SUPPLEMENTARY MATERIAL
MATERIAL AND METHODS
Animals and treatments
Rats were implanted subcutaneously with an Alzet osmotic minipump (Alza Corp., Palo Alto,
CA) which delivered 0.5 µl per hour during 3 or 7 days, depending on the experimental
process. The minipumps were filled with vehicle (10% ethanol, 50% propylenglycol, 40%
saline) or 1.5 mg/kg/day RS67333 (1-(4-amino-5-chloro-2-methoxyphenyl)-3-[1-butyl-4piperidinyl]-1-propanone hydrochloride), Tocris (Spain). For the behavioural tests fluoxetine
(Fagron, Spain) was administered in the drinking water (10 mg/kg/day) for 7, 14 or 21 days.
All animals were killed at the end of the treatment with RS67333 in order to perform the
herein reported studies. To analyze cell proliferation, animals received 5-bromo-2’deoxyuridine (BrdU; 2 x 100 mg/kg every 12 h, i.p.; Sigma, Spain) in sterile 0.9% NaCl
solution the last day of antidepressant treatment. All other chemicals used were of analytical
grade.
Immunohistochemistry
Twenty-four hours after the first BrdU injection, rats were anaesthetized with sodium
pentobarbital (50 mg/kg, i.p.) and transcardially perfused with 4% paraformaldehyde in PBS.
Brains were cryoprotected with 30% sucrose and 6 sagittal series (40 µm) of the brains were
obtained through the entire hippocampus. Sections were incubated for 2 h in 50%
formamide/2x SSC at 65ºC, followed by incubation in 2 N HCl for 30 min. Then sections
were incubated for 10 min in 0.1 M borate buffer. After washing in PBS, sections were
incubated in 1% H2O2 for 30 min, blocked with 5% donkey normal serum for 30 min and then
incubated with monoclonal mouse anti-BrdU (1:600; Roche Diagnostics, Barcelona, Spain)
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overnight at 4ºC followed by biotinylated donkey anti-mouse IgG incubation (1:200; Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA), and by amplification with ABC
Vectastain Kit (Vector Laboratories, Burlingame, CA). BrdU positive cells were labeled using
DAB + Ni as chromogen (Vector Laboratories, Burlingame, CA) and counterstained with
eosin.
To quantify the number of cells with -catenin cytoplasmatic accumulations, DAB
immunohistochemistry and immunofluorescence colocalization were carried out. After
several rinses in PBS, sections were boiled at 90ºC in 10 mM citric acid, pH 6.0 for 20 min,
blocked with 5% donkey normal serum and then, incubated overnight at 4ºC with an anti-βcatenin monoclonal IgG (1:500; Santa Cruz Biotechnology, Inc. Heidelberg, Germany) and
subsequently with a biotinylated donkey anti-mouse IgG (1:200) followed by ABC Vectastain
Kit. The immunohistochemical signal was reveled with DAB or AlexaFluor 594 tyramide
(Invitrogen, Barcelona, Spain).
The phenotype of neural progenitors expressing β-catenin was addressed by colocalization
with antibodies against GFAP (1:1000; Dako, Denmark), Sox2 (1:200; Santa Cruz
Biotechnology, Inc. Heidelberg, Germany), BLBP (1:500; Chemicon, Temecula, CA) and
DCX (1.1000; Abcam, Cambridge, UK) incubated with their respective FITC and Cy5
conjugated secondary antibodies (1.200; Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA).
The different cell types were classified according to its morphology and the expression of
phenotype markers. Quiescent neural progenitors (QNPs) present a triangular soma with
processes that cross the granule cell layer and express GFAP, Sox2 and BLBP. However,
amplifying neural progenitors (ANPs) express Sox2 and BLBP but not GFAP and have very
short processes. Immature neuroblasts are positive for DCX and their differentiated state was
achieved by the length and arborization of their processes and Sox2 expression.
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Sections were evaluated under a Light Microscope (Carl Zeiss Axioskop 2 Plus) and a
confocal microscope (Carl Zeiss LSM 510) by an observer blinded to the study code. Cells in
the subgranular zone of the dentate gyrus of the hippocampus (DG) were counted in one each
6 sections corresponding to lateral stereotaxic coordinates ranging 0.1-3.9 mm (Paxinos and
Watson, 1998) and multiplied by 6 to obtain the total number of cells in each hippocampus.
Data are expressed as a percentage of the vehicle group values (100%) and are presented as
mean ± S.E.M.
Western blot
Animals were killed by decapitation, and their brains were removed from skulls. Under a
dissecting microscope, each hippocampus was bisected and the dorsomedial half was divided
into four slabs cut perpendicular to the long axis of the hippocampus. Using anatomic
landmarks, each slab was dissected further into samples containing the DG, CA3 or CA1
subfields of the hippampus; CA3 was isolated with a cut connecting the ends of the inner and
outer blades of the DG, and then the DG and CA1 were separated along the hippocampal
fissure. Samples were weighed, frozen on dry ice, and stored at -80ºC. Every sample was
thawed, homogenized and processed in order to obtain the total cell lysate (TCL). Protein
quantification was performed according to the Lowry method (Lowry et al., 1951). About
thirty-fifty µg of protein were resolved on 10 or 12.5% SDS-PAGE and transferred to PVDF
(non-phosphorylated proteins) or to nitrocellulose (phosphorylated proteins) membranes.
Membranes were incubated in the following primary antibodies: mouse anti-β-catenin
(1:1000), mouse anti-GAPDH (1:2000), mouse anti-AKT1 (1:1000) and rabbit anti-BDNF
(1:500), from Santa Cruz Biotechnology, Inc. Heidelberg, Germany; rabbit anti-CREB
(1:800) and rabbit anti-pCREB (1:800), from Abcam plc, Cambridge, UK. After extensive
washings in TBS-T (TBS/0.05% Tween 20) membranes were incubated with horseradish
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peroxidase conjugated secondary antibodies. Secondary antibodies were detected with ECL
Advance kit (GE Healthcare Europe GmbH, Munich, Germany). Blot quantitations were
performed by densitometric scanning using Scion Image Software. The densitometry values
were normalized with respect to the values obtained with anti-GAPDH antibody. Data for
every sample was the mean of at least two independent experiments. Data are expressed as a
percentage of the vehicle group values (100%) and are presented as the mean ± S.E.M.
In situ hybridization
Rats were killed by decapitation and brains were quickly removed, frozen in dry ice and
stored at -80C until sectioning. Tissues were fixed with 4% paraformaldehyde in phosphatebuffered saline (PBS) for 5 minutes, acetylated with 0.25% acetic anhydride in 0.1 M
triethanolamine buffer for 10 minutes, dehydrated in a series of ethanol washes (70%, 80%,
95% and 100%), incubated chloroform for 10 min, and finally rehydrated with 100% and 95%
ethanol. The sections were air-dried and stored at -20ºC until use.
The labeled oligonucleotide probe was added to each section (250000 cpm/section) in the
hybridization buffer (50% formamide (v/v), 4xSSC (saline sodium citrate buffer), 10 mM
sodium phosphate, pH 7 0.1 mM sodium pyrophosphate, 5x Denhardt’s Solution, 0.2 mg/ml
salmon sperm DNA, 10% (w/v), 20 mM dextran sulphate, 0.1 mg/ml polyadenylic acid, 0.12
mg/ml heparin and 20 mM DTT added fresh). After incubation at 42C in humidity chambers
for 16-20 h, the slides were washed twice in 2x SSC containing 4 mM DTT, at 50oC followed
by 5 min washes in 1x SSC, 0.1 x SSC, ethanol (80%) and 1 min ethanol (96%) at room
temperature. Sections were then air-dried and exposed to films (Biomax MR, Kodak, Madrid,
Spain) together with
C microscales (Amersham) at 4C for 3 weeks. Controls included
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hybridization of sections with an excess of unlabeled probe. The abundance of mRNA in
hippocampus was analyzed and quantified using a computerized image analysis system
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(Scion Image, Scion Corporation, Maryland). Data are expressed as a percentage of the
vehicle group values (100%) and are presented as the mean ± S.E.M.
Adenylate cyclase assay
Hippocampi were homogenized in membrane buffer (MB) (5.33 mM KCl, 0.44 mM KH2PO4,
4.17 mM NaHCO3, 137.9 mM NaCl, 0.34 mM Na2HPO4, 5.56 mM D-glucose, 5 mM
HEPES, pH 7.4 containing 1mg/ml BSA, 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 100
U/ml creatine phosphokinase, 10 mM phosphocreatine), using a motor-driven Teflon and
glass tissue grinder (10 strokes at 800 rotations per minute). The homogenate was centrifuged
at 500 xg for 5 min at 4ºC. The pellet was discarded and the supernatant was centrifuged at
12000 xg for 5 min at 4ºC. The supernatant was discarded and the pellet was resuspended
(1:160 w/v) in stimulation buffer (SB) (MB buffer containing 10 mM ATP magnesium salt).
The membranes were used immediately.
The protein concentration used per assay was around 0.9-1 µg/µl. The incubation reaction
consisted in 5 µl of membrane homogenate and 5 µl of SB alone (basal condition) and
containing different concentrations of the 5-HT4 agonist zacopride 10-6 to 10-3 M (stimulated
condition). The conditions were made in triplicate. Measurement of cAMP amount was
performed using an AlphaScreen cAMP Assay kit (Perkin Elmer), following manufacturer’s
instructions. Membrane protein content was determined with the Bio-Rad Protein Assay Kit
(Bio-Rad, Munich, Germany), using -globulin as standard. Data are expressed as a
percentage of the basal cAMP accumulation (100%) for each experimental group. Data are
presented as the mean ± S.E.M. Statistical comparisons between Emax and EC50 values in
vehicle and RS67333 treated groups for both 3 and 7 days, were made using an unpaired
Student’s t-test with GraphPad Prism 5.01 (GraphPad Software, Inc. San Diego, CA). The
level of significance was chosen at p<0.05.
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Behavioural tests
Novelty suppressed feeding (NSF) test was performed following published protocols
(Bodnoff et al., 1989) with minor modifications. Rats were placed into the testing apparatus,
an open field arena (100x100x50cm; luminance 40-50 lux), from the corner after 24 h food
deprivation. The first latency to eat a single food pellet placed in the center during a 10 min
session was recorded (defined as the rat biting the food). Immediately after this test, the
animal was transferred to its home cage, and the latency to feed as well as food consumption
in each rat was measured within 5 min. Data are expressed as percentage of the vehicle group
values (100%) and are presented as mean ± S.E.M.
Forced-swimming test (FST) was performed as described previously (Porsolt et al., 1977).
Rats were placed individually into a cylinder (25cm diameter, 65cm height) with water at a
depth of 45cm (23-25°C). The total immobility time was measured during a 5 min.
Immobility period was regarded as the time spent by the rat floating in the water without
struggling and making only those movements necessary to keep its head above.
These behavioural tests were performed in rats receiving RS67333 (1.5 mg/kg/day
subcutaneously for 3 or 7 days) or fluoxetine HCl (10 mg/kg/day in drinking water for 7 and
14 days). Data are expressed as percentage of the vehicle group values (100%) and are
presented as mean ± S.E.M.
Sucrose consumption after chronic corticosterone administration: the paradigm of chronic
corticosterone administration as a model of animal depression and described by (Gourley and
Taylor, 2009) was employed. Corticosterone (50 µg/ml; 4-pregnen-1121-DIOL-3 20 DIONE
21-hemisuccinate; Steraloids Inc., Newport, RI) was dissolved in tap water and given in place
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of drinking water for 14 days. After a weaning period (3 days with 25 µg/ml, 3 days with 12.5
µg/ml and 3 days with tap water), rats received RS67333 (1.5 mg/kg/day subcutaneously for 3
or 7 days) or fluoxetine HCl (10 mg/kg/day in drinking water for 21 days). Then, rats were
habituated to sucrose replacing drinking water by a 5% sucrose solution for 2 days. The
following days the animals were submitted to 4, 14 and 19 h of water deprivation. Then,
water and sucrose were provided and individual sucrose consumption during 1-hr period was
measured in the home cage. Data are expressed as mean ± S.E.M. of the volume (ml) of
sucrose consumption.
REFERENCES
Bodnoff SR, Suranyi-Cadotte B, Quirion R, Meaney MJ (1989). A comparison of the
effects of diazepam versus several typical and atypical anti-depressant drugs in an animal
model of anxiety. Psychopharmacology 97, 277-279.
Gourley SL, Taylor JR (2009). Recapitulation and reversal of a persistent depression-like
syndrome in rodents. Current Protocols in Neuroscience Chapter 9:Unit 9.32., 1-14.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the
Folin phenol reagent. Journal of Biological Chemistry 193, 265-275.
Paxinos G, Watson C (1998). The Rat Brain in Stereotaxic Coordinates. 4th edition.
Academic Press Inc., USA.
Porsolt RD, Le Pichon M, Jalfre M (1977). Depression: a new animal model sensitive to
antidepressant treatments. Nature 266, 730-732.
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FIGURE LEGENDS
Figure S1. Behavioural effects of fluoxetine treatment on the forced swimming test-FST (a)
and the novelty suppressed feeding paradigm-NSF (b). Note that 14 days of treatment are
required to obtain a positive effect in these tests. Data are mean ± S.E.M. of 8-9 animals per
group. * p<0.05 vs vehicle group by Student’s t-test.
Figure S2. Effect the administration of 3 (A) and 7 (B) days fluoxetine treatment on BDNF
mRNA expression in the rat hippocampus. BDNF mRNA expression was not modified in the
hippocampus. Data are mean ± S.E.M. of 6-7 animals per group.
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