Neuron, volume 62

Supplemental Data

Behavioral Effects of Fluoxetine in an Animal Model of

Anxiety/Depression Are Mediated by Both Neurogenesis-Dependent
and -Independent Mechanisms
Denis J. David, Benjamin Adam Samuels, Quentin Rainer, Jing-Wen Wang, Douglas
Marsteller, Indira Mendez, Michael Drew, Douglas A. Craig, Bruno P. Guiard, Jean-
Philippe Guilloux, Roman P. Artymyshyn, Alain M. Gardier, Christophe Gerald, Irina A.
Antonijevic, E. David Leonardo, and René Hen

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

X-Ray irradiation

Mice were anesthetized with ketamine and xylazine (100 mg/ml ketamine; 20 mg/ml

xylazine), placed in a stereotaxic frame and exposed to cranial irradiation using a

Siemens Stabilopan X-ray system operated at 300 kVp and 20 mA. Animals were

protected with a lead shield that covered the entire body, but left unshielded a 3.22 X

11-mm treatment field above the hippocampus (interaural 3.00 to 0.00) exposed to X-

Ray. Dosimetry was done using a Capintec Model PR06G electrometer ionization

chamber and Kodak Readypack Radiographic XV films. The corrected dose rate was

approximately 1.8 Gy per min at a source to skin distance of 30 cm. The procedure

lasted 2 min and 47 sec, delivering a total of 5 Gy. Three 5 Gy doses were delivered on

days 1, 4 and 8.

Immunohistochemistry
BrdU labeling for proliferation and survival study

The effects of a chronic corticosterone treatment in presence or absence of fluoxetine

treatment were assed on cell proliferation or cell survival. Mice were administered with

BrdU (150 mg/kg, i.p. dissolved in saline), 2h before sacrifice or twice a day during 3

days before the start of the corticosterone treatment for cell proliferation and cell survival

respectively. After anesthesia with ketamine and xylazine (100 mg/ml ketamine; 20

mg/ml xylazine),, mice were perfused transcardially (cold saline for 2 min, followed by

4% cold paraformaldehyde at 4°C). The brains were then removed and cryoprotected in

30% sucrose and stored at 4°C. Serial sections (35 µM) were cut through the entire

hippocampus (plate 41-61; Franklin and Paxinos, 1997) on a cryostat and stored in PBS

with 0.1% NaN3. For DAB staining, sections were mounted on slides and boiled in citric

acid (pH 6.0) for 5 min, rinsed with PBS, and treated with 0.01% trypsin in Tris/CaCl2 for

10 min. Brain sections were incubated for 30 min with 2N HCl and blocked with 5%

NGS. Sections were then incubated overnight at room temperature with anti-mouse

BrdU (1:100). After washing with PBS, sections were incubated for 1 hr with secondary

antibody (1:200 biotinylated goat anti-mouse) followed by amplification with an avidin-

biotin complex. The staining was visualized with DAB. For the quantification of BrdU

labeling, a stereological procedure was used as previously described (Malberg et al.,

2000).

Doublecortin (DCX) labeling for maturation index study

For doublecortin staining, the procedure consisted of the following steps (Wang et al.,
2008): sections were rinsed in PBS, treated with 1% H2O2 in 1:1 PBS and methanol for

15 min to quench endogenous peroxidase activity (and to enhance dendritic staining),

incubated in 10% normal donkey serum and 0.3% Triton X-100 for 30 min, and then

incubated overnight at 4°C in primary antibody for doublecortin (goat;1:500; Santa Cruz

Biotechnology, Santa Cruz, CA, USA). The secondary antibody was biotinylated donkey

anti-goat (1:500) (Jackson ImmunoResearch, PA, USA) in PBS for 2 hr at room

temperature. Sections were developed using avidin-biotin complex (Vector, CA, USA)

and DAB kit. Bright-field images were taken with a Zeiss (Oberkochen, Germany)

Axioplan-2 upright microscope. Stereological procedure was used to quantify labeled

cells (Wang et al., 2008). DCX+ cells were subcategorized according to their dendritic

morphology: DCX+ cells with no tertiary dendritic processes and DCX+ cells with

complex, tertiary dendrites. The maturation index was defined as the ratio of DCX+ cells

possessing tertiary dendrites over the total DCX+ cells.

Transcription analysis

Tissue preparation:

Animals were sacrificed by cervical dislocation. Selected brain regions were dissected

and placed in tubes containing RNAlater (Ambion, TX, USA), incubated at 4o overnight

and stored at –80o until processing.

RNA extraction and cDNA preparation

Brain regions (10-20mg) were homogenized for 20 sec at medium speed in 1.25ml lysis

/ denaturation buffer (Ambion, TX, USA) using an Autogizer (Tomtec, CT, USA). Total

RNA was isolated from 100-300ul aliquots of the homogenate using the RNAqueous 96

automated kit (Ambion, TX, USA) according to the manufacturer’s protocol. A second

DNase I digestion was incorporated after elution of the RNA from the Ambion filter plate

to remove residual genomic DNA. Digestion was performed for 1 hr at room temperature

using DNase I (Invitrogen, CA, USA) and the buffer supplied with the enzyme. After

inactivation of the Dnase with EDTA and heat, the RNA was desalted with a Multiscreen

filter plate (Millipore, MA, USA) and stored at –80o. Conversion of total RNA into first

strand cDNA was accomplished with Superscript II enzyme (Invitrogen, CA, USA)

followed by desalting over a Multiscreen plate. Approximately 1ug of total RNA was

used for each cDNA reaction. The yield of cDNA was determined using Quant-iT

Oligreen reagent (Invitrogen, CA, USA). Prior to the Oligreen assay, total RNA carried

over from the cDNA reaction was hydrolyzed with NaOH and heat, followed by

neutralization with Tris buffer. This treatment eliminates any contribution of the RNA to

the Oligreen signal. The unknown cDNA samples were compared to a standard curve

derived using a 18mer oligonucleotide. Replica cDNA plates containing 3ng of cDNA per

well were prepared using an Evolution P3 workstation (PerkinElmer, MA, USA). Each

animal in a given experiment was represented by one well on each plate and each plate

always contained the control and treatment groups.

QPCR analysis

Quantitative PCR (qPCR) was carried out in 25ul reactions using Full Velocity enzyme

(Stratagene, CA, USA). Plates were run on either a Stratagene MX3000P or an Applied

Biosystems 7900 HT instrument. The cycling parameters were set based on

recommendations from the enzyme manufacturer. One gene expression profile was

analyzed per PCR plate and duplicate plates were run for each gene. Two

housekeeping genes, cyclophilin and GAPDH, were included in the gene list and were

used to normalize the expression results obtained from the other genes of interest (see

data analysis section). The sequences of the primers and probes for each gene are

listed in supplemental table 1. Duplicate cycle thresholds (Ct values) were obtained for

each gene/region and averaged. The values for cyclophilin and GAPDH were combined

and used to normalize the expression values from the other genes by employing the

delta Ct method. After converting delta Ct values to percentage, the mean and SEM of

each animal group (controls and experimental) was calculated.

Behavioral testing

Novelty Suppressed Feeding

The testing apparatus consisted of a plastic box (50x50x20 cm), the floor of which was

covered with approximately 2 cm of wooden bedding. 24 hours prior to behavioral

testing, all food was removed from the home cage. At the time of testing, a single pellet

of food was placed on a white paper platform in the center of the box. An animal was

placed in a corner of the box, and a stopwatch was immediately started. The latency to
eat (defined as the mouse sitting on its haunches and biting the pellet with the use of

forepaws) was timed. Immediately afterwards, the animal was transferred to its home

cage, and the amount of food consumed by the mouse in the subsequent 5 min was

measured. Each mouse was weighed before food deprivation and before testing to

assess the percentage of body weight loss (data not shown).

Light-dark test

The light/dark test was conducted in an open field chamber measuring 43.2 x

43.2 cm (Med Associates, Vermont, USA), a white floor and clear walls, with a dark

plastic box insert opaque to visible light but transparent to infrared covering half of the

area of the chamber. Infrared tracking beams and data collection were controlled by a

computer running the software Activity Monitor (Med Associates). Based on the

modifications proposed by David et al. (2007), the open field box was divided into two

equal areas with an opening located in the center of the dark wall at floor level, allowing

passage between the light and dark chambers. The light compartment was brightly

illuminated with an 8 W fluorescent tube (400 lux). The test was performed in a quiet,

darkened room, and the mice were kept in this room at least 1 h before the test.

Between each trial, the light/dark compartments were cleaned. At the beginning of the

test, the mouse was placed in the dark compartment and allowed to freely explore both

chambers for 6 minutes. During the test, the total entries in the light compartments and

the total ambulatory distance in the dark were recorded.

Elevated plus maze

The elevated plus maze was performed as previously described (Lira et al., 2003).

Briefly, animals were placed into the central area facing one closed arm and allowed to

explore the maze for 5 min. Testing took place in bright ambient light conditions (800–

900 lux). An observer blind to mouse genotype scored the videotapes.

Tail suspension test

Tail suspension testing was performed as previously described (Mayorga et al., 2001

and Steru et al., 1985). Mice were recorded by a video camera and scored by a highly

trained observer blind to treatment.

Changes in coat state

The state of the coat was assessed at the end of the corticosterone regimen in the

presence or absence of 3-weeks of fluoxetine treatment. The total score resulted from

the sum of the score of five different body parts: head, neck, dorsal/ventral coat, tail,

fore-/hindpaws. For each body area, a score of 0 was given for a well-groomed coat and

1 for an unkempt coat (Griebel et al., 2002; Santarelli et al., 2003).

Splash test
The grooming latency was assessed at the end of the corticosterone regimen (end of

seventh week) in the presence or absence of 3-weeks of fluoxetine treatment (Yalcin et

al., 2008). This test consisted in squirting 200 ul of a 10% sucrose solution on the

mouse’s snout. The grooming frequency was then recorded.

Mouse body weight

Mouse body weight for each animal was followed once a week during the 4-weeks of

corticosterone treatment.

Food consumption

Food consumption was followed once a week during the 4-weeks of corticosterone

treatment in each cage.

Drinking consumption

Drinking consumption was followed once a week during the 4-weeks of corticosterone

treatment in each cage.

Stress evoked increase of corticosterone levels

Adult male C57BL/6Ntac mice were exposed to a 6 minutes swim stress. Mice were

placed into plastic buckets (19 cm diameter, 23 cm deep, filled with 23-25°C water) and

sacrified 12 min after the end of the test. Blood was collected into ice-chilled tubes

containing EDTA and centrifuged at 3000 rpm for 10 min (at 4 oC) for separation of
plasma, and plasma samples were stored at –80 oC until assayed. Plasma

corticosterone levels were determined with a commercially available RIA kit (Rat

Corticosterone RIA, DSL-80100; Diagnostic Systems Laboratories, Inc. Webster, TX,

USA; sensitivity limit: 20 ng/ml). ACTH was measured directly in plasma using an

ImmuChemTM Double Antibody hACTH 125I RIA kit (No. 07-106101; MP Biomedicals,

LLC, NY, USA) with a sensitivity limit ~ 5.7 pg/ml. All samples were measured

simultaneously to reduce inter-assay variability.

Home cage activity

Home cage activity was quantified using the ActiV-Meter (Bioseb, Vitrolles, France) over

a 24 hours period. During the experiment, food and water were provided ad libitum.

Various parameters such as activity time (sec), ambulatory distance (cm) and inactivity

duration (calculated from the difference between immobility and motionless activity

duration while the animal is eating or scratching) were recorded.

Supplemental Data
Supplemental table 1 : Oligonucleotides sequences (5’→3’) for the QPCR analysis

Gene oligo sequence (5' - 3')

ARRB1 F CCACCAGACAGTTCCTCATGTC

ARRB1 R CATTGACGCTGATGGGTTCTC

ARRB1 T CCCTGCACCTTGAGGCATCTCTGGATA

ARRB2 F TCCGCTATGGCCGAGAAG

ARRB2 R CCTGGTAGGTGGCGATGAAC

ARRB2 T ATGTACTGGGCCTGTCTTTCCGCAAA

CREB F TCAAGCTGCCTCTGGTGATG

CREB R GGAGGACGCCATAACAACTCC

CREB T AAACATACCAGATTCGCACAGCACCCA

Cyclo F TTTCGCCGCTTGCTGC

Cyclo R CTCGTCATCGGCCGTGA

Cyclo T CATGGTCAACCCCACCGTGTTCTTC

Erk1 F CAAGTACATACACTCGGCCAATG

Erk1 R TCGCAGGTGGTGTTGATAAGC

Erk1 T ACCGGGACCTGAAGCCCTCCAAT

GAPDH F CAAATTCAACGGCACAGTCAAG

GAPDH R ACCCCATTTGATGTTAGTGG

GAPDH T TCATCAACGGGAAGCCCATCACCATCT
 Gi2 F ACCATGGTGTGCAAGCCTG

Gi2 R GGTAGTAAGCGGCTGAGTCATTG

Gi2 T TTGGCCGCTCACGGGAATATCAA

MR F TGTCCTCCTCCACAGCTAGCTT

MR R GCATGTCAGTGAGGTTCCTTGA

MR T CAGTTTCCCAGTGCACAGTCCCATCA

F = forward primer
R = reverse primer
T = TaqMan Probe
 Supplemental table 2 : complete statistical summary analysis for behavioral data

Behavioral Degrees of
Measurement Statistical Test Comparison Statistics p Fig.
paradigm freedom

Factor 1- Pre-
F=24.75 1, 78 <0.01**
treatment

2-way ANOVA Factor 2

F=3.82 2, 78 <0.01**
Treatment
Interaction
Total Time in F=8.32 2, 78 <0.01**
(F1 x F2) 1A
the center
CORT vs Veh <0.01**

PLSD
CORT vs CORT/Flx <0.01##
Open Field Post-hoc test

CORT vs CORT/Imi <0.01##

Factor 1- Pre-
F=2.11 1, 78 <0.08
treatment
Factor 2
2-way ANOVA F=0.413 2, 78 <0.062
Ambulatory Treatment
1B
Distance Interaction
F=4.49 2, 78 <0.01**
(F1 x F2)
PLSD
CORT vs CORT/Flx <0.01#
Post-hoc test
Factor 1- Pre-
F=11.2 1, 80 <0.01**
treatment
Factor 2
2-way ANOVA F=1.48 2, 80 <0.23
Treatment
Novelty
Latency to Interaction
Suppressed F=1.80 2, 80 <0.15 1C
feed (F1 x F2)
Feeding
CORT vs Veh <0.01**
PLSD
Post-hoc test
CORT vs CORT/Flx <0.01##

The Forced Mobility Factor 1- Pre-

F=0.01 1, 75 >0.92 1D
Swim test duration treatment
Factor 2
2-way ANOVA F=12.32 2, 75 <0.01**
Treatment
Interaction
F=0.03 2, 75 >0.97
(F1 x F2)
PLSD
Flx vs Veh <0.01**
Post-hoc test
 CORT/Flx vs Veh <0.01**

CORT/Flx vs CORT <0.01##

Factor 1- Pre-
F=877.23 1, 42 <0.01**
treatment
Factor 2
2-way ANOVA F=7.49 1, 42 <0.01**
Treatment
Interaction
F=13.93 1, 42 <0.01**
(F1 x F2)
Coat State 1E
CORT vs Veh <0.01**

PLSD
CORT/Flx vs Veh <0.01**
Post-hoc test

CORT vs CORT/Flx <0.05#

Factor 1- Pre-
F=12.13 1, 42 <0.01**
treatment
Factor 2
2-way ANOVA F=3.05 1, 42 <0.05*
Treatment
Frequency of Interaction
Splash Test F=1.59 1, 42 <0.21 1F
grooming (F1 x F2)

CORT vs Veh <0.01**

PLSD
Post-hoc test
CORT vs CORT/Flx <0.01##

F=6.74 2, 34 <0.01**
Elevated plus Total entries in
One-way ANOVA 1G
maze the Open arms
CORT vs CORT/Flx <0.01**

One-way ANOVA F=8.61 2, 34 <0.01**

Tail
Mobility
Suspension CORT vs CORT/Flx <0.01** 1H
duration
Test PLSD
Post-hoc test
CORT vs
<0.05*
CORT/Rbx

3-way repeated
Time in the Factor 1 Pre-
Open Field measures F=0.148 1, 230 <0.70 3A
center treatment
ANOVA
Factor 2
F=9.45 2, 230 <0.01**
Treatment
Factor 3
F=0.092 5, 230 <0.76
Time
 Interaction
F=1.27 10, 230 <0.27
(F1 x F2 x F3)
SHAM/Flx vs
<0.05*
PLSD SHAM/Veh
Post-hoc test XRAY/Flx vs
<0.05*
SHAM/Veh

Factor 1- Pre-
F=0.148 1, 46 <0.7
treatment

2-way ANOVA Factor 2

F=9.45 1, 46 <0.05*
Treatment
Interaction
Total Time in F=0.092 1, 46 <0.76
(F1 x F2) 3B
the center
SHAM/Flx vs
<0.05*
SHAM/veh
PLSD XRAY/Flx vs
<0.05*
Post-hoc test SHAM/Veh
XRAY/Flx vs
<0.05#
XRAY/Veh
Factor 1- Pre-
F=1.245 1, 46 <0.27
treatment
Factor 2
2-way ANOVA F=2.682 1, 46 <0.10
Treatment
Total Entries in Interaction
F=0.067 1, 46 <0.79 3C
the enter (F1 x F2)
SHAM/Flx vs
<0.01**
Planned sham/Veh
comparisons test XRAY/Flx vs
<0.05*
SHAM/Veh
Factor 1- Pre-
F=0.01 1, 46 <0.92
treatment
Factor 2
2-way ANOVA F=45.56 1, 46 <0.01**
Treatment
Ambulatory Interaction
F=1.55 1, 46 <0.21
Distance in the (F1 x F2)
3D
center/Total SHAM/Flx vs
distance <0.01**
SHAM/veh
PLSD XRAY/Flx vs
<0.01**
Post-hoc test SHAM/Veh
XRAY/Flx vs
<0.01##
XRAY/Veh
Novelty Latency to Factor 1- Pre-
Suppressed 2-way ANOVA F=1.64 1, 49 <0.2 3E
feed treatment
Feeding
Factor 2
F=2.69 1, 49 <0.10
Treatment
 Interaction
F=6.82 1, 49 <0.01**
(F1 x F2)
PLSD SHAM/Flx vs
<0.01**
Post-hoc test SHAM/veh
Kaplan–Meier
<0.10 3G
survival analysis
Factor 1- Pre-
F=0.37 1, 49 <0.54
treatment
Food Factor 2
2-way ANOVA F=1.74 1, 49 <0.19
consumption Treatment 3F
Interaction
F=0.016 1, 49 <0.89
(F1 x F2)
Factor 1- Pre-
F=0.10 1, 32 <0.74
treatment
Factor 2
2-way ANOVA F=28.25 1, 32 <0.01**
Treatment
The Forced Mobility Interaction
F=1.43 1, 32 <0.23 3H
Swim test duration (F1 x F2)
SHAM/Flx vs
<0.01**
PLSD SHAM/veh
Post-hoc test XRAY/Flx vs
<0.01**
SHAM/Veh
Factor 1’’- Pre-
F=8.76 1, 59 <0.01**
treatment

2-way ANOVA Factor 2

F=1.59 1, 59 <0.22
Time in the Treatment
5A
center Interaction
F=2.94 1, 59 <0.09
(F1 x F2)
PLSD WT/Flx vs
<0.05§
Post-hoc test βArr2KO/Flx
Open Field
Factor 1’’- Pre-
F=7.17 1, 59 <0.01**
treatment
Factor 2
Total 2-way ANOVA F=0.12 1, 59 >0.72
Treatment
ambulatory 5B
Distance Interaction
F=1.58 1, 59 >0.21
(F1 x F2)
PLSD WT/Flx vs
<0.05§
Post-hoc test βArr2KO/Flx
Total entries Factor 1’’- Pre-
Light Dark test F=2.45 1,32 >0.12 5C
into the light treatment
Factor 2
2-way ANOVA F=1.91 1,32 >0.16
Treatment
Interaction
F=4.30 1,32 <0.05*
(F1 x F2)
 PLSD WT/Flx vs
<0.05§
Post-hoc test βArr2KO/Flx
Factor 1’’- Pre-
F=0.51 1,32 >0.47
Total treatment
ambulatory Factor 2
2-way ANOVA F=0.02 1,32 >0.87 5D
distance into Treatment
the dark (cm)
Interaction
F=0.01 1,32 >0.91
(F1 x F2)
Factor 1- Pre-
F=17.108 1, 59 <0.01**
treatment
Factor 2
2-way ANOVA F=4.781 1, 59 <0.05*
Treatment
Interaction
F=1.749 1, 59 >0.19
(F1 x F2)
Latency to WT/Veh vs
<0.05* 5E
feed βArr2KO/Veh
WT/Flx vs
Novelty <0.01$$
PLSD βArr2KO/Flx
Suppressed
Post-hoc test
Feeding WT/Veh vs WT/Flx <0.05*

βArr2KO/Veh vs
>0.49
βArr2KO/Flx
Factor 1- Pre-
F=2.82E-4 1, 59 >0.98
treatment
Food Factor 2
2-way ANOVA F=0.008 1, 59 >0.92 5F
consumption Treatment
Interaction
F=.523 1, 59 >0.47
(F1 x F2)
Factor 1- Pre-
F=0.025 1, 59 >0.65
treatment
Factor 2
F=15.35 1, 59 <0.01**
Treatment
The Forced Total mobility Interaction
2-way ANOVA F=0.22 1, 59 >0.63 5G
Swim test duration (F1 x F2)

WT/Veh vs WT/Flx <0.05*

βArr2KO/Veh vs
<0.01##
βArr2KO/Flx
Frequency of Factor 1- Pre-
Splash test 2-way ANOVA F=14.24 1, 33 <0.01** 5H
grooming treatment
Factor 2
F=3.43 1, 33 >0.07
Treatment
Interaction
F=2.45 1, 33 >0.12
(F1 x F2)
 WT/Veh vs WT/Flx <0.05*

WT/Flx vs
<0.01§§
βArr2KO/Flx
Factor 1’- Pre-
F=2.78 2, 210 <0.05*
treatment
2-way repeated
measures Factor 2
F=4.98 5, 230 <0.01**
Time in the ANOVA Treatment
S2A
center Interaction
F=1.87 10, 230 <0.05*
(F1 x F2)
PLSD CORT 35ug vs Veh
<0.01**
Post-hoc test (t5, t10, t15, t20)

Open Field one-way ANOVA F=2.97 2, 42 <0.05*

Total Time in
S2B
the center PLSD
CORT 35ug vs Veh <0.01**
Post-hoc test
Amb. Dist
center/Total one-way ANOVA F=1.918 2, 42 <0.10 S2C
Amb (in %)

Total one-way ANOVA F=4.26 2, 42 <0.01**

ambulatory S2D
distance PLSD CORT 35ug vs
<0.05#
Post-hoc test CORT 7ug

one-way ANOVA F=4.01 2, 42 <0.05*

CORT 7ug vs Veh 2, 42 <0.05*

PLSD
S2E
Latency to Post-hoc test
Novelty CORT 35ug vs Veh 2, 42 <0.01**
feed
Suppressed
Feeding PLSD SHAM/Flx vs
<0.01**
Post-hoc test SHAM/veh
Kaplan–Meier
<0.01** S2G
survival analysis
Food
one-way ANOVA F=1.52 2, 42 <0.23 S2F
consumption
The Forced Total mobility
one-way ANOVA F=1.59 2, 42 <0.21 S2H
Swim test duration
Mouse body Factor 1- Pre-
F=5.65 1, 232 <0.01** S3B
weight treatment
2-way repeated
Factor 2
measures F=205.88 4, 232 <0.01**
Time
ANOVA
Interaction
F=47.67 4, 232 <0.01**
(F1 x F2)
PLSD CORT wk3 vs Veh
<0.01**
Post-hoc test wk3
 CORT wk4 vs Veh
<0.01**
wk4
Factor 1- Pre-
F=6.21 1, 16 <0.01**
treatment
2-way repeated
Factor 2
measures F=2.55 4, 16 <0.01**
Time
ANOVA
Interaction
F=1.60 4, 232 <0.01**
Food (F1 x F2)
S3C
consumption CORT wk2 vs Veh
<0.01**
wk2
PLSD CORT wk3 vs Veh
<0.01**
Post-hoc test wk3
CORT wk4 vs Veh
<0.01**
wk4
Factor 1- Pre-
F=40.8 1, 16 <0.01**
treatment
2-way repeated
Factor 2
measures F=2.25 4, 16 <0.1
Time
ANOVA
Interaction
F=4.40 4, 232 <0.01**
(F1 x F2)
Drinking CORT wk1 vs Veh
<0.01** S3D
consumption wk1
CORT wk2 vs Veh
<0.01**
PLSD wk42
Post-hoc test CORT wk3 vs Veh
<0.01**
wk3
CORT wk4 v Veh
<0.01**
wk4
Factor 1- Pre-
F=320.43 1, 38 <0.01**
treatment
Factor 2
2-way ANOVA F=4.97 2, 38 <0.01**
Treatment
Stress evoked Interaction
F=4.68 2, 38 <0.01**
increase of (F1 x F2)
S3E
corticosterone
levels CORT vs Veh <0.01**

PLSD
CORT/Flx vs Veh <0.01**
Post-hoc test

CORT/Imi vs Veh <0.01**

Ratio
ambulatory
Home cage distance during PLSD
activity Unpaired -test t=2.817 1,7 <0.01** S4A
the dark phase Post-hoc test
over the light
phase
 Ambulatory
PLSD
distance during Unpaired -test t=5.45 1, 7 <0.01** S4B
Post-hoc test
the dark phase
Ambulatory
PLSD
distance during Unpaired -test t=1.62 1, 7 <0.14 S4C
Post-hoc test
the light phase
Factor 1- Pre-
F=11.83 1, 7 <0.01**
treatment
Factor 2
2-way ANOVA F=0.65 1, 7 <0.44
Treatment
Ambulatory Interaction
F=0.031 1, 7 <0.86 S4D
distance (F1 x F2)

CORT vs Veh <0.01**

PLSD
Post-hoc test
CORT/Flx vs Veh <0.01**

Factor 1- Pre-
F=55.6 1, 7 <0.01**
treatment
Factor 2
2-way ANOVA F=0.25 1, 7 <0.6
Treatment
Inactivity Interaction
F=0.089 1, 7 <0.72 S4E
duration (F1 x F2)

CORT vs Veh <0.01**

PLSD
Post-hoc test
CORT/Flx vs Veh <0.01**

2-way repeated
Factor 1’-
measures F=5.75 1, 130 <0.05*
Treatment
ANOVA
Factor 2
Time in the F=8.45 5, 130 <0.01**
Time S5A
center
Interaction
F=1.83 5, 130 <0.11
(F1 x F2)
PLSD CORT 35ug vs Veh
Open Field <0.01**
Post-hoc test (t20, t25, t30)
Total Time in PLSD
Unpaired t-test T=2.398 <0.05* S5B
the center Post-hoc test
Amb. Dist
PLSD
center/Total Unpaired t-test T=2.66 <0.05* S5C
Post-hoc test
Amb (in %)
Total
PLSD
ambulatory Unpaired t-test T=-1.50 <0.14 S5D
Post-hoc test
distance
Novelty PLSD
Suppressed Unpaired t-test T=-2.13 <0.05* S5E
Latency to Post-hoc test
Feeding feed Kaplan–Meier
<0.05* S5G
survival analysis
 Food
one-way ANOVA F=1.34 <0.19 S5F
consumption
The Forced Mobility PLSD
Unpaired t-test T=-0.614 <0.54 S5G
Swim test duration Post-hoc test
Factor 1- Pre-
F=24.79 1, 390 <0.01**
treatment
Factor 2
3-way repeated F=3.82 2, 390 <0.01**
Treatment
measures
ANOVA Factor 3
F=4.52 5, 390 <0.01**
Time
Time in the Interaction
F=1.35 10, 390 0.2 S7A
center (F1 x F2 x F3)

CORT vs Veh <0.01**

PLSD
CORT vs CORT/Flx <0.01##
Post-hoc test

Open Field CORT vs CORT/Imi <0.01##

Factor 1- Pre-
F=1.69 1, 78 <0.19
treatment
Factor 2
2-way ANOVA F=3.74 2, 78 <0.05*
Treatment
Interaction
Amb. Dist F=1.46 2, 78 <0.23
(F1 x F2)
center/Total S7B
Amb (in %) CORT vs Veh <0.05*

PLSD
CORT vs CORT/Flx <0.01##
Post-hoc test

CORT vs CORT/Imi <0.05#

Latency to Kaplan–Meier
<0.01** S7C
Feed survival analysis
Factor 1- Pre-
Novelty F=2.1 1, 80 <0.14
treatment
Suppressed
Feeding Food Factor 2
2-way ANOVA F=1.16 2, 80 <0.30 S7D
consumption Treatment
Interaction
F=0.65 2, 80 <0.52
(F1 x F2)
Open Field Time in the 3-way repeated Factor 1- Pre-
measures F=8.76 1, 295 <0.01** S7E
Paradigm center treatment
ANOVA
Factor 2
F=1.50 1, 295 >0.22
Treatment
Factor 3
F=14.79 5, 295 <0.01**
Time
 Interaction
F=0.80 5, 390 >0.52
(F1 x F2 x F3)
WT/Flx vs WT/Veh,
<0.05*
PLSD t15
Post-hoc test CORT vs
<0.05*
CORT/Flx, t30
Factor 1’’- Pre-
F=7.98 1, 59 <0.01**
treatment
Factor 2
2-way ANOVA F=0.69 1, 59 >0.40
Entries in the Treatment
S7F
enter Interaction
F=2.73 1, 59 <0.1
(F1 x F2)
PLSD WT/Flx vs
<0.01$$
Post-hoc test βArr2KO/Flx
Latency to Kaplan–Meier
Novelty <0.01** S7G
Feed survival analysis
Suppressed
Feeding Food
one-way ANOVA F=1.52 2, 42 <0.23 S7H
consumption
Factor 1 - pretreatment : Vehicle or Corticosterone ;Factor 1’- pretreatment : SHAM or XRAY; Factor 1’’-
pretreatment : SHAM or XRAY
Factor 2- treatment : Vehicle, fluoxetine, imipramine
Legend : CORT : corticosterone ; Imi ; imipramine ; Flx : fluoxetine ; Rbx : reboxetine ; MR :
mineralocorticoid receptor; WT: wild-type; βArr2KO: β−Arrestin 2 Knock Out mice
 Supplemental table 3 : complete statistical summary analysis for morphological
data

Behavioral Degrees of
Measurement Statistical Test Comparison Statistics p Fig.
paradigm freedom

Neurogenesis Factor 1- Pre-

F=3.90 1, 12 <0.07
treatment

2-way ANOVA Factor 2

F=6.76 1, 12 <0.05*
Treatment
Interaction
F=9.70 1, 12 <0.01**
(F1 x F2)

Proliferation CORT vs Veh <0.05* 2A

CORT/Flx vs Veh <0.05*

PLSD
Post-hoc test
CORT/Flx vs CORT <0.01##

CORT/Flx vs Flx <0.05§

Factor 1- Pre-
F=0.0007 1, 20 <0.99
treatment
Factor 2
2-way ANOVA F=4.34 1, 20 <0.05*
Treatment
Interaction
Survival F=0.487 1, 20 <0.49 2B
(F1 x F2)

CORT/Flx vs Veh <0.05*

PLSD
Post-hoc test CORT/Veh vs
<0.05#
CORT/Flx
Factor 1- Pre-
F=3.12 1,16 <0.09
treatment
Factor 2
2-way ANOVA F=19.53 1, 16 <0.01**
Treatment
Interaction
Total F=3.20 1, 16 <0.09
(F1 x F2)
doublecortine 2G
positive cells CORT/Flx vs Veh <0.01**

PLSD CORT/Veh vs
<0.01##
Post-hoc test CORT/Flx

CORT/Flx vs Flx <0.05§

Doublecortine Factor 1- Pre-

positive cells 2-way ANOVA F=3.85 1,16 <0.06 2H
treatment
with tertiary
dendrites Factor 2
F=23.05 1, 16 <0.01**
Treatment
 Interaction
F=3.11 1, 16 <0.09
(F1 x F2)

Flx vs Veh <0.05*

CORT/Flx vs Veh <0.01**

PLSD
Post-hoc test
CORT/Flx vs Flx <0.05$

CORT/Veh vs
<0.01##
CORT/Flx
Factor 1- Pre-
F=3.62 1,16 <0.01**
treatment
Factor 2
2-way ANOVA F=22.76 1, 16 <0.77
Treatment
Interaction
F=2.06 1, 16 <0.17
Maturation (F1 x F2)
2I
index
Flx vs Veh <0.05*

PLSD
CORT/Flx vs Veh <0.01**
Post-hoc test
CORT/Veh vs
<0.01##
CORT/Flx
Factor 1 - pretreatment : Vehicle or Corticosterone
Factor 2- treatment : Vehicle, fluoxetine, imipramine
Legend : CORT : corticosterone ; Flx : fluoxetine
 Supplemental table 4 : complete statistical summary analysis for gene expression

Behavioral Degrees of
Measurement Statistical Test Comparison Statistics p Fig.
paradigm freedom

One-way ANOVA F=3.59 3, 27 <0.01**

β-arrestin 1 CORT vs Veh <0.05* 4A

Newman-Keuls
Post-hoc test CORT/Flx vs
<0.05#
CORT/veh
Gene One-way ANOVA F=3.61 3,22 <0.05
expression in β-arrestin 2 4B
the Newman-Keuls CORT/Flx vs
hypothalamus <0.05#
Post-hoc test t CORT/veh

One-way ANOVA F=3.88 3, 27 <0.01**

Giα2 CORT vs Veh <0.05* 4C

Newman-Keuls
Post-hoc test CORT/Flx vs
<0.05#
CORT/veh

One-way ANOVA F=3.02 3, 27 <0.01**

β-arrestin 1 4D
Newman-Keuls
CORT vs Veh >0.05
Post-hoc test

Gene β-arrestin 2 One-way ANOVA F=3.04 3,21 >0.05 4E

expression in
the amygdala One-way ANOVA F=4.88 <0.01**

Giα2 CORT vs veh <0.05* 4F

Newman-Keuls
Post-hoc test
CORT/Flx vs /veh <0.05*

β-arrestin 1 One-way ANOVA F=2.20 3, 27 >0.05 4G

Gene One-way ANOVA F=3.09 3,27 >0.051

expression in
β-arrestin 2 4H
the Newman-Keuls
hippocampus CORT/Flx vs veh <0.05*
Post-hoc test

Giα2 One-way ANOVA F=2.61 >0.05 4I

Gene MR receptor One-way ANOVA F=2.75 3, 27 >0.05 S6A

expression in
the
hypothalamus Creb1 One-way ANOVA F=2.25 3, 27 >0.05 S6B
 Gene MR receptor One-way ANOVA F=0.33 3, 27 >0.05 S6C
expression in
the amygdala Creb1 One-way ANOVA F=0.26 3, 27 >0.05 S6D

Gene MR receptor One-way ANOVA F=2.15 3, 27 >0.05 S6E

expression in
the
hippocampus Creb1 One-way ANOVA F=0.17 3, 27 >0.05 S6F

β-arrestin 1 One-way ANOVA F=1.89 3, 28 >0.05 S8A

Giα2 One-way ANOVA F=2.00 3, 29 >0.05 S8B

Gene Creb1 One-way ANOVA F=1.10 3, 31 >0.05 S8C

expression in
the
hypothalamus MR receptor One-way ANOVA F=0.61 3, 28 >0.05 S8D

One-way ANOVA F=5.79 3, 29 <0.05*

Erk-1 S8E
Newman-Keuls
WT/Veh vs WT/Flx <0.05*
Post-hoc test

β-arrestin 1 One-way ANOVA F=3.00 3, 29 <0.05* S8F

One-way ANOVA F=11.54 3, 29 <0.01**

Giα2 WT/Veh vs WT/Flx <0.05* S8G

Newman-Keuls
Post-hoc test WT/Veh vs
Gene <0.01**
βArr2KO/veh
expression in
the amygdala Creb1 One-way ANOVA F=1.10 3, 32 >0.05 S8H

MR receptor One-way ANOVA F=2.57 3, 32 >0.05 S8I

One-way ANOVA F=2.75 3, 31 <0.05*

Erk-1 S8J
Newman-Keuls WT/Veh vs
<0.01**
Post-hoc test βArr2KO/veh
Gene
expression in One-way ANOVA F=4.78 3, 33 <0.01**
the β-arrestin 1 S8K
hippocampus Newman-Keuls WT/Veh vs
<0.01**
Post-hoc test βArr2KO/Flx

Giα2 One-way ANOVA F=1.81 3, 32 >0.05 S8L

Creb1 One-way ANOVA F=6.38 3, 32 <0.01** S8M

 Newman-Keuls
WT/Veh vs WT/Flx <0.01**
Post-hoc test

One-way ANOVA F=2.57 3, 32 >0.05

MR receptor S8N
Newman-Keuls WT/Veh vs
<0.01**
Post-hoc test βArr2KO/Flx

Erk-1 One-way ANOVA F=2.75 3, 31 >0.05 S8O

Legend : CORT : corticosterone ; Flx : fluoxetine ; MR : mineralocorticoid receptor; βArr2KO : β−Arrestin 2

Knock Out mice
Supplemental figures

Supplemental figure 1: Experimental timeline

In a first set of experiments (supplemental figure 1A), in place of normal drinking water,

grouped-housed male C57BL/6Ntac mice were presented during 7 weeks with vehicle

(0.45% hydroxypropyl-β-cyclodextrin) or corticosterone (35 ug/ml) in the presence or

absence of an antidepressant (imipramine, 40 mg/kg/day, fluoxetine, 18 mg/kg/day or

reboxetine 20 mg/kg/day) during the last three weeks of the corticosterone regimen. We

investigated whether the behavioral changes during chronic corticosterone were

reversed by antidepressant treatment. The same animal was successively tested in the

Open Field (OF) paradigm, the Novelty Suppressed Feeding (NSF), the Forced Swim

Test (FST) and then sacrificed for neurogenesis or transcription analysis.

In another set of experiments (supplemental figure 1B), a focal X-irradiation of the

hippocampus was employed to assess whether the mechanisms underlying the

restoration of a normal mouse phenotype by antidepressants in corticosterone-treated

animals were neurogenesis-dependent. X-radiation (5 Gy) was delivered on days 1, 4,

and 8 before the start of the corticosterone treatment. All animals (Sham or X-irradiated)

received 7 weeks of corticosterone (35 ug/ml) regimen in presence or absence of

fluoxetine (18 mg/kg/day) during the last three weeks of the 3 weeks regimen.

Supplemental figure 2: 4 weeks corticosterone treatment (7 or 35 ug/ml per day)

induced behavioral changes in the Open Field paradigm (A-D), the Novelty-
Suppressed Feeding test (E-G), but not the Forced Swim test (H) in C57BL/6Ntac

mice.

(A-D) Effects of corticosterone (7 or 35 ug/ml/day) regimen on anxiety behaviors in the

Open-Field paradigm. Anxiety, measured for various parameters in the center of OF

paradigm, is expressed as mean total of the time-spent (in seconds) for each 5 min

period (A), for the entire session (B) and also for the ambulatory distance in the center

over total (C). Locomotor activity is reported as ambulatory distance traveled for the all

session. Values plotted are mean ± SEM (n = 11-15 per group). PSLD post hoc test:

**p<0.01 versus vehicle group).

(E and G) Effects of 4 weeks of corticosterone regimen (7 or 35 ug/ml/day) on anxiety-

and depression related behaviors in the Novelty Suppressed Feeding paradigm. Results

are expressed as mean of latency to feed (in seconds) (E) or cumulative survival with

percentage of animals that have not eaten over 10-min (F). The feeding drive of each

mouse was assessed by returning the animal to the familiar environment of the home

cage, immediately after the test, and measuring the amount of food consumed over a

period of 5 min (mg/g of mouse) (F). Values plotted are mean ± SEM (n = 11-15 per

group). PSLD post hoc test: *p<0.05, **p<0.01 versus vehicle group) (Kaplan–Meier

survival analysis, Mantel–Cox log-rank test **p<0.01).

(H) Effects of 4 weeks of corticosterone regimen (35 ug/ml/day) on depression-like

behavior in the Mouse Forced Swim Test. Results are expressed as mean of mobility
duration (in seconds). Values plotted are mean ± SEM (n = 12-15 per group). No

statistical difference was observed between groups.

Supplemental figure 3: 4 weeks corticosterone treatment (35 ug/ml per day)

induced deterioration of the coat state (A), increased mouse body weight (B), food

(C), drinking consumption (D) and altered HPA axis function.

(A) Photos of the coat state in C57BL/6Ntac mice in controls and corticosterone treated

animals.

(B-D) The effects of corticosterone regimen (35 ug/ml/day) on mean mouse body weight

(in g) (B), food consumption (in mg/g of mouse/day) (C) and drinking consumption (ml/g

of mouse/day) (D) ± SEM (n = 12-15 per group) were calculated over 4- weeks of

treatment. PSLD post hoc test: **p<0.01 versus control group.

(F) Effects of chronic antidepressant treatment (IMI: imipramine, 40 mg/kg/day; FLX:

fluoxetine, 18 mg/kg/day) on corticosterone levels after an acute stressor. Values plotted

are mean ± SEM (n = 8-9 per group). PSLD post hoc test: **p<0.01 versus vehicle group

for corticosterone levels.

Supplemental figure 4: 4 weeks corticosterone treatment (35 ug/ml per day)

decreased home cage activity and flattened circadian rhythm is not reversed by

chronic antidepressant treatment

(A) The effects of corticosterone (35 ug/ml/day) regimen on the mean dark/total distance

traveled during the light phase ratio ± SEM (n = 15 per group) were calculated over a 24
hour period in the home cage. A 4-weeks corticosterone treatment flattened circadian

rhythm since the dark/total distance traveled during the light phase ratio is decreased

(unpaired t-test, **p<0.01).

(B-C) The effects of corticosterone (35 ug/ml/day) regimen on the mean distance

traveled during the dark phase (in cm) (B), during light phase (in cm) (C) ± SEM (n = 15

per group) were calculated over a 24 hours period in the home cage. A 4-weeks

corticosterone treatment decreased ambulatory distance traveled during the dark phase

but not the light phase (unpaired t-test, **p<0.01).

(D) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment after corticosterone (35

ug/ml/day) regimen induced decrease of the mean total distance traveled (in cm) ± SEM

(n = 15 per group) were calculated over a 24-hours period in the home cage. PSLD post

hoc test: **p<0.01 versus control group.

(E) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment after corticosterone (35

ug/ml/day) regimen induced increase of the inactivation duration (in seconds) ± SEM (n

= 15 per group) were calculated over a 24-hours period in the home cage. PSLD post

hoc test: **p<0.01 versus control group.

Supplemental figure 5: 4 weeks corticosterone treatment (35 ug/ml per day)

induced behavioral changes in the Open Field paradigm (A-D), the Novelty-

Suppressed Feeding test (E-G), but not the Forced Swim test (H) in CD1 mice.
(A-D) Effects of corticosterone (35 ug/ml/day) regimen on anxiety behaviors in the

OpenField paradigm (OF). Anxiety, measured for various parameters in the center of OF

paradigm, is expressed as mean total of the time-spent (in seconds) for each 5 min

period (A), for the entire session (B) and also for the ambulatory distance in the center

over total (C). Locomotor activity is reported as ambulatory distance traveled for the all

session. Values plotted are mean ± SEM (n = 12-15 per group). Unpaired t-test: *p<0.05

versus vehicle group).

(E and G) Effects of 4-weeks of corticosterone regimen (35 ug/ml/day) on anxiety- and

depression related behaviors in the Novelty Suppressed Feeding paradigm. Results are

expressed as mean of latency to feed (in seconds) (E) or cumulative survival with

percentage of animals that have not eaten over 10-min (F). The feeding drive of each

mouse was assessed by returning the animal to the familiar environment of the home

cage, immediately after the test, and measuring the amount of food consumed over a

period of 5 min (mg/g of mouse) (F). Values plotted are mean ± SEM (n = 12-15 per

group). Unpaired t-test: *p<0.05 versus vehicle group). (Kaplan–Meier survival

analysis,Mantel–Cox log-rank test *p<0.05).

(H) Effects of 4-weeks of corticosterone regimen (35 ug/ml/day) on depression-like

behavior in the Mouse Forced Swim Test. Results are expressed as mean of mobility

duration (in seconds). Values plotted are mean ± SEM (n = 12-15 per group). No

statistical difference was observed between groups.

Supplemental figure 6: Effects of 3 weeks fluoxetine regimen in combination with

corticosterone on mineralocorticoid receptor and Creb-1 gene expression in

mouse hypothalamus, amygdala and hippocampus.

(A-B) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment in combination with

corticosterone (35 ug/ml/day) regimen on mean mineralocorticoid receptor (A) and Creb-

1 gene (B) expression (in % cyclophilin and GAPDH genes expression) ± SEM (n = 10-

12 per group) were calculated in the mouse hypothalamus. The levels of expression of

mineralocorticoid receptor (A) and Creb-1 gene (B) were unchanged by chronic

corticosterone alone or in combination with fluoxetine treatment.

(C-D) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment in combination with

corticosterone (35 ug/ml/day) regimen on mean mineralocorticoid receptor (A) and Creb-

1 gene (B) expression (in % cyclophilin and GAPDH genes expression) ± SEM (n = 10-

12 per group) were calculated in the mouse amygdala. The levels of expression of

mineralocorticoid receptor (C) and Creb-1 gene (D) were unchanged by chronic

corticosterone alone or in combination with fluoxetine treatment.

(E-F) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment in combination with

corticosterone (35 ug/ml/day) regimen on mean mineralocorticoid receptor (A) and Creb-

1 gene (B) expression (in % cyclophilin and GAPDH genes expression) ± SEM (n = 10-

12 per group) were calculated in the mouse hippocampus. The levels of expression of

mineralocorticoid receptor (A) and Creb-1 gene (B) were unchanged by chronic

corticosterone alone or in combination with fluoxetine treatment.

Supplemental figure 7: 3 week antidepressant treatment blocked corticosterone-

induced behavioral changes in the Open Field paradigm (A-B), the Novelty-

Suppressed Feeding test (C-D), but not in β-arrestin 2 knock-out mice (E-H).

(A-B) We examined the effects of 3 weeks of antidepressant treatment (IMI: imipramine,

40 mg/kg/day; FLX: fluoxetine, 18 mg/kg/day), started after 4-weeks of corticosterone

(35 ug/ml/day), on anxiety behaviors in the Open Field paradigm (OF). Anxiety,

measured by various parameters in the OF paradigm, was expressed as mean total of

the time spent in the center (in seconds) for each 5 min period (A), and also for the

ambulatory distance in the center over total (B). PSLD post hoc test: **p<0.01,

##p<0.01, significant difference versus control group for the respective time point and

corticosterone/vehicle group respectively.

(C-D) Effects of chronic antidepressant treatment (IMI: imipramine, 40 mg/kg/day; FLX:

fluoxetine, 18 mg/kg/day), after 7 weeks of corticosterone regimen (35 ug/ml/day), on

anxiety- and depression-like behaviors in the Novelty Suppressed Feeding paradigm.

Results are expressed as or cumulative survival with percentage of animals that have

not eaten over 10-min (C). The feeding drive of each mouse was assessed by returning

the animal to the familiar environment of the home cage, immediately after the test, and

measuring the amount of food consumed over a period of 5 min (mg/g of mouse) (D).

Values plotted are mean ± SEM (n = 10-12 per group). Kaplan–Meier survival analysis,

Mantel–Cox log-rank test **p<0.01.

(E-F) We examined the effects of 4 weeks of fluoxetine treatment (18 mg/kg/day), in β-

arrestin 2 knock-out mice (βArr2-KO) and their littermates, on anxiety behaviors in the

Open Field paradigm (OF). Anxiety, measured by various parameters in the OF

paradigm, was expressed as mean total of the time spent in the center (in seconds) for

each 5 min period (E), and also for the ambulatory distance in the center over total (F).

PSLD post hoc test: *p<0.05, §§p<0.01, significant difference versus control group for

the respective time point and vehicle/fluoxetine group respectively.

(G-H) The effects of chronic fluoxetine in β-arrestin 2 knock-out mice and their

littermates were examined in the Novelty Suppressed Feeding paradigm. Results are

expressed as cumulative survival with percentage of animals that have not eaten over

10-min (G). The feeding drive of each mouse was assessed by returning the animal to

the familiar environment of the home cage immediately after the test and measuring the

amount of food consumed over a period of 5 min (mg/g of mouse) (H). Values plotted

are mean ± SEM (n = 15-18 per group). Kaplan–Meier survival analysis, Mantel–Cox

log-rank test, *p<0.05.

Supplemental figure 8: Effects of 3 weeks fluoxetine regimen in β-arrestin 2

knock-out mice on β-arrestin 1, Gi alpha2, Creb-1, mineralocorticoid receptor and

Erk-1 gene expression in mouse hypothalamus, amygdala and hippocampus.

(A-E) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment in β-arrestin 2 knock-out

mice on β-arrestin 1 (A), Gi alpha2 (B), Creb-1 (C), mineralocorticoid receptor (D) and
Erk-1 (E) expression (in % cyclophilin and GAPDH genes expression) ± SEM (n = 10-12

per group) were calculated in the mouse hypothalamus. In contrast to Erk-1 (E) in

controls littermates, the levels of expression of β-arrestin 1, Gi alpha2, mineralocorticoid

receptor and Creb-1 gene were unchanged by chronic fluoxetine treatment. No

statistical difference was observed between groups.

(F-J) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment in β-arrestin 2 knock-out

mice on β-arrestin 1 (F), Gi alpha2 (G), Creb-1 (H), mineralocorticoid receptor (I) and

Erk-1 (J) expression (in % cyclophilin and GAPDH genes expression) ± SEM (n = 10-12

per group) were calculated in the mouse amygdala. In contrast to Gi alpha2 (G) in

controls littermates, the levels of expression of β-arrestin 1, mineralocorticoid receptor,

Creb-1 gene and Erk-1 were unchanged by chronic fluoxetine treatment. In β-arrestin 2

knock-out mice, the levels of Gi alpha2 (G) and Erk-1 were increased (J). Newman-

Keuls post hoc: *p<0.05, **p<0.01 versus control group.

(K-O) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment in β-arrestin 2 knock-out

mice on β-arrestin 1 (K), Gi alpha2 (L), Creb-1 (M), mineralocorticoid receptor (N) and

Erk-1 (O) expression (in % cyclophilin and GAPDH genes expression) ± SEM (n = 10-12

per group) were calculated in the mouse hippocampus. In contrast to Creb-1 for controls

littermates (M) and β-arrestin 1 (K), mineralocorticoid receptor (N) in β-arrestin 2 knock-

out mice, the levels of expression of Gi alpha2 (L) and Erk-1 (O) were unchanged by

chronic fluoxetine treatment. Newman-Keuls post hoc: **p<0.01 versus control group.
SUPPLEMENTAL FIGURE 1
A

0 1 4 7

weeks

Vehicle

Vehicle Vehicle + Antidepressant

Corticosterone 35 ug/ml

Corticosterone 35 ug/ml Corticosterone 35 ug/ml + Antidepressant

Start of chronic Behavior

Start of chronic
antidepressant treatment Neurogenesis study
Corticosterone treatment
Gene analysis

-1 0 4 7
weeks

X-irr / Sham Corticosterone 35ug/ml

X-irr / Sham Corticosterone 35 ug/ml Corticosterone 35 ug/ml + Antidepressant

Start of chronic Behavior

Start of chronic Neurogenesis study
Corticosterone treatment antidepressant treatment
SUPPLEMENTAL FIGURE 2
Open Field
A B
100 500
90

Total time in the center (sec)

Time in the center (sec)

80 400
70
60 300
50 **
40 200
30
20 ** ** ** ** 100
10
0 0
5 10 15 20 25 30 Time (min)
Corticosterone ug/ml/d - 7 35
Vehicle Corticosterone 35ug/ml/d
Corticosterone 7ug/ml/d
C D
25
22,5 4500
Amb. dist center/Total Amb (in %)

Total ambulatory distance (cm)

20 4000
17,5 3500
15 p=0.058 3000
12,5
2500
10
2000
7,5
1500
5
2,5
1000
0 500
0
Corticosterone ug/ml/d - 7 35 Corticosterone ug/ml/d - 7 35

Novelty Suppressed Feeding

E F

500 7
Food Consumption (mg/g of mouse)

* **
6
400
Latency to feed (sec)

5
300 4

200 3

2
100
1

0 0

Corticosterone ug/ml/d - 7 35 Corticosterone ug/ml/d - 7 35

G H
Forced Swim Test
1 75
Total mobility duration (sec)

.8

50
.6
Cum. Survival

.4
25
.2

0 0
0 100 200 300 400 500
Latency to feed (sec) Corticosterone ug/ml/d - 7 35
Vehicle Corticosterone 35 ug/ml/d
Corticosterone 7ug/ml/d
SUPPLEMENTAL FIGURE 3

B
A
30
**
Vehicle-treated animal Corticosterone-treated animal

Mouse body weight (g)

28
**
26

24

22

20
0 1 2 3 4 weeks

Vehicle Corticosterone 35 ug/ml/d

C D

Drinking consumption (in ml/g of mouse/day)

Food consumption (in mg/g of mouse/day)

0.2 0.3

0.25
**
0.15
** ** 0.2
** ** ** **
0.1 0.15

0.1
0.05
0.05

0 0
1 2 3 4 0 weeks
0 weeks 1 2 3 4

Vehicle Corticosterone 35 ug/ml/d Vehicle Corticosterone 35 ug/ml/d

E
800
Corticosterone (ng/ml)

600

400

200

** ** **
0
Drug VEH IMI FLX VEH IMI FLX
Corticosterone 35ug/ml/d - - - + + +
SUPPLEMENTAL FIGURE 4
Home cage activity
A
2.5

the dark phase over the light phase

Ratio ambulatory distance during
2.25
2
1.75
**
1.5
1.25
1
.75
.5
.25
0
Corticosterone 35 ug/ml/d - +

B
C
16000
30000
during the dark phase (in cm)

during the light phase (in cm)

14000
25000
Ambulatory distance

Ambulatory distance
12000
20000 10000
** 8000
15000
6000
10000
4000
5000 2000

0 0
+ Corticosterone 35 ug/ml/d - +
Corticosterone 35 ug/ml/d -

D E
50000
** **
70000 45000
Inactivation duration (sec)
Ambulatory distance (cm)

60000 40000
35000
50000
30000
40000
25000
30000 20000
** ** 15000
20000
10000
10000
5000
0 0
Drug VEH FLX VEH FLX Drug VEH FLX VEH FLX
Corticosterone 35 ug/ml/d - - + + Corticosterone 35 ug/ml/d - - + +
 SUPPLEMENTAL FIGURE 5 Open Field

A B
100
Total time in center in the center (sec)

90 500
80

Total time in the center (sec)

70 400
60
Vehicle
50 300
Corticosterone 35 ug/ml/day
40 *
30 200
** ** Vehicle
20
** 100 Corticosterone
10 35 ug/ml/d
0
5 10 15 20 25 30 0
Time (min)

C D

20 5000
4500

Total ambulatory distance (cm)

18
Amb. dist center/Total Amb (in %)

16 4000
14 * 3500
12 Vehicle 3000
Vehicle
10 Corticosterone 2500
Corticosterone
35 ug/ml/d 2000
8 35 ug/ml/d
6 1500
4 1000
2 500
0 0

Novelty Suppressed Feeding

E F
500 6
Food Consumption (mg/g of mouse)
Latency to Feed (in sec)

400 5

300
* 4

Vehicle 3 Vehicle
200 Corticosterone Corticosterone
35 ug/ml/d 35 ug/ml/d
2

100
1

0 0

G H
Forced Swim Test
1
45

.8 40
35
Cum. Survival

Mobility duration (sec)

.6 30
25
.4
20
Vehicle
.2 15 Corticosterone
35 ug/ml/d
10
0
5
0 100 200 300 400 500 0
Latency to feed (sec)
Vehicle Corticosterone 35 ug/ml/d
SUPPLEMENTAL FIGURE 6 HYPOTHALAMUS

A B
Mineralocorticoid receptor
Creb-1
125 125

100
Expression %
100

Expression %
75
75
50
50
25
25
0
Drug VEH FLX VEH FLX 0
- - + + Drug VEH FLX VEH FLX
Corticosterone 35 ug/ml/d
Corticosterone 35 ug/ml/d - - + +

AMYGDALA

C Mineralocorticoid receptor D Creb-1

125
125

Expression %
100 100
Expression %

75 75

50 50

25 25

0 0
Drug VEH FLX VEH FLX Drug VEH FLX VEH FLX
Corticosterone 35 ug/ml/d - - + + Corticosterone 35 ug/ml/d - - + +

HIPPOCAMPUS

E Mineralocorticoid receptor F Creb-1

125 125
Expression %

100 100
Expression %

75 75

50 50

25 25

0 0
Drug VEH FLX VEH FLX Drug VEH FLX VEH FLX
Corticosterone 35 ug/ml/d - - + + Corticosterone 35 ug/ml/d - - + +
SUPPLEMENTAL FIGURE 7 Open Field in corticosterone-treated animals

A B ##
60
1800
50 #
1600
Time in the center (sec)

Amb. dist center/Total Amb (in %)

40 1400
1200
30
1000
20 ##
800 *
10
** 600
** ** ** ** ** 400
0 Time (min)
5 10 15 20 25 30 200
Vehicle, Vehicle Corticosterone 35 ug/ml, Vehicle 0
Vehicle, Imipramine 40 mg/kg/d Corticosterone 35 ug/ml, Imipramine 40 mg/kg/d Drug VEH IMI FLX VEH IMI FLX
Vehicle, Fluoxetine 18 mg/kg/d Corticosterone 35 ug/ml, Fluoxetine 18 mg/kg/d Corticosterone 35 ug/ml/d - - - + + +

Novelty Suppressed Feeding in corticosterone-treated animals

C D 6

Food Consumption (mg/g of mouse)

1
5
.8
4
Cum. Survival

.6
3
.4
2
.2
1
0
0
0 100 200 300 400 500 600 700 Drug VEH IMI FLX VEH IMI FLX
Latency to Feed (sec) Corticosterone 35 ug/ml/d- - - + + +
Vehicle, Vehicle Corticosterone 35 ug/ml/d, Vehicle
Vehicle, Imipramine 40 mg/kg/d Corticosterone 35 ug/ml/d, Imipramine 40 mg/kg/d
Vehicle, Fluoxetine 18 mg/kg/d Corticosterone 35 ug/ml/d, Fluoxetine 18 mg/kg/d

Open Field in β-arrestin 2 KO mice

E
F 3,5
Amb. dist center/Total Amb (in %)

6 3
Time in the center (sec)

5 2,5

4 2
3 *
1,5
2
* 1
1 §§
,5
0

Time (min)
0
5 10 15 20 25 30 WT BArr2 KO

WT, Vehicle WT, Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18mg/kg/day

Arr2-KO, Vehicle Arr2-KO, Fluoxetine 18 mg/kg/d

Novelty Suppressed Feeding in β-arrestin 2 KO mice

G H
1
6
Food Consumption (mg/g of mouse)

,8 5
Cum. Survival

,6 4

,4 3

,2 2

0 1
0 100 200 300 400 500 600 700
Latency to Feed (sec) 0
WT βArr2 KO
WT, Vehicle βArr2 KO, Vehicle
WT, Fluoxetine 18 mg/kg/d βArr2 KO, Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18mg/kg/day
SUPPLEMENTAL FIGURE 8 HYPOTHALAMUS
A
Expression %
150 β-arrestin 1 B 150 Gi alpha2

100 100

Expression %
50 50

0 0
WT β-arr2 KO WT β-arr2 KO
Vehicle Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18 mg/kg/d
E Erk-1
C D 150
150 150 Mineralocorticoid receptor *
Creb-1

Expression %

Expression %
100
Expression %

100 100

50 50 50

0 0 0
WT WT β-arr2 KO WT β-arr2 KO
β-arr2 KO
Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18 mg/kg/d
Vehicle

F AMYGDALA
β-arrestin 1 G Gi alpha2
150
150
* **
Expression %

Expression %

100
100

50
50

0
WT β-arr2 KO 0
WT β-arr2 KO
Vehicle Fluoxetine 18 mg/kg/d
Vehicle Fluoxetine 18 mg/kg/d

H Creb-1 I Mineralocorticoid receptor

150 150 J 200 Erk-1
**
150
Expression %

Expression %

Expression %

100
100
100

50 50
50

0 0
0 WT β-arr2 KO
WT β-arr2 KO WT β-arr2 KO
Vehicle Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18 mg/kg/d
Vehicle Fluoxetine 18 mg/kg/d

L HIPPOCAMPUS
K 150 β-arrestin 1 150 Gi alpha2
**
100 100
Expression %
Expression %

50 50

0 0
WT β-arr2 KO WT β-arr2 KO
Vehicle Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18 mg/kg/d
M Creb-1 N Mineralocorticoid receptor O Erk-1
200 150 150
**
**
150
Expression %

100
Expression %

100
Expression %

100

50 50
50

0 0 0
WT β-arr2 KO WT β-arr2 KO WT β-arr2 KO
Vehicle Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18 mg/kg/d Vehicle Fluoxetine 18 mg/kg/d
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