline 5 times (n = 8-12

slices from 4 mice/group, mPFC: t20 = -1.12, P = 0.275; BLA: t14 = -0.63, P = 0.539). (G-I) Distribution of

c-Fos-positive neurons in the mPFC and BLA in mice treated with quinpirole or saline 7 times (n = 8-12

slices from 4 mice/group, mPFC: t22 = -1.67, P = 0.108; BLA: t14 = -0.87, P = 0.4). Average c-Fos-positive

neurons per 0.04 mm² imaging area. Scale bars = 50 μm. Data are means ± SEM. Unpaired t test for (C),

(F), and (I).

A CSaline Quinpirole

I

FD

B

Injection 3 times mPFC BLA

mPFC BLA

mPFC BLAc-

Fos

posi

tive

neur

on n

umbe

r

0

5

10

15

c-Fo

s po

sitiv

e ne

uron

num

ber

0

5

10

15

c-Fo

s po

sitiv

e ne

uron

num

ber

0

5

10

15

SalineQuinpirole

SalineQuinpirole

SalineQuinpirole

E

G H

c-Fos c-Fos

c-Fos c-Fos

c-Fos c-Fos

mPFC mPFC

mPFC mPFC

mPFC mPFC

BLA

CeA

BLA

CeA

BLA

CeA

BLA

CeA

BLA

CeA

BLA

CeA

Saline Quinpirole

Saline Quinpirole Saline Quinpirole

Saline Quinpirole Saline Quinpirole

Injection 5 times

Injection 7 times

14

B

A

C

lBLA-lmPFC

rBLA-rm

PFC-0.2

0.0

0.5

1.0

Mea

n fu

nctio

nalc

onne

ctiv

ity

*** *

Saline

Quinpirole

Saline

Quinpirole

Quinpirolevs

Saline

Left BLA

Right BLA

Saline

Quinpirole

Quinpirolevs

Saline

-3 3-2 2

mPFC

L R

L R

L R

L

L

L

R

R

R mPFC

mPFC

mPFC

mPFC

mPFC

mPFC

mPFC

mPFC

mPFC

mPFC

mPFC

15

Fig. S4. Brain areas with significant differences in functional connectivity of the BLA in OCD mice. (A and

B) fMRI images showing brain areas with significant differences in functional connectivity with the left

BLA (A) and the right BLA (B) in quinpirole-treated mice compared with saline-treated mice. The t values

are indicated by the color bars shown at the bottom of the image. The red rectangle in each image represents

the region that contains the mPFC. (C) The mean functional connectivity strength between the BLA and

mPFC in indicated groups (n = 8 mice/group, lBLA-lmPFC: t14 = -5.29, P < 0.001; rBLA-rmPFC: t14 = -

2.85, P = 0.013). L, left; R, right; lBLA, left BLA; rBLA, right BLA; lmPFC, left mPFC; rmPFC, right

mPFC. Data are means ± SEM. *P < 0.05, ***P < 0.001. Unpaired t test for (C).

16

Fig. S5. Mapping presynaptic inputs onto glutamate neurons and GABA neurons in the mPFC. (A)

Schematic diagram for Cre-dependent retrograde trans-monosynaptic virus injection in CaMKII-Cre mice

to retrogradely trace the input neurons (red) to mPFC glutamatergic neurons. (B) Typical examples of

A B

mPFC

BLA

Day 1AAV-DIO-TVA-GFP

AAV-DIO-RVG

Day 21RV-EvnA-DsRed

mice

D

S2

S1BF

CL

MDL

MDC

MDM

Po

AM

VM

VL

VA

mt

mt

VM

VA

Cl

AcbSh

Cl

LSDDP

LSI PVAPT

LGP

AIV

DEn

HDB

MCPO

MHb

LHb

MD

LPMR

Sub

V2MM

LH

V2MM

MHb

LHbPVP

LPLR

fr

PF

Po

pv

VTASuM

vHPC VM

C

mPFC

BLA

Day 1AAV-DIO-TVA-GFP

AAV-DIO-RVG

Day 21RV-EvnA-DsRed

CaMKII -Cre

miceGAD2 -Cre

17

DsRed-expressing input neurons in the claustrum (Cl); anteromedial thalamic nucleus (AM);

mammillothalamic tract (mt); ventromedial thalamic nucleus (VM); ventrolateral thalamic nucleus (VL);

ventral anterior thalamic nucleus (VA); central mediodorsal thalamic nucleus (MDC); lateral mediodorsal

thalamic nucleus (MDL); centrolateral thalamic nucleus (CL); posterior thalamic nuclear group (Po);

accumbens nucleus shell (AcbSh); S1BF; secondary somatosensory cortex (S2). Scale bars = 50 μm. (C)

Schematic diagram for Cre-dependent retrograde trans-monosynaptic virus injection in GAD2-Cre mice to

retrogradely trace the input neurons (red) to mPFC GABAergic neurons. (D) Typical examples of DsRed-

expressing input neurons in the dorsal peduncular cortex (DP); dorsolateral septal nucleus (LSD);

intermediate lateral septal nucleus (LSI); anterior paraventricular thalamic nucleus (PVA); paratenial

thalamic nucleus (PT); lateral globus pallidus (LGP); VTA; supramammillary nucleus (SuM); Cl; dorsal

endopiriform nucleus (DEn); ventral agranular insular cortex (AIV); lateral hypothalamic area (LH);

nucleus of the horizontal limb of the diagonal band (HDB); magnocellular preoptic nucleus (MCPO);

mediodorsal thalamic nucleus (MD); mediorostral lateral posterior thalamic nucleus (LPMR); ventral

hippocampus (vHPC); submedius thalamic nucleus (Sub); VM; mediomedial secondary visual cortex

(V2MM); posterior paraventricular thalamic nucleus (PVP); periventricular fiber system (pv);

parafascicular thalamic nucleus (PF); Po; laterorostral lateral posterior thalamic nucleus (LPLR). Scale bars

= 50 μm.

18

Fig. S6. Local GABAergic circuits in the mPFC. (A) Schematic of mPFC injection of AAV-DIO-ChR2-

mCherry in GAD2-Cre mice and the recording configuration in acute slices. (B and C) Typical IPSC traces

(B) and summarized data (C, n = 7 neurons, t6 = 4.04, P = 0.007) recorded at 0 mV in mCherry- neurons

after photostimulation (blue bar) before and after bath application of 10 μM bicuculline in the mPFC. (D

and E) Photostimulation of mPFCGABA terminals in the mPFC decreased the spikes of mPFCGlu neurons (n

= 5 neurons, t4 = 6.0, P = 0.004). Data are means ± SEM. **P < 0.01. Paired t test for (C) and (E).

A

Control

Bicuculline

100

pA

50 ms

LightmPFC

GluGABA

R

IPSC

s am

plitu

de (p

A)

400

800

0

**

CB

AAV-DIO-ChR2-mCherry

mice

473nm light

GAD2-Cre

IPSCs

Control

Bicucullin

e

200 pA0Fi

ring

rate

(spi

kes/

s) 30

20

10

Control

Light

**

40 m

V

100 ms

200 pA

LightControl

D E

19

Fig. S7. HPLC chromatograms of different concentrations of glutamate. All elution profiles contained one

peak that eluted at 5.2000 min, which was the component of glutamate.

6.79671 μM16.9918 μM33.9836 μM50.9753 μM67.9671 μM

8

Curr

ent (

nA)

6

4

2

0

0 5 10 15 2025Time (min)

0

Components

20

40

60

80

[%]

Glutamate

20

Fig. S8. Excitability of mPFC-projecting BLA neurons in mice treated with quinpirole or saline 3, 5, and 7

times. (A) Representative traces of voltage responses recorded from FG+ BLA neurons in slices from mice

treated with quinpirole or saline 3 times. (B) Summarized data showing firing rates of evoked action

potentials in the groups as indicated in (A) (n = 18 neurons/group, F1,34 = 0.02, P = 0.9). (C) Representative

traces of voltage responses recorded from FG+ BLA neurons in slices from mice treated with quinpirole or

saline 5 times. (D) Summarized data showing firing rates of evoked action potentials in the groups as

indicated in (C) (n = 20-21 neurons/group, F1,39 = 0.59, P = 0.448). (E) Representative traces of voltage

responses recorded from FG+ BLA neurons in slices from mice treated with quinpirole or saline 7 times. (F)

Summarized data showing firing rates of evoked action potentials in the groups as indicated in (E) (n = 19-

20 neurons/group, F1,37 = 0.04, P = 0.85). Data are means ± SEM. Two-way repeated-measures ANOVA

with Bonferroni post hoc analysis for (B), (D), and (F).

B

A C

D

E

F

Injected current (pA)40 120 200 280

40 m

V

100 ms

Saline

Quinpirole

Injection 3 times Injection 5 times Injection 7 times

240 pA80 pA 240 pA80 pA240 pA80 pA

40 m

V

100 ms

Saline

Quinpirole

40 m

V

100 ms

Saline

Quinpirole

0

40

Firin

gra

te (s

pike

s/s)

30

20

10

0

40

Firin

gra

te (s

pike

s/s)

30

20

10

0

40

Firin

gra

te (s

pike

s/s)

30

20

10

0 80 160 240Injected current (pA)

40 120 200 2800 80 160 240Injected current (pA)

40 120 200 2800 80 160 240

SalineQuinpirole

SalineQuinpirole

SalineQuinpirole

21

Fig. S9. Excitability of mPFC-projecting BLA neurons in mice without or with 30-day environmental

training. (A) Representative traces of voltage responses recorded from FG+ BLA neurons in slices from mice

without or with 30-day environmental training. (B) Summarized data showing firing rates of evoked action

potentials in the groups as indicated in (A) [n = 18-20 neurons/group, (quinpirole, ET-) vs. (quinpirole, ET+),

F1,36 = 10.57, P = 0.003]. (C and D) Distribution of c-Fos-positive neurons in the BLA in mice without or

with 30-day environmental training [n = 6-8 slices from 3 mice/group, (quinpirole, ET-) vs. (quinpirole,

ET+), F1,12 = 211.54, P < 0.001). Average c-Fos-positive neurons per 0.04 mm² imaging area. Scale bars =

50 μm. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way repeated-measures ANOVA

with Bonferroni post hoc analysis for (B); one-way ANOVA with Bonferroni post hoc analysis for (D).

A

D

B

ET-

SalineQuinpirole

C

240 pA80 pA

100 ms

40 m

V

40 m

V

100 ms

240 pA80 pA

*

*

**

*

**

**

0

40

Firin

gra

te (s

pike

s/s)

30

20

10

Injected current (pA)40 120 200 2800 80 160 240

Quinpirole, ET+

Quinpirole, ET-Saline, ET-

Saline, ET+Saline, ET-

Quinpirole, ET-

Saline, ET+

Quinpirole, ET+

Quinpirole, ET+Quinpirole, ET-Saline, ET- Saline, ET+

c-Fos BLA

CeA CeA

BLA

CeA CeA

BLA BLA

c-Fo

s po

sitiv

e ne

uron

num

ber

0

5

10

15

20

25

ET+

***

22

Fig. S10. Excitability of mPFC-projecting BLA neurons in naïve mouse brain slices by perfusion of

quinpirole. (A) Representative traces of voltage responses recorded from FG+ BLA neurons in slices by

perfusion of 0.98 μM quinpirole. (B) Summarized data showing firing rates of evoked action potentials in

the groups as indicated in (A) (n = 7 neurons, control vs. quinpirole, F1,12 = 0.04, P = 0.853). Data are means

± SEM. Two-way repeated-measures ANOVA with Bonferroni post hoc analysis for (B).

BA

40 m

V

100 ms

Control

Quinpirole

Wash

ControlQuinpiroleWash

0

25

Firin

gra

te (s

pike

s/s)

20

15

10

5

Injected current (pA)40 120 200 2800 80 160 240

240 pA80 pA

23

Fig. S11. Response of BLA neurons to optogenetic stimuli. (A) Schematic of the injection of AAV-DIO-

eNpHR3.0-eYFP virus into the BLA of CaMKII-Cre mice and the recording configuration in acute slices.

(B) Representative image of the injection sites and viral expression in the BLA. Scale bar = 500 μm. (C)

Voltage response of eNpHR+ neurons (n = 8 neurons, t7 = 5.91, P = 0.0006) in the BLA after

photostimulation (yellow bar). (D) Schematic of the injection of AAV-DIO-ChR2-mCherry virus into the

BLA of CaMKII-Cre mice and the recording configuration in acute slices. (E) Representative image of

injection sites and viral expression in the BLA. Scale bar = 500 μm. (F) Light-evoked currents were recorded

at -70 mV from the ChR2+ neuron after photostimulation (blue bars). Data are means ± SEM. ***P < 0.001.

Paired t test for (C).

i

D

R

BLA

Glu

Light

400 ms

20 Hz

BLA

AAV-DIO-ChR2-mCherry

***

ΔV(m

V)

0

-40

-80

100 ms

Light

594nm light

AAV-DIO-eNpHR3.0-eYFP

BLA

Glu

473nm light

BLA

mice

R

A B C

E F

CaMKII-Cre

miceCaMKII-Cre

80 m

V80

0 pA

24

Fig. S12. The effects of CNO on synaptic transmission. (A) A representative trace (left) from a whole-cell

current-clamp electrophysiological recording showing that bath application of CNO (10 μM) hyperpolarizes

mPFCGABA neurons and statistics (right) showing the average magnitude of hyperpolarization (n = 5 neurons,

t4 = 4.30, P = 0.013). (B) A representative trace (left) that bath application of CNO (10 μM) depolarizes

mPFCGlu and statistics (right) showing the average magnitude of depolarization (n = 5 neurons, t4 = -3.32,

P = 0.029). (C) A representative trace (left) from a whole-cell current-clamp electrophysiological recording

showing that bath application of CNO (10 μM) hyperpolarizes mPFCGlu and statistics (right) showing the

average magnitude of hyperpolarization (n = 5 neurons, t4 = 5.56, P = 0.005). (D) A representative trace

C D

2 m

V

2 min

0

-2

-4

-6

-8CNO 10 μM**

ΔV

(mV)

ΔV

(mV)

8

6

4

2

0

*

2min

8 m

V

CNO 10 μM

CaMKII -Cre GAD2-Cre:: AAV-DIO-hM4Di-mCherry :: AAV-DIO-hM3Dq-mCherry

GAD2-Cre :: AAV-DIO-hM4Di-mCherry

4 m

V

2 min 2 min2

mV

CNO 10 μM CNO 10 μM

CaMKII -Cre :: AAV-DIO-hM3Dq-mCherryA B

0

-2

-4

-6

-8

ΔV

(mV)

*

ΔV

(mV)

6

4

2

0

*

F G

0

10

20

30

40

50

CNOBaseline

mCherryhM4Di

**

50

0

100

150

200

250

CNOBaseline

Drin

k tim

e (s

/h)

mCherryhM4Di

*

Freq

uenc

y (t

imes

/h)

H I

0

10

20

30

40

50

mCherryhM3Dq

CNOBaseline

Freq

uenc

y (t

imes

/h)

Drin

k tim

e (s

/h)

mCherryhM3Dq

CNOBaseline

*

50

0

100

150

200

250*

E

10 days 14-daytraining

1 dayCNO injection

AAV-DIO-hM4Di-mCherry or

AAV-DIO-hM3Dq-mCherry

::

::

mPFC

GAD2-Cre

CaMKII -Cre

25

(left) showing that bath application of CNO (10 μM) depolarizes mPFCGABA and statistics (right) showing

the average magnitude of depolarization (n = 5 neurons, t4 = -3.63, P = 0.022). (E) Experimental timeline

for chemogenetic manipulations. (F and G) Chemogenetic inhibition of mPFCGlu neurons produced an

increase in sucrose drinking frequency and duration in mice injected with quinpirole 7 times (n = 4-5

mice/group, frequency: F1,7 = 14.79, P = 0.006; drink time: F1,7 = 5.99, P = 0.044). (H and I) Chemogenetic

activation of mPFCGABA neurons produced an increase in sucrose drinking frequency and duration in mice

injected with quinpirole 7 times (n = 5 mice/group, frequency: F1,8 = 6.40, P = 0.035; drink time: F1,8 = 9.48,

P = 0.015). Data are means ± SEM. *P < 0.05, **P < 0.01. Paired t test for (A), (B), (C), and (D); one-way

ANOVA with Bonferroni post hoc analysis for (F), (G), (H), and (I).

26

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