SUPPLEMENTARY INFORMATION

Ketamine produces antidepressant-like effects through phosphorylation-dependent

nuclear export of histone deacetylase 5 in rats

Miyeon Choi, Seung Hoon Lee, Sung Eun Wang, Seung Yeon Ko, Mihee Song, June-Seek

Choi, Yong-Seok Kim, Ronald S. Duman and Hyeon Son

LIST OF SUPPLEMENTARY MATERIALS

Fig S1. Ketamine transiently and dose-dependently activates neuronal signalings in rat

hippocampal neurons.

Fig S2. Subcellular localization of HDAC5 upon ketamine stimulation in rat hippocampal neurons.

Fig S3. Subcellular localization of HDAC5 upon ketamine stimulation in rat hippocampal neurons

expressing GFP-HDAC5-S/A.

Fig S4. Ketamine regulates Arc, Klf6 and Nurr77 genes via MEF2D in hippocampal neurons.

Fig S5. Expression of Hdac5 mRNA in hippocampus upon ketamine treatment.

Fig S6. Expression of HDAC5 phosphorylation in hippocampus upon a single dose of ketamine

injection and following behavioral tests.

Fig S7. Effects of ketamine on the behavioral deficits caused by CUS exposure.

Fig S8. Expression of Egr1 mRNA in hippocampus upon ketamine treatment.

Fig S9. Ketamine stimulates HDAC5 phosphorylation at Ser279 in rat hippocampal neurons.

Fig. S10. Glutamate AMPA receptor antagonist NBQX blocks ketamine-induction of HDAC5

phosphorylation in hippocampus.

Fig S11. MC1568, a class II HDAC inhibitor, produces fast antidepressant-like effects.

Table S1. Primer Sequences for RT-PCR

Supplementary Materials and Methods

References

Fig S1. Ketamine transiently and dose-dependently activates neuronal signalings in rat

hippocampal neurons. (A) Dose-dependent activation, determined 30 min after ketamine

administration, of phospho-ERK (p-ERK), phospho-4E-BP1 (p-4E-BP1), phospho-CREB (p-

CREB), phospho-CaMKII (p-CaMKII), phospho-AKT (p-AKT), and phospho-PKD (p-PKD) in

whole cellular extracts determined by Western blot analysis. Levels of total ERK, CREB, CaMKII,

AKT, PKD and β-actin were also determined. (B) Values represent mean ± SEM (n = 4 independent

experiments; *p < 0.05; **p < 0.01, Student’s t-test).

A

Fold

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of

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42KDa 44KDa

Ctl 0.01 0.1 10 50 (µM)

Ctl 0.01 0.1 10 50 (µM) Ctl 0.01 0.1 10 50 (µM)

Ctl 0.01 0.1 10 50 (µM)

Ctl 0.01 0.1 10 50 (µM)

β-actin

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* **

** ** **

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** * **

*

B

Figure-S1 (Choi)

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f

p-P

KD

/PK

D * **

0

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Ctl 0.01 0.1 10 50 (µM)

PKD

p-PKD

AKT

p-AKT

CaMKII

p-CaMKII

CREB

p-CREB

p-4EBP1

ERK

p-ERK

Fig S2. Subcellular localization of HDAC5 upon ketamine stimulation in rat hippocampal

neurons. Hippocampal neurons were exposed to ketamine for 30 min at 100 nM. Accumulation

of p-HDAC5 (S259) was shown in the cytosol of ketamine treated neurons. VEGF, a positive

control. Nuclear extracts were determined by lamin B1, a nuclear marker.

Figure-S2 (Choi)

Cytosol Nucleus

Ctl Ketamine VEGF Ctl Ketamine VEGF

p-HDAC5 (S259)

HDAC5

LaminB1

GAPDH

Fig S3. Subcellular localization of HDAC5 upon ketamine stimulation in rat hippocampal

neurons expressing GFP-HDAC5-S/A. Representative images are hippocampal neurons

transfected with pCI-neo-GFP-HDAC5-S/A. Twenty-four hours after transfection, cells were

treated with ketamine for the time indicated in each image. Ketamine did not induce HDAC5

nuclear export. Images were captured at a magnification of ×60, using a fluorescence microscope.

Scale bar, 25 μm.

0 min

30 min

1 h

3 h

12 h

24 h

48 h

72 h

DAPI GFP Merge DAPI GFP Merge

25 ㎛

Figure-S3 (Choi)

Fig S4. Ketamine regulates Arc, Klf6 and Nurr77 genes via MEF2D in hippocampal neurons.

(A) Increased binding of MEF2D to the Arc, Klf6 and Nurr77 promoters in the hippocampal

neurons. Effect of ketamine on association of MEF2D with gene promoters was analyzed by

chromatin immunoprecipitation (ChIP) assay and the result was shown by quantitative PCR (n =

2 cultures). (B) Ketamine-induced increase in MEF2D protein in hippocampus. Representative

Western blot analyses of MEF2D in the hippocampus of rats injected with ketamine (n = 3

animals). Student’s t test, * p < 0.05, ** p < 0.01.

Fig S5. Expression of Hdac5 mRNA in hippocampus upon ketamine treatment. qRT-PCR

analysis shows the expression of Hdac5 mRNA in hippocampus after ketamine treatment (10

mg/kg, i.p.) for various times. Results of Hdac5 mRNA levels were normalized with the level of

GAPDH. The mRNA levels at each time point were depicted relative to the level of the vehicle-

treated control and are shown as fold changes relative to the value at 0 h. Values represent mean ±

SEM (n = 4 animals).

HD

AC

5 m

RN

A leve

l

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0.9

0.3

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0 0.5 3 6 24 48

Time after ketamine treatment (h)

Figure-S4 (Choi)

Fig S6. Expression of HDAC5 phosphorylation in hippocampus upon a single dose of

ketamine injection and following behavioral tests. (A) Experimental design. Rats were exposed

to home cage during whole procedure. (B) HDAC5 phosphorylation in hippocampus of rats

exposed to ketamine (10 mg/kg) and behavioral tests for 5-6 consecutive days. Representative

immunoblots of p-HDAC5 (S259) and HDAC5 are shown.

Fig S7. Effects of ketamine on the behavioral deficits caused by CUS exposure. (A)

Experimental design. Rats were split into two experimental groups and exposed to home cage or

CUS for 35 d. Each of the two cohorts was split into two experimental groups for vehicle and

ketamine treatments, respectively. (B) NSFT. Main effect of ketamine: F3, 56 = 12.13, p < 0.001;

main effect of stress: F3, 56 = 0.04, p > 0.05; interaction: F3, 56 = 0.77, p > 0.05 (n = 21, 21, 9, 9).

Further analysis indicates that a significant decrease in the latency to feed was shown by ketamine

and home caged and CUS animals (* p < 0.05). (C) FST. Main effect of ketamine: F3, 56 = 27.20, p

< 0.0001; main effect of stress: F3, 56 = 18.65, p < 0.0001; interaction: F3, 56 = 4.72, p < 0.05 (n =

21, 21, 9, 9). Further analysis indicates that a significant decrease in immobility was shown by

ketamine in home caged animals (** p < 0.01) and CUS animals (*** p < 0.001). (D) LHT. Main

effect of ketamine: F3, 24 = 28.50, p < 0.001; main effect of stress: F3, 24 = 5.42, p < 0.05; interaction:

F3, 24 = 4.60, p < 0.05 (n = 6, 7, 7, 8). Further analysis indicates that ketamine decreased escape

failures in NO CUS animals (* p < 0.05) and CUS animals (*** p < 0.001). (E) SPT. Main effect of

ketamine: F3, 44 = 7.98, p < 0.01; main effect of stress: F3, 44 = 0.04, p > 0.05; interaction F3, 44 =

2.44, p > 0.05 (n = 16, 16, 7, 9). Further analysis indicates that ketamine increased sucrose

preference only in CUS animals (* p < 0.05). (F) There was no difference in the home cage food

intake. Main effect of ketamine: F3, 56 = 0.26, p > 0.05; main effect of stress: F3, 56 = 1.44, p > 0.05;

interaction F3, 56 = 1.16, p > 0.05. (G) Total consumption. Main effect of ketamine: F3, 44 = 0.217,

p > 0.05; main effect of stress: F3, 44 = 0.256, p > 0.05; interaction F3, 44 = 0.627, p > 0.05. (H)

Total distance moved in the box between groups. Main effect of ketamine: F3, 36 = 3.247, p > 0.05;

main effect of stress: F3, 36 = 2.363, p > 0.05; interaction F3, 36 = 0.525, p > 0.05. Data are the mean

± SEM. Two-way ANOVA followed by LSD post hoc analysis. * p < 0.05, ** p < 0.01, *** p <

0.001.

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28 35 d 27

SPT FST NSFT LMA LH

1 Biochemistry

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Home cage CUS Home cage CUS Home cage CUS

Home cage CUS Home cage CUS Home cage CUS

Figure-S6 (Choi)

To

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Fig S8. Expression of Egr1 mRNA in hippocampus upon ketamine treatment. qRT-PCR

analysis shows the expressions of Egr1 mRNAs in hippocampus after ketamine treatment for 30

min-24 h. Results of Egr1 mRNA levels were normalized with the level of GAPDH. The mRNA

levels at each time point were depicted relative to the level of the vehicle-treated control and are

shown as fold changes relative to the value at 0 h. Values represent mean ± SEM (n = 4 animals)

0

0.5

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Egr1

mR

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vel

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Time after ketamine treatment (h)

Figure-S7 (Choi)

Fig S9. Ketamine stimulates HDAC5 phosphorylation at Ser279 in rat hippocampal neurons.

(A) Hippocampal neurons were exposed to ketamine for 30 min for various concentrations (n = 4

independent experiments). (B) Neurons were pretreated with the KN-62 (30 µM) or Gö6976 ( 1

µM) for 30 min, and then exposed to ketamine (100 nM) for 6 h. Quantitative data of HDAC5

phosphorylation normalized with the level of HDAC5 are shown as fold changes relative to the

vehicle-treated control (Ctl) value. Values represent mean ± SEM (n = 4 independent

experiments). * p < 0.05, ** p < 0.01.

p-HDAC5 (S279)

Ctl

β-actin

Ctl 0.01 0.1 10 50 (µM) 0

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+ - - - - - Vehicle

p-HDAC5 (S279)

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- + - - + + Ketamine - - + - + - KN62 - - - + - + Gö6976

+ - - - - - Vehicle

0.01 0.1 10 50 (µM)

** * *

* **

Figure-S8 (Choi)

Fig S10. Glutamate AMPA receptor antagonist 2, 3-dihydroxy-6-nitro-7-sulfamoyl-

benzolquinoxaline-2, 3-dione (NBQX) blocks ketamine-induction of HDAC5

phosphorylation in hippocampus. (A, B Top panel) Experimental paradigm. (Left)

Representative Western blot analysis shows that NBQX (10 mg/kg) blocks ketamine stimulation

of the HDAC5 phosphorylation 3 h (A) or 6 h (B) after ketamine administration i.p.. (Right) Levels

of p-HDAC5 S259 were quantified by densitometry. Values represent mean ± SEM (n = 3

animals/group). * p < 0.05.

Fig S11. MC1568, a class II HDAC inhibitor, produces fast antidepressant-like effects. (Top

panel) Experimental paradigm. (Graph) Immobility of rats in the FST after treatment with

MC1568 (50 mg/kg). Single injection of MC1568 (50 mg/kg, (1)) significantly reduced

immobility, indicating an antidepressant-like response, at 6 h and 9 h, compared to vehicle

treatment. Independent groups of rats were used at each time point. Values represent mean ± SEM

(n = 3 animals/group). * p < 0.05, ** p < 0.01.

Table S1. Primer Sequences for RT-PCR

Table S1 (Choi)

Arc F 5’-GGG ACC TGT ACC AGA CAC-3’

R 5’-GGT CCT GTC ACT GGC TAC-3’

Nur77 F 5’-CGG AGA TGC CCT GTA TCC-3’

R 5’-ATG GTG GGC TTG CTG AAC-3’

Egr1 F 5’-CTT CGC TCA CTC CAC TAT CC-3’

R 5’-GAT GAG TTG GGA CTG GTA GG-3’

KLF6 F 5’-TTG AAA GCA CAT CAG CGC ACT CAC-3’

R 5’-ACC GGT ATG CTT TCG GAA GTG TCT-3’

HDAC5 F 5’-ATG GGA TTC TGC TTC TTC AA-3’

R 5’-TGT CCT TCA ACA GCA TCA AA-3’

Supplementary Materials and Methods

Preparation of hippocampal neurons Primary hippocampal neurons were prepared and processed

as described previously (2). Hippocampi from E16.5 Sprague-Dawley rat embryos were rapidly

and aseptically dissected from each brain in ice-cold Ca2+/Mg2+-free Hank's balanced salt solution

(HBSS; Gibco, Carlsbad, CA, USA), followed by removal of meninges and mincing into small

pieces. The hippocampal tissue was then digested in 0.25 % EDTA–trypsin (Worthington

Biochemical, Lakewood, NJ, USA), and dissolved in Ca2+/Mg2+-free HBSS for 10 min at 37 °C in

a humidified atmosphere of 5 % CO2 and 95 % air. The tissue was transferred to chilled Ca2+/Mg2+-

free HBSS and triturated through a siliconized fire-polished Pasteur pipette. Undissociated tissue

fragments were allowed to settle for 5 min, the supernatant was transferred to a new tube and it

was centrifuged at 200 ×g for 1 min. The pelleted cells were gently resuspended in culture medium

and plated at 40,000–50,000 cells per cm2 on poly-L-lysine (25 mg/ml in phosphate-buffered saline

(PBS); Sigma-Aldrich, St Louis, MO, USA) and laminin (10 mg/ml in PBS, Invitrogen, Carlsbad,

CA, USA) coated glass coverslips. Hippocampal cultures were grown for 1 day in neurobasal (NB)

medium (Gibco, Carlsbad, CA, USA) containing 10 % fetal bovine serum (FBS), 75 mmol/L L-

glutamine and 0.1 % penicillin–streptomycin. The medium was changed the following day to NB

supplemented with 0.02 % B27 serum-free supplement, 75 mmol/L L-glutamine, and 0.1 %

penicillin–streptomycin antibiotic mixture. Half of the medium was replaced every 3 days and

AraC (2 μM) was added on day 3. Cultures were maintained for 10-12 days at 37 °C in a 5 %

CO2/95 % air humidified incubator. The neurons were used after 10 to 14 days. Animal care and

experiments were conducted in accordance with the 2004 Guide for the Care and Use of

Laboratory Animals (Korea National Institute of Health) and Hanyang University Veterinary

committee.

Drugs The following compounds were used: KN-62 (Sigma, St. Louis, MO, USA), Gö6976

(Tocris Bioscience) and ketamine. KN-62 and Gö6976 were solubilized in dimethyl sulfoxide

(DMSO), and the same volume of DMSO (final concentration 0.02 %) was added into the medium

of non-treated cultures as vehicle. NBQX (10 mg/kg; Tocris Bioscience, Bristol, UK) was injected

i.p..

Reverse transcription (RT)-PCR and quantitative real-time RT-PCR Total RNA was prepared

from in vitro hippocampal neurons and whole hippocampus using the phenol-free total RNA

isolation kit, RNAqueous (Ambion, Austin, TX, USA). Whole hippocampus were obtained from

the following groups of rats 24 h after the last injection of ketamine and 24 h after the last

behavioral test: CUS plus one dose of saline, CUS plus one dose of ketamine, nonstressed controls

plus saline and nonstressed controls plus one dose of ketamine. RNA was processed as described

previously (2). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an

internal control. Real-time PCR was performed using SYBR Green (Bio-Rad) and iCycler/iQ

detection system (Bio-Rad, Hercules, CA, USA). The primer pairs used are listed (Table S1). The

analysis of relative mRNA levels was performed using a delta-CT (ΔΔCt) relative quantification

model with GAPDH as a reference gene. Each value is expressed as fold change relative to the

mRNA level of the control value. Real-time PCR reactions, run in triplicate for each

condition/brain sample, were performed independently at least four times.

Chromatin immunoprecipitation (ChIP) assay ChIP assays were performed following a

procedure provided by a ChIP Assay Kit (Upstate Biotechnology, Lake Placid, NY). The following

antibodies were used: anti-MEF2D (Santa Cruz, Dallas, TX). Immunoprecipitated DNA samples

were resuspended in H2O and fractions used for semiquantitative PCR (TC-312, TECHNE, UK)

or real-time PCR (iCycler, Bio-Rad, Hercules, CA). Input or total DNA (non-immunoprecipitated)

and immunoprecipitated DNA was PCR amplified in triplicate in the presence of SYBR Green

(Applied Biosystems, CA). Ct values for each sample were obtained using the Sequence Detector

1.1 software. Real-time PCR reactions, run in triplicate for each brain sample, were repeated at

least twice independently. The primer sequences are: Arc, F: 5-agaaccttgcaggagcctta-3, R: 5-

atggaggaacctcaacatgg-3; Nurr77, F: 5-gtccgggactgactgggaaa-3, R: 5-gtcccggggtccgaaataac-3;

Klf6, F: 5’- agtggtatctctagagatcgca-3’, R: 5’-actccatgttgttttgcttcct-3’.

Histone isolation and immunoblotting analyses Hippocampal neurons were cultured without or

with ketamine for the indicated times. The cells were recovered by centrifugation and core histone

proteins were extracted as previously described (3). The extracts were boiled in SDS sample buffer

(62.5 mM Tris-Cl pH 6.8, 2 % SDS, 10 % glycerol, 50 mM DTT, 0.1 % bromophenol blue) to

denature the histones. The samples were then electrophoresed on 10 % polyacrylamide gels and

transferred to nitrocellulose membrane filters (Amersham Pharmacia Biotech, Arlington Heights,

IL, USA). The blots were blocked with 5 % non-fat milk in Tris-buffered saline with Triton-X100

(TBST) for 1 h and incubated overnight at 4 °C with primary antibody in TBST with 5 % non-fat

milk. The primary antibodies recognizing acetylated and total histones H3 and H4 were from

Upstate Biotechnology (Lake Placid, NY, USA): mouse anti-Histone H3 (1:1000), rabbit anti-

acetyl Histone H3 (Lys-14, 1:1000), rabbit anti-Histone H4 (1:1000), and rabbit anti-acetyl Histone

H4 (Lys-5/Lys-8/Lys-12/Lys-16, 1:1000), mouse anti-β-actin (1:1000, Santa Cruz, Dallas, TX,

USA). After washing with TBST, the membranes were incubated with anti-rabbit or anti-mouse

horseradish peroxidase-linked secondary antibody (1:2000, Santa Cruz) for 1 h at room

temperature. They were washed with TBST and processed for chemiluminescence detection using

a horseradish peroxidase substrate and the enhanced chemiluminescence (ECL) detection kit

(GenDEPOT, Barker TX, USA). The intensity of signals was captured on film.

HDAC5 subcellular localization immunofluorescence study Hippocampal neurons grown on

glass coverslips were transfected after 3 days in vitro with a plasmid encoding a green fluorescent

protein (GFP)-HDAC5 fusion protein (4) (GFP-HDAC5-WT; Addgene plasmid #32211) and a

phosphorylation-defective (Ser259/498Ala) mutant of GFP-HDAC5 (5) (GFP-HDAC5-S/A;

Addgene plasmid #32218) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA)

according to the manufacturer’s instructions. The cells were stimulated with ketamine for the

indicated time and the localization of HDAC5 was categorized as cytoplasmic, nuclear, or both

(evenly distributed across nucleus and cytoplasm) for neuron as described previously (6) under

experimenter-blind conditions. The cells were fixed with 4 % paraformaldehyde and

immunostained with mouse monoclonal anti-GFP (1:400, Roche) followed by 2 h incubation with

Alexa488-conjugated secondary antibodies (1:400, Invitrogen). Neurons were then mounted in

Vectashield mounting medium containing DAPI (Vector Laboratories). Images were captured

using the Leica TCS SP5 (magnification: ×60, Leica Microsystems, Wetzlar, Germany). To

characterize the subcellular localizations of HDAC5, GFP immunofluorescence intensities were

quantified with ImageJ software in the cytoplasmic and nuclear compartments of transfected

neurons, as described previously (7). All experiments were independently replicated at least three

times.

MEF2 Luciferase assay The pCI-neo-HDAC5-WT and pCI-neo-HDAC5-S/A expression

plasmids were generated by subcloning the coding sequence from HDAC5-WT (Addgene plasmid

#32211) and HDAC5-S/A (Addgene plasmid #32218) into the pCI-neo (Promega, Madison, WI,

USA), respectively. Hippocampal neurons were cotransfected with p3xMEF2-Luc (Addgene

plasmid #32967), Renilla luciferase expression plasmid pGL3-Luc and when indicated with pCI-

neo-HDAC5-WT or pCI-neo-HDAC5-S/A 5 h after plating using Lipofectamine 2000 reagent

(Invitrogen). Cells were stimulated for 6 h before harvesting, and dual luciferase reporter assays

were performed according to the manufacturer’s instructions (Promega, Madison, WI, USA). All

transfections were performed in triplicate and represent the mean of at least 4 independent

experiments.

Western blot analysis Whole-cell proteins were extracted as described previously (7). Nuclear and

cytoplasmic proteins were extracted using NE-PER nuclear and cytoplasmic extraction reagents

(Thermo Scientific). Western blot analysis was performed, as previously described (7), with rabbit

anti-HDAC5 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-phosphorylated HDAC5

(1:1000, Abcam), rabbit anti-phosphorylated CaMKII (Thr286) (1:1000, Cell Signaling, Danvers,

MA, USA), rabbit anti-CaMKII (1:1000, Cell Signaling), rabbit anti-phosphorylated PKD

(1:1000, Cell Signaling), rabbit anti-PKD (1:1000, Cell Signaling), mouse anti-phosphorylated

p44/p42 ERK (Thr202/Tyr204) (1:1000, Cell Signaling), rabbit anti-p44/p42 ERK (1:2000, Cell

Signaling), rabbit anti-phosphorylated CREB (Ser133) (1:1000, Cell Signaling), rabbit anti- CREB

(1:1000, Cell Signaling), rabbit anti-phosphorylated AKT (Ser473) (1:1000, Cell Signaling), rabbit

anti-AKT (1:1000, Cell Signaling), rabbit anti-phosphorylated 4E-BP1(Thr37/46) (1:1000, Cell

Signaling), rabbit anti-phosphorylated 4E-BP1(Thr37/46) (1:1000, Cell Signaling), goat anti-

MEF2D (1:1000, Santa Cruz), mouse anti-β-actin (1:1000, Santa Cruz), rabbit anti-lamin B1

(1:1000, Abcam), antibodies. Then blots were treated with anti-mouse or anti-rabbit IgG

conjugated with peroxidase (1:2000, Santa Cruz). Bands were visualized with an ECL detection

kit (GenDEPOT). The total densitometric value of each band was quantified with ImageJ software

(http://rsbweb.nih.gov/ij/), normalized to the corresponding β-actin level, and expressed as fold

change relative to the control value.

Immunohistochemistry Slices were prepared and processed as described previously (2).

Antibodies used were as follows: anti-GFP monoclonal (1:300, Roche). Slices were placed in

secondary antibody conjugated to Alexa 488 (1:300, Invitrogen) then mounted in vectashield

mounting medium (Vector Laboratories, Burlingame, CA, USA) for fluorescence and

photographed with a fluorescent microscope (Nikon, Tokyo, Japan).

Viral production and purification The virus was generated using a triple-transfection, helper-free

method, and purified with a published protocol (Invitrogen). Briefly, HEK 293T cells (ATCC,

Manassas, VA, USA) were cultured in five 150 25 mm cell culture dishes and transfected with

ViraPower lentiviral expression systems (Invitrogen) using Lipofectamine (Invitrogen). Cells were

collected, pelleted and resuspended in buffer (0.15 M NaCl and 50 mM Tris, pH 8.0) 66-70 h after

transfection. After two freeze-thaw cycles to lyse the cells, benzonase was added (50 U/ml, final)

and the mixture was incubated at 37 °C for 30 min. The lysate was added to PEG-it solution and

processed to manufacturer’s protocol (System Biosciences, Inc., Mountain View, CA, USA). The

final purified virus was stored at -80 °C.

Behavioral experiments

Animals, drug administration, stereotaxic surgery and infusions. Animals, drug administration,

stereotaxic surgery and infusions Adult male Sprague-Dawley rats (8-10 weeks old; Charles

River Laboratories, Wilmington, MA, USA) weighing 200 to 250 g were pair-housed and

maintained on a 12-h light-dark cycle with access to food and water ad libitum. All procedures

were in strict accordance with Institutional Animal Care and Use Committee (IACUC) guidelines

and Use of Laboratory Animals were approved by the Hanyang University Animal Care and Use

Committee. All animals were randomly assigned to each experimental group. For behavioral

experiments, rats were injected intraperitoneally (i.p.) with ketamine (10 mg/kg body weight),

MC1768 (50 mg/kg; Tocris Bioscience, Bristol, UK) or saline and then analyzed 24 h after the

injection or indicated time points. Stereotaxic surgery and infusions were conducted as previously

described (2). Rats were anesthetized with Rompun (25 mg/kg) and Zoletil (50 mg/kg). Bilateral

viral injections were performed with coordinates -4.1 mm (anterior/posterior), ±2.4 mm (lateral),

and -4.6 mm (dorsal/ventral) relative to the bregma. A total of 6 µl of purified virus was delivered

at a rate of 0.1 µl/min followed by 10 min of rest. Needles were removed and the scalp incision

was closed with wound clips. Members of the same cage were randomly assigned to different

experimental groups for behavioral studies and the order of testing was distributed across groups.

After the behavioral testing was performed, animals were perfused with PBS. The brain was kept

overnight in 4 % paraformaldehyde and then transferred to 30 % sucrose. Brain sections (30 µm

thickness) were cut using a microtome for visualization of EGFP. We assessed gene-transfer

efficacy with immunofluorescent staining 4 - 8 weeks after surgery and observed EGFP staining

predominantly in dentate granule cells surrounding the infusion site in each hemisphere.

Chronic unpredictable stress (CUS) procedure CUS is an experimental procedure in which

animals are exposed to a variable sequence of mild and unpredictable stressors. Our CUS

procedure was successfully used in the laboratory to produce behavioral changes as well as

alteration of corticosterone levels (8, 9). The CUS animals were subjected to exactly the same

sequence of 12 stressors (2 per day for 35 days) described in Banasr et al (10): cage rotation, light

on, light off, cold stress, isolation, crowding, cold swim stress, food and water deprivation, wet

bedding, stroboscope, cage tilt and odor exposure. Rats in the ketamine treatment groups received

one dose of the drug on day 28 of CUS. The day after the injection, the behavioral consequences

of the CUS were tested with continued CUS (total 35 days).

Forced swim test (FST) After the test, animals were dried under a lamp for 30 min. All FST

experiments were filmed by a camcorder and the first 5 min or 15 min of swim was scored offline

for the duration of immobility. Immobility was defined as floating or remaining motionless without

leaning against the wall of the cylinder (11). All behavioral tests were analyzed by an experimenter

blinded to the study code.

Novelty Suppressed Feeding (NSF) Animals were food deprived for 12 h and on the test day

were placed in an open field (76.5 cm × 76.5 cm × 40 cm, Plexiglas) with eight pellets of food in

the center. The animals were given 8 min to approach the food and eat. The test was stopped as

soon as the animal took the first bite. The latency to eat was recorded in seconds. Home cage food

intake was also measured as a control.

Sucrose preference test (SPT) The SPT consisted of a 48 period of exposure to sucrose solution

(1 %; Sigma) for acclimation, followed by 4 h of water deprivation and a 1 h exposure to two

identical bottles, one filled with sucrose solution and the other with water. Sucrose and water

consumption was determined by measuring the change in volume of fluid consumed. Sucrose

preference was defined as the ratio of the volume of sucrose vs. total volume of sucrose and water

consumed during the 1 h test.

Learned helplessness paradigm The learned helplessness procedure was performed in commercial

shuttle boxes divided into two equal compartments by a central barrier (Gemini Avoidance System,

San Diego Instruments, San Diego, CA, USA), as previously described (2). A computer-operated

guillotine door built into the central barrier allowed passage between compartments. On day 1,

inescapable footshock (IES) was administered at one side of the shuttle box with the guillotine

door closed (60 footshocks, 0.85 mA intensity, 15 sec average duration, 60 sec average intershock

interval). Active avoidance testing consisted of 30 trials of escapable footshock (0.65 mA intensity,

35 sec maximum duration, 90 sec average intertribal interval) with the guillotine door open. Each

trial used a fixed-ratio 1 schedule, during which one shuttle crossing by rats terminated the shock.

Shock was terminated automatically if rats did not escape after 35 sec. A computer automatically

recorded the number of escape failures. Results are expressed as number of escape failures, that

is, the number of times that the animal did not terminate the footshock.

Locomotor Activity test (LMA) The general locomotor activity of the rats in the open field test

was measured by an automatic video tracking system (SmarTrack®, Smartech, Madison, WI).

Rats were placed in the central part of the square-shaped arena (76.5 cm x 76.5 cm x 40 cm) and

allowed to explore it for 10 min. Total distance traveled (locomotion activity), time spent in central

part of the arena (center duration), distance moved in central part of the arena (center distance)

were recorded for 10 min.

Statistical analysis The appropriate statistical test was determined based on the number of

comparisons being done. Student's t tests were used for comparison of two groups, in the analysis

of biochemical results. Statistical differences for behavioral experiments consisted of four

experimental groups were determined by analysis of the variance (ANOVA; StatView 5, SAS

Software, Cary, NC, USA) followed by LSD post hoc analysis. The F-values, group and

experimental degrees of freedom are included in the legends of the figures. Experimental sample

sizes as indicated in the figure legends were determined to give the reported SEM values that were

sufficiently low to allow meaningful interpretation of the data. Animals with incorrect viral

injection placement as determined by GFP immunostaining were excluded from analyses. To

minimize the chance of including false behavioral responses, animals with solution leakage in SPT

were excluded from analyses. Data replication was observed in instances of repeated experiments.

Sample sizes are similar to those reported in previous publications in this field (2, 8-10, 12-14).

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