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
Incre
ase
of
p-E
RK
/ER
K
0
0.5
1
2
1.5
2.5
0
1
2
1.5
2.5
0.5
Fold
Incre
ase
of
p-C
RE
B/C
RE
B
0
0.5
1
2
1.5
2.5
Fo
ld In
cre
ase
of
p-A
KT
/AK
T
0
Fold
In
cre
ase
of
p-C
aM
Kll/
Ca
MK
ll
0.5
1
2
1.5
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
2
2.5
0
1
1.5
0.5
2
2.5
Fo
ld I
ncre
ase
of
p-4
EB
P1
/β-a
ctin
Ctl 0.01 0.1 10 50 (µM)
* **
** ** **
**
** * **
*
B
Figure-S1 (Choi)
Fo
ld I
ncre
ase o
f
p-P
KD
/PK
D * **
0
1
1.5
0.5
2
2.5
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
0
0.9
0.3
1.2
0.6
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.
To
tal d
ista
nce
(m
)
Vehicle Ketamine
0
15
5
25
35
Locomoter activity
Ho
me
ca
ge f
oo
d in
take
(g
)
0
0.9
1.8
2.7
0
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4
14
12
10
8
6
B C D
F G H
Late
ncy t
o fe
ed (
s)
0
100
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300
Home cage CUS
NSFT
* *
Su
cro
se
pre
fere
nce
(%
)
0
20
40
60
80
SPT
*
0
Imm
ob
ility
(s)
150
300
450
FST
** ***
E
Fa
ilure
s o
f e
sca
pe
0
5
10
15
20
LH
***
A
CUS
Ketamine or Vehicle
28 35 d 27
SPT FST NSFT LMA LH
1 Biochemistry
*
Home cage CUS Home cage CUS Home cage CUS
Home cage CUS Home cage CUS Home cage CUS
Figure-S6 (Choi)
To
tal con
su
mp
tion
(m
l)
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
1
1.5
2
Egr1
mR
NA
le
vel
*
**
*
0 0.5 3 6 24 48
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
Fold
Incre
ase
of
p-H
DA
C5
/HD
AC
5
0.5
1
2
1.5
HDAC5
- + - - + + Ketamine - - + - + - KN62 - - - + - + Gö6976
+ - - - - - Vehicle
p-HDAC5 (S279)
β-actin
HDAC5
B A
0
Fo
ld I
ncre
ase o
f
p-H
DA
C5/H
DA
C5
0.5
1
2
1.5
- + - - + + 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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