TITLE PAGE

Title: A role for nuclear β-catenin in SNRI antidepressant-

induced hippocampal cell proliferation.

Authors: Ricardo Mostany1 PhD, Elsa M. Valdizán MD PhD, Angel

Pazos MD PhD.

Authors’ addresses: Departamento de Fisiología y Farmacología,

Instituto de Biomedicina y Biotecnología de Cantabria

(IBBTEC), Universidad de Cantabria-CSIC-IDICAN, 39011

Santander, Cantabria, Spain.

1 Present address: Department of Neurology, University of

California, Los Angeles. Reed Neurological Research Center,

710 Westwood Plz. Los Angeles, CA 90095 USA.

Corresponding author: Dr. Angel Pazos.

Department of Physiology and Pharmacology. Universidad de

Cantabria. Av. Cardenal Herrera Oria s/n, 39011 Santander

(Cantabria), Spain.

Tel: +34 942 201 985; Fax: +34 942 201 903; e-mail:

pazosa@unican.es

Angel Pazos 1

Running title____________________________45 characters

Abstract 250/248 words

Introduction: 750/434

Methods 1500/1459

Text 4197 words

Figures 8

Tables 1

Angel Pazos 2

Abstract sin apartados tienes el anterior después de este

Background: Increasing evidences have been accumulated

suggesting a role for antidepressant drugs (ADs) as

hippocampal neurogenesis enhancers, but the information about

the mechanisms involved in this response is limited. We have

studied in the hippocampus the effects of chronic treatment

with the dual reuptake inhibitor (SNRI) venlafaxine on both

cellular proliferation rate and expression of key effectors of

several signaling pathways.

Methods: Adult Wistar rats were chronically (14 days) treated

either with subcutaneous saline or venlafaxine (10 and 40

mg/kg). Hippocampal cell proliferation was studied by BrdU

immunohistochemistry. -catenin localization was analyzed by

both cellular immunohistochemistry and immunoelectron

microscopy. Protein levels of -catenin, AKT1, ERK1/2,

pERK1/2, CREB and pCREB were determined by Western blot

analysis.

Results: Increased cell proliferation in subgranular zone was

achieved after chronic treatment with venlafaxine (40

mg/kg/day). However, significant increases in the

immunoreactivity of hippocampal β-catenin in SGZ were already

Angel Pazos 3

detected after administration of a lower dose (10 mg/kg/day).

Western blot and immunoelectron microscopy studies

demonstrated an increased presence of β-catenin at the nuclear

level. Cytosolic AKT levels were also increased in

venlafaxine-treated animals. Furthermore, p-ERK2/ERK2 ratio

was increased in the hippocampus of treated animals, while no

differences in the levels of CREB and p-CREB were observed.

Conclusions: Chronic venlafaxine induces a hippocampal

proliferative effect, which involves the activation of

intracellular signaling through Wnt and AKT/PKB pathways,

resulting in an increase of the expression of cell cycle

regulator genes. These results illustrate the complexity of

the intracellular events underlying the neurogenetic responses

of ADs.

Angel Pazos 4

Abstract

Increasing evidences have been accumulated during recent years

suggesting a role for antidepressant drugs (ADs) as

hippocampal neurogenesis enhancers, but the information about

the transductional mechanisms involved in this response is

very limited. We have studied in the adult rat hippocampus the

effects of chronic treatment with the dual reuptake inhibitor

(SNRI) venlafaxine on both cellular proliferation rate and

expression of key effectors of several signaling pathways.

Increased cell proliferation (BrdU incorporation) in

subgranular zone (SGZ) was achieved after chronic treatment

with a high dose (40 mg/kg/day) of venlafaxine. However,

significant increases in the immunoreactivity of hippocampal

β-catenin in SGZ were already detected after administration of

a lower dose of the drug (10 mg/kg/day). Western blot and

immunoelectron microscopy studies demonstrated an increased

presence of β-catenin at the nuclear level. An increase in

cytosolic AKT levels was also observed in venlafaxine-treated

animals. These results suggest that the hippocampal

proliferative effect of chronic venlafaxine, only evident when

both 5-HT and NE reuptake systems are inhibited, requires a

Angel Pazos 5

strong activation of intracellular signaling through Wnt (β-

catenin translocation) and AKT/PKB pathways. This activation

would probably result in an increase of the expression of cell

cycle regulator genes. Furthermore p-ERK2/ERK2 rate was also

increased in the hippocampus of AD-treated animals, while no

differences in the levels of CREB and p-CREB were observed.

These results illustrate the complexity of the intracellular

events underlying the neurogenetic responses of ADs. They also

support the relevance of such effects for the therapeutic

effects of these drugs.

Keywords: venlafaxine, β-catenin, AKT, neurogenesis,

subgranular zone, hippocampus.

Angel Pazos 6

INTRODUCTION

The most common first step in the therapeutic action of

antidepressant drugs (ADs) is the increase in the monoamines

serotonin (5-HT; 5-hydroxytrytamine) and/or norepinephrine

(NE) extracellular levels (Richelson, 1991). This immediate

effect contrasts with the onset of appreciable clinical

improvement, which requires several weeks of AD treatment.

Trying to clarify this apparent mismatch, a large number of

studies about the trophic effects of AD drugs and

electroconvulsive therapy (ECS) have been reported in the

recent years (Altar, et al

2003; Altar, et al 2004; Russo-Neustadt, et al 2004). In this

sense, it is well documented that chronic AD treatment

enhances cell proliferation in adult rodent and nonhuman

primate subgranular zone (SGZ) of hippocampus and that the

time required for the differentiation and maturation of

newborn neurons correlates well with the appearance of

clinical response to the AD treatment (Malberg, et al 2000; D'Sa

and Duman, 2002; Perera, et al 2007). Furthermore, the existence

of a decrease in the hippocampal volume, restored after AD

treatment (Vermetten, et al 2003) has been reported in depressed

Angel Pazos 7

patients by some, but not all authors (Sheline, et al 1996;

Vythilingam, et al 2004). However, the molecular pathways that

control these processes are still unclear. Transduction

pathways promoting cell proliferation are the main targets for

studying the possible mediators for the effect of these drugs

in neurogenesis. Thus, the cAMP cascade has been suggested to

be involved in the neuroproliferative responses induced by ADs

but contradictory results have been reported on this issue

(Nibuya, et al 1996; Manier, et al 2002). In a similar way, the

role of MAPK pathway is under discussion (Tiraboschi, et al 2004;

Fumagalli , et al 2005) and no clear data are available with

respect to AKT/PKB signaling pathway. An emerging candidate to

play a key role in these processes is the Wnt pathway.

Activation of the canonical Wnt pathway leads to the

inhibition of GSK-3, allowing β-catenin to be stabilized in

the cytosol and translocated to the nucleus, where activates

transcription of target genes (Logan and Nusse, 2004). It has

been recently shown that this signaling pathway, a main

regulator of the hippocampal neurogenesis in the adult brain

(Lie, et al 2005), is modulated by chronic ECS treatment (Madsen,

et al 2003).

Angel Pazos 8

The degree of stimulation of cell proliferation varies with

the type of AD drug used (Malberg, et al 2000). The dual reuptake

inhibitor (5-HT and NE, SNRI) venlafaxine blocks both reuptake

processes, with a higher potency for the 5-HT component

(Bëíque, et al 1998) so it is possible to selectively act on the

5-HT transport or to affect both aminergic systems, depending

on the doses assayed for this AD. The goal of the present

study has been to evaluate, in the adult rat hippocampus, the

effects of chronic treatment with venlafaxine at two different

doses on both the hippocampal cellular proliferation and the

expression and cellular distribution of β-catenin, as well as

of other key effectors of several main signaling pathways

(cAMP, MAPK and PKB/AKT).

MATERIALS AND METHODS

Animals

Adult male albino Wistar rats (Harlan, Barcelona, Spain)

weighing 175-200 g were used for the present study. The

animals were housed at 22 ºC with a 12:12 light-dark cycle.

Food and water were provided ad libitum. All experimental

procedures were done according to the Spanish legislation and

Angel Pazos 9

the European Communities Council Directive on “Protection of

Animals Used in Experimental and Other Scientific Purposes”

(86/609/EEC).

Antidepressant treatment and BrdU administration

Rats were implanted subcutaneously with an osmotic minipump

Alzet 2002 (Alza Corp., Palo Alto, CA) which delivered 0.5 µl

per hour during 14 days. Twenty-two animals per group were

treated either with saline, 10 mg/kg/day venlafaxine, or 40

mg/kg/day venlafaxine. Venlafaxine HCl was extracted from

Vandral 75 (Wyeth-Farma, S.A., Madrid, Spain). Minipumps were

removed the fourteenth day of treatment.

For immunohistochemical analysis of cell proliferation,

animals received 5-bromo-2’-deoxyuridine (BrdU; 4 x 75 mg/kg

every 2 hours, i.p.; Sigma, Madrid, Spain) in sterile 0.9%

NaCl solution the last day of antidepressant treatment.

Immunohistochemistry

Twenty-four hours after the last BrdU injection, rats were

deeply anaesthetized with sodium pentobarbital (100 mg/kg,

i.p.) and transcardially perfused with saline followed by 4%

Angel Pazos 10

cold paraformaldehyde in PBS. Brains were removed, postfixed

overnight at 4 ºC and transferred to 30% sucrose in PBS at 4

ºC. Serial coronal sections (45 µm) of the brains were

obtained through the entire hippocampus on a cryostat and

stored at –20 ºC in cryoprotectant solution (25% glycerol, 25%

ethylene glycol in PBS, pH 7.4).

BrdU inmunohistochemistry was carried out following a

previously described protocol (Malberg, et al 2000). Free-

floating sections from animals from the three experimental

groups (n = 9) were incubated for 2 h in 50% formamide/2x SSC

at 65 ºC, 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 to remove endogenous peroxidases. After several rinses in

PBS, sections were incubated in PBS/0.2% Triton X-100/5% goat

serum (PBS-TS) for 30 min and then incubated with monoclonal

mouse anti-BrdU (1:600; Roche Diagnostics, Barcelona, Spain)

overnight at 4 ºC. After several rinses in PBS-TS, sections

were incubated for 2 h with biotinylated horse anti-mouse IgG

secondary antibody (4 µg/ml; Vector Laboratories, Burlingame,

CA), followed by amplification with avidin-biotin complex

Angel Pazos 11

(Vector Laboratories). BrdU positive (BrdU+) cells were labeled

using DAB as chromogen (Vector Laboratories).

For quantification of BrdU+ cells, every sixth section

throughout the hippocampus was processed and mounted on coded

slides. BrdU+ cells in the SGZ of the dentate gyrus were

counted by an experimenter blinded to the study code under a

light microscope (Carl Zeiss Axioskop 2 Plus) at 400x

magnification. The total number of BrdU+ cells per section was

determined and multiplied by 6 to obtain the total number of

BrdU+ cells per hippocampus. Data were analyzed using ANOVA and

Student-Newman-Keuls post hoc test using GraphPad Prism

(GraphPad Software, Inc. San Diego, CA). Statistical

significance was set at p < 0.05.

β-catenin immunohistochemistry was carried out in adjacent

sections of those used in the BrdU labeling studies (n = 9).

After several rinses in PBS, sections were boiled in

microwaves in 10 mM citric acid, pH 6.0 for 10 min. Following

several rinses in PBS, sections were incubated in methanol and

3% H2O2. Then sections were transferred to blocking buffer

(PBS-TS) for 30 min and incubated for 2 d at 4 ºC with mouse

anti-β-catenin (1:500; BD Biosciences, Madrid, Spain). After

Angel Pazos 12

washes in PBS-TS sections were incubated with biotinylated

horse anti-mouse IgG secondary antibody followed by peroxidase

detection with DAB or FITC conjugated donkey anti-mouse IgG

secondary antibody (1:250; Jackson ImmunoResearch

Laboratories, Inc., West Grove, PA), followed by propidiun

iodide (Sigma) counterstain.

The immunostaining of β-catenin after peroxidase method was

evaluated by an observer blinded to the study code in at least

four sections from each animal corresponding to interaural

stereotaxic coordinates ranging 4.48-5.70 mm (Paxinos and

Watson, 1982). For this purpose, the section with the highest

number of positive clusters in the SGZ was arbitrarily

considered as containing the maximal level of expression. The

remaining sections were ranged according to the following: +,

number of positive clusters < 25% of that observed in the

section with the maximal number; ++, ranging 25-50%; +++,

ranging 50-75%; ++++, > 75% with respect the section with the

highest density.

For BrdU/β-catenin double labeling, frozen sections, 14 µm

thick, were mounted on gelatine-coated slides and fixed in

absolute ethanol for 20 min at –20 ºC (n = 5). Then, sections

Angel Pazos 13

were boiled in microwaves in citric acid followed by several

PBS rinses. Sections were blocked with PBS-TS for 30 min and

then incubated with monoclonal rat anti-BrdU (1:400; Serotec

Ltd., Oxford, UK) overnight at 4 ºC. Sections were incubated

with Texas Red conjugated donkey anti-rat (1:250; Jackson

ImmunoResearch Laboratories, Inc.) secondary antibody for 2 h.

After exhaustive washes in PBS-TS, sections were incubated

overnight with monoclonal mouse anti-β-catenin (1:500) primary

antibody at 4 ºC and then with FITC conjugated donkey anti-

mouse secondary antibody (1:250) for 2 h, followed by DAPI

(Roche Diagnostics) counterstain.

Subcellular fractionation and Western blot

Animals from each experimental group (n= 5) were killed by

decapitation, their brains being removed from skulls and

hippocampi dissected and rapidly frozen at -80 ºC. Every

sample was thawed and homogenized (1:15, 500-600 µl aprox.) by

using a Potter homogenizer provided with a loosely fitting

Teflon pestle in homogenization buffer (10 mM Hepes-HCl, pH

7.9, 1.5 mM MgCl2, 10 mM KCl) containing the following protease

and phosphatase inhibitors: 1 mM PMSF; 10 µl/ml aprotinin; 10

Angel Pazos 14

µg/ml leupetin; 10 µg/ml pepstatin A; 10 µg/ml antipain; 10

µg/ml chymostatin; 5 µg/ml trypsin inhibitor; 1 mM NaV; 1 mM

NaF; 1 mM cantharidin; and 10 µM E-64. After homogenization,

250 µl of homogenate were lysated in lysis buffer

(homogenization buffer containing 1% Igepal; 0.1% sodium

deoxicholate, 0.2% SDS, and 0.1% Triton X-100) 30 min on ice

for the total cell lysate (TCL). The remaining homogenate

(250-300 µl) was processed for subcellular fractionation.

Homogenate was centrifuged at 1000 g for 10 min at 4 ºC, and

the resulting supernatant (S1) and pellet (P1) separated. The

S1 was ultra-centrifuged at 100 000 g for 15 min at 4 ºC,

resulting in a supernatant (S2) cytosolic fraction, that was

lysated and a pellet P2 membrane fraction, resuspended in

buffer containing detergents and protease and phosphatase

inhibitors and centrifuged 10 min at 14 000 g. Nuclear

proteins were isolated by high salt extraction from P1

fraction. P1 fraction was homogenized in 20 mM Hepes pH 7.9,

0.45 M NaCl, 1 mM EDTA containing protease and phosphatase

inhibitors and incubated in ice for 30 min. Solubilized

proteins were recovered in the supernatant after

centrifugation at 14 000 g for 10 min at 4 ºC. Protein

Angel Pazos 15

quantification was performed according to the Lowry method

(Lowry, et al 1951).

Thirty-fifty µg of protein were resolved on 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--catenin (1:1000), mouse anti-GAPDH (1:2000), mouse

anti-histone H1 (1:200), mouse anti-AKT1 (1:1000) and rabbit

anti-ERK1/2 (1:2000), from Santa Cruz Biotechnology, Inc.

Heidelberg, Germany; mouse anti-pERK1/2 (1:10 000) and rabbit

anti-actin (1:2000) from Sigma; rabbit anti-CREB (1:1000) and

rabbit anti-pCREB (1:1000), from Upstate, Charlottesville, VA.

After extensive washings in TBS-T (TBS/0.05% Tween 20)

membranes were incubated with horseradish peroxidase

conjugated secondary antibodies. Secondary antibodies were

detected with ECL Advance kit (GE Healthcare Europe GmbH,

Munich, Germany). Anti-GAPDH (cytosolic marker) and anti-

histone H1 (nuclear marker) antibodies were used to discard

subcellular fraction contaminations. Blot quantitations were

performed by densitometric scanning using Scion Image

Software. The densitometry values were normalized with respect

Angel Pazos 16

to the values obtained with anti-actin antibody. Data for

every sample was the mean of at least two independent

experiments. Statistical analysis was performed using ANOVA

following Student-Newman-Keuls post hoc test.

Immunogold electron microscopy

Vibratome brain sections containing dentate gyrus (400 µm)

were obtained from transcardially perfused (4%

paraformaldehyde) animals from the three experimental groups

(n=3). The tissue samples were then washed with 0.1 M

cacodylate buffer, dehydrated in increasing concentrations of

methanol at –20 ºC, embedded in Lowicryl K4M at -20 ºC and

polymerized under ultraviolet irradiation. Ultrathin sections

were mounted on nickel grids and sequentially incubated with

0.1 M glycine in PBS for 15 min, 5% BSA in PBS for 30 minutes

and mouse anti--catenin (1:50) antibody (diluted in 50 mM

Tris HCl, pH 7.6, containing 1% BSA and 0.1 M glycine) for 1h

at 37 ºC. After washing, the sections were incubated with goat

anti-mouse antibody coupled to 15 nm gold particles (British

BioCell International, Cardiff, UK; 1:50 in PBS containing 1%

BSA). After immunogold labeling, the grids were stained with

Angel Pazos 17

lead citrate and uranyl acetate and examined with a Philips

EM208 electron microscope operated at 60kV.

RESULTS

Effect of venlafaxine on cell proliferation

The analysis of the number of BrdU+ cells showed that chronic

(14 d) venlafaxine treatment produces a significant increase

in cell proliferation in SGZ of dentate gyrus, only when a

high dose (40 mg/kg/day) of this AD was administered (4888 ±

266; 44.8 ± 8.4% increase vs saline group, 3374 ± 355; p <

0.01; Figure 1). No statistically significant changes in cell

proliferation rate were observed in the animals receiving 10

mg/kg/day of venlafaxine (3621 ± 335; 7.3 ± 10.6% increase vs

saline group). Furthermore, significant differences in the

cell proliferation rates were observed when comparing 10 and

40 mg/kg/day venlafaxine dose groups (34.9 ± 7.9%; p < 0.05).

Modifications in the expression patterns of main efector

proteins of signal transduction pathways after venlafaxine

treatment

Angel Pazos 18

Measurement of β-catenin expression in hippocampal total

homogenates revealed significant higher levels of this protein

after chronic treatment with either doses of venlafaxine

compared with saline treated rats (73 ± 13%, p < 0.05 and 175

± 36%, p < 0.001 increases for 10 and 40 mg/kg/day doses

respectively; Figure 2a and 2b). After cellular fractionation,

β-catenin was detected in the membrane-associated and nuclear

fractions. In both of them, significant increases in the

levels of this protein were found after chronic treatment at

either doses of venlafaxine when compared with saline rats,

these increases being more pronounced in the nuclear fraction.

Chronic treatment with 40 mg/kg/day resulted in amounts of

this protein higher than those observed for 10 mg/kg/day in

the two cellular fractions studied (62 ± 25%, p < 0.05 in

membrane-associated fraction, 88 ± 9%, p < 0.001 in nuclear

fraction).

Detectable AKT protein levels were obtained in the cytosolic

and nuclear fractions, as well as in the TCL (Figure 2b and

2c). A dose-dependent increase in the levels of expression of

this protein was observed in the total homogenate (28 ± 11%, p

= n.s.; 84 ± 31%, p < 0.05, for 10 and 40 mg/kg/day doses

Angel Pazos 19

respectively). In the cytosolic subfraction, increased levels

of AKT were found in tissues from animals chronically treated

at either venlafaxine doses (47 ± 13%, p < 0.05 and 69 ± 23%,

p < 0.05). In contrast, a significant dose-dependent decrease

in the amount of AKT was detected in the nuclear fraction (-22

± 6%, p < 0.05 and -38 ± 8%, p < 0.01).

No significant changes in the levels of expression of both

CREB and p-CREB were observed in the nuclear fractions of

animals chronically treated with either dose venlafaxine when

compared with saline group (Figure 3). Phosphorylated

CREB/CREB ratio was not affected either.

Immunoblot analysis of the expression of the two isoforms of

ERK protein (ERK1, ERK2) in total homogenates (Figure 4)

showed no change after chronic treatment with 10 or 40

mg/kg/day venlafaxine with respect to saline administered

rats. On the other hand, increased levels of the

phosphorylated form (activated form) of both isoforms of this

protein were observed in TCL after the AD treatment (96 ± 8%,

p < 0.05 and 141 ± 49%, p < 0.01 for p-ERK1 for 10 and 40

mg/kg/day, respectively. 80 ± 12%, p < 0.05 and 101 ± 38%, p <

0.05 for p-ERK2 for 10 and 40 mg/kg/day, respectively). In

Angel Pazos 20

this sense, p-ERK1/ERK1 and p-ERK2/ERK2 ratios showed at least

a 1.5 fold change after venlafaxine chronic treatment.

Increased immunoreactivity of β-catenin in the SGZ after

chronic treatment with venlafaxine

β-Catenin staining throughout the hippocampus presented a

distribution pattern characterized by a weak immunostaining

over the GCL and a positive immunostained clusters detected in

the SGZ of the dentate gyrus. Saline group presented levels of

immunostaining under 25% with respect to the highest

immunoreactivity found. Animals treated with 10 mg/kg/day

venlafaxine showed a mean number of immunopositive clusters

per section ranging 50-75% with respect the highest

immunoreactivity found. Mean number of immunopositive clusters

for β-catenin in the SGZ of animals treated with 40 mg/kg/day

venlafaxine was over 75% with respect the highest

immunoreactivity found (Table 1 and Figure 5).

The clusters of β-catenin immunolabeling observed in the SGZ

were studied in order to detect nuclear β-catenin presence. No

presence of this protein could be detected in nucleus from SGZ

cells when they were observed under confocal microscopy

Angel Pazos 21

(Figure 6) in any sample from animals from the three

experimental groups. GCL cells nuclei neither were β-catenin

positive when examined.

No nuclear BrdU+/β-catenin+ colocalization could be observed in

any sample analyzed, but all BrdU+ nuclei observed showed high

β-catenin labeling surrounding them and were embedded in β-

catenin clusters (Figure 6).

Detection of nuclear β-catenin in SGZ cells by immunoelectron

microscopy

β-catenin presence in nuclei from SGZ cells could be observed

by immunoelectron microscopy. β-catenin presence and

distribution appeared in DNA euchromatinic regions, with no

presence in nucleoli or heterochromatin, compatible with a

transcription factor distribution pattern. The low density of

gold particles observed in nucleus from SGZ and CGL cells from

animals of saline group strongly contrasts with those observed

after chronic treatment with venlafaxine (Figure 7) showing an

increased number of gold particles together with an increase

in the number of particles per cluster.

Angel Pazos 22

DISCUSSION

The present study demonstrates for the first time that chronic

treatment with the SNRI venlafaxine increases both the

hippocampal cell proliferation and the translocation rate to

the nucleus of β-catenin, a main effector of the Wnt pathway.

The positive effects reported in the last years for AD drugs

and ECS on adult hippocampal cell proliferation (Malberg, et al

2000; Madsen, et al 2000; Czéh, et al 2001; Sairanen, et al 2005;

Dranovsky and Hen, 2006) support a new hypothesis about the

mechanisms through AD exert their therapeutic actions.

Fluoxetine has been the AD drug more widely used in these

studies (Malberg, et al 2000; Sairanen, et al 2005; Malberg and

Duman, 2003; Santarelli, et al 2003). However, it is being

suggested that the use of dual reuptake inhibitors, such as

venlafaxine, could be of special interest in the treatment of

some major depression cases, under certain clinical conditions

(Tzanakaki, et al 2000; Stahl, et al 2002; Poirier and Boyer, 1999;

Mehtonen, et al 2000). Taking into account that venlafaxine shows

a relative preferential pattern of 5-HT transport inhibition

Angel Pazos 23

(Bëíque, et al 1998), we studied the state of cell proliferation

rate after 14 days of treatment, using two doses: 10

mg/kg/day, which mainly acts on 5-HT transporter, and 40

mg/kg/day, a dose that inhibits the reuptake process through

both 5-HT and NE transporters (Bëíque, et al 1998). A

significant increase in cell proliferation in SGZ was observed

in the animals administered with 40 mg/kg/day, while no effect

was found following the administration of 10 mg/kg/day. These

results are in contrast with previous data (Khawaja, et al 2004),

which showed augmentation of cell proliferation in rats

treated for 14 days with 10 mg/kg/day venlafaxine. Two

methodological differences in the experimental design could

explain this discrepancy. First, in Khawaja et al.’s study

(Khawaja, et al 2004) venlafaxine was administered i.p. while we

used s.c. route (osmotic minipumps). Second, in the mentioned

study BrdU was injected along four consecutive days following

the last administration of venlafaxine, while in the present

work the cell proliferation analysis was carried out 24 h

after removing the osmotic minipump. These results suggest

that, depending on several factors (type of drug, route of

administration, duration) the activation of the serotonergic

Angel Pazos 24

system might be not enough to elicit by itself a proliferative

response, thus requiring the concomitant inhibition of NE

transport. Furthermore, they are in good agreement with the

reported ability of venlafaxine to prevent the decrease in

hippocampal cell proliferation induced by chronic stress (Xu,

et al 2006).

These results strongly support that AD drugs are potential

enhancers of hippocampal cell proliferation and neurogenesis.

In order to address the signaling pathways involved, we

studied the effects of venlafaxine on the amount and cellular

distribution of main effector proteins of several signaling

pathways involved in cell proliferation. β-Catenin expression

was increased in both TCL and purified nuclear fraction in

venlafaxine-treated animals in a dose-dependent manner,

suggesting an increase in translocation of β-catenin to cell

nucleus. This finding correlates well with that reported in a

previous study using ECS therapy (Madsen, et al 2003). The

increase in β-catenin immunoreactivity observed in the

proliferating area SGZ after chronic venlafaxine treatment,

together with the fact that BrdU+ cells were embedded in

immunopositive β-catenin clusters further supports the

Angel Pazos 25

relevance of this protein in hippocampal cell proliferation.

Interestingly, GSK-3 inhibitors, which allow stabilization of

-catenin, have been reported to exert antidepressant-like

effects. (Kaidanovich-Beilin, et al 2004; Gould, et al 2006). The

role of β-catenin in affecting gene expression would be

mediated by the interaction with TCF/LEF DNA-binding proteins

(Logan and Nusse, 2004; van de Wetering, et al 1997). Target

genes would include, in addition to cell cycle regulator

genes, as myc and cyclinD1 (He, et al 1998; Shtutman, et al

1999), Wnt signaling genes, thus leading to a fine feedback

regulation of this pathway (Logan and Nusse, 2004). Taking into

account that the adult hippocampal stem/progenitor cells

(AHPs) express receptors for Wnt proteins (Madsen, et al 2003),

it could be suggested that chronic venlafaxine would result in

a double -catenin-dependent activation of AHPs: a direct

activation of neural precursors in SGZ to re-enter in cell

cycle, and an indirect regulation of surrounding

differentiated cells, such as astrocytes, in order to secret

Wnt proteins, that would act on hippocampal progenitor cells,

leading to a further increase in neurogenesis (Lie, et al 2005).

Activated AHP cells are able to divide through mechanisms that

Angel Pazos 26

involve the expression of CyclinD genes, producing daughter

cells that can differentiate to neurons (neurogenesis) or

astrocytes (gliogenesis) (Fig. 8). In this regard, it has been

recently shown that chronic treatment with antidepressants

induces an increase of symmetric divisions of an early

progenitor cell class in the dentate gyrus (Encinas, et al 2006).

However, while the increase in nuclear β-catenin was observed

in animals receiving either doses of venlafaxine (10 and 40

mg/kg/day), we could not detect any clear increase in cell

proliferation after treatment with the lower dose of the drug.

This suggests that a high level of induction of the Wnt

pathway is required to affect cell proliferation.

Alternatively, these results could indicate the involvement of

other signaling pathways in this response. No information

about hippocampal β-catenin expression in tissues from

depressed patients is available, although no changes have been

found in cortical samples (Beasley, et al 2002).

We tried to detect the presence of nuclear β-catenin in SGZ

and GCL cells, as well as in BrdU+ nuclei from SGZ, by

immunofluorescence microscopy techniques. A clear perinuclear

β-catenin staining was detected surrounding BrdU+ nuclei, but

Angel Pazos 27

no nuclear staining was demonstrated as previously reported in

the literature (Madsen, et al 2003; Lucas, et al 1999; Lucas, et al

2001) where the use of immunoelectron microscopy has been

suggested to overcome this limitation (Lucas, et al 2001): our

immunoelectron microscopy studies confirmed the presence of β-

catenin in the nucleus of SGZ cells in all experimental

groups. The pattern of distribution found, in euchromatin and

no nucleolar presence, confirms the role of this protein in

the regulation of target genes expression. In venlafaxine

treated animals, nuclear β-catenin density was higher than in

the saline group, and gold particles usually appeared forming

patches of several units. This is, to our knowledge, the first

report of an increased level of nuclear β-catenin following AD

treatment.

Taking into account that β-catenin is also a component of the

cadherin/catenin complex, stabilizing the actin cytoskeleton

and mediating cell adhesion (Cadigan and Nusse, 1997), the

increased levels of β-catenin detected after venlafaxine

chronic treatment in the membrane-associated fraction could

suggest a positive effect in stabilizing synapses and

increasing dendritic arborization. This agrees with recent

Angel Pazos 28

reports demonstrating, first, that the intracellular level of

cadherin/catenin complex is a limiting factor in dendritic

morphogenesis (Yu and Malenka, 2003) and, second, that AD

treatment increases the formation of dendritic spines in rat

hippocampus (Hajszan, et al 2005 ).

The AKT/PKB signaling is strongly involved in the regulation

of several cell responses, such as cell proliferation, growth

and cell survival (Brazil and Hemmings, 2001). Interestingly,

it has been recently reported the existence of a significantly

lower activity of this protein in occipital cortex of

depressed suicide subjects (Hsiung, et al 2003). Our results

regarding AKT levels after venlafaxine treatment (increase in

cytosolic and decrease in nuclear fractions) suggest that this

protein is being accumulated in the cytosol, in order to be

subsequently activated in the cell membrane. Since GSK-3 is

directly inhibited by AKT (Cross, et al 1995), this recruitment

of cytosolic AKT would lead to a strong inhibition of this

kinase, resulting in further stabilization of β-catenin.

Our results show that CREB levels were not affected by chronic

venlafaxine treatment. A tendency to the decrease in cortical

CREB levels from depressed patients has been reported

Angel Pazos 29

(Dowlatshahi, et al 1998; Yamada, et al 2003), although no study

has been performed in hippocampus. In line with that, several

reports indicate that AD treatment could increase CREB

function, although the level of response appears to depend on

several factors (Tiraboschi, et al 2004; Frechilla, et al 1998;

Thome, et al 2000). Fluoxetine has been reported to be more

effective in increasing the expression and phosphorylation of

hippocampal CREB, when compared to ADs affecting NE uptake

(reboxetine, desipramine) (Tiraboschi, et al 2004; Frechilla, et

al 1998). Furthermore, a reduction in the ability to

phosphorylate CREB in human neuroblasts has been described

following reboxetine and desipramine administration, but not

after venlafaxine treatment (Manier, et al 2002). These results

illustrate the variability of the modifications induced by ADs

on CREB expression, which strongly depend on the particular

drug used.

Few data are available about the effect of ADs on the

components of MAPK pathway (Tiraboschi, et al 2004; Fumagalli ,

et al 2005). Our study agrees with most previously published

results, showing no consistent change in the levels of ERK1/2

in hippocampus after venlafaxine treatment, supporting that

Angel Pazos 30

their expression is not affected by ADs. Regarding pERK1 and

pERK2, data in the literature are contradictory: while no

changes have been reported after treatment with imipramine,

decreases (nuclear pERK1/2), increases (nuclear pERK1) and

lack of changes have been observed as a consequence of

fluoxetine treatment (Tiraboschi, et al 2004; Fumagalli , et al

2005). Although caution is needed when analyzing these results,

our data support the activation of the MAPK pathway following

chronic administration with venlafaxine, and are in agreement

with results from postmortem studies showing a decrease in

brain pERK1/2 levels in depressed patients (Hsiung, et al 2003).

In conclusion, our results indicate that chronic treatment

with venlafaxine induces nuclear translocation of β-catenin,

probably trough activation of Wnt and AKT pathways. This

effect could be mediating the increased hippocampal cell

proliferation observed, thus reinforcing the role of

neurotrophic mechanisms in the therapeutic activity of ADs.

Angel Pazos 31

Disclosure/conflicts of interes

Dr. Pazos reports having received research funding and lecture

fees from Faes Farma S.A.. Drs. Mostany and Valdizán report no

biomedical financial interests or conflicts of interest.

Conferencias??

Angel Pazos 32

Acknowledgements:

We are grateful to FAES FARMA, S.A. (Mr. Antonio Toledo) for

the extraction of venlafaxine. We wish to thank Ms. María

Josefa Castillo, Ms. Paula Díez del Valle, Ms. Rebeca

Madureira, Ms Lourdes Lanza, Mr. Germán Pérez and Ms. Isabel

Ruiz for their technical assistance. We are deeply in debt

with Prof. Miguel Lafarga and Prof. María Teresa Berciano, of

the Departmento de Anatomía y Biología Celular of Universidad

de Cantabria, by their invaluable help in scientific and

technical aspects of confocal and electron microscopy. R.M.

received a postdoctoral fellowship from Fundación Pública

“Marqués de Valdecilla”-IFIMAV. This work has been supported

by CICYT (SAF04-00941), IFIMAV (API 05/23), Alicia Koplowitz

Foundation, the Spanish Ministry of Health, Instituto de Salud

Carlos III, RETICS RD06/0011(REM-TAP Network and FAES FARMA,

S.A.-Universidad de Cantabria contract.

Angel Pazos 33

References

Altar CA, Laeng P, Jurata LW, Brockman JA, Lemire A, Bullard J

et al. (2004). Electroconvulsive seizures regulate gene

expression of distinct neurotrophic signaling pathways. J

Neurosci 24: 2667-2677.

Altar CA, Whitehead RE, Chen R, Wortwein G, Madsen TM, (2003).

Effects of electroconvulsive seizures and antidepressant drugs

on brain-derived neurotrophic factor protein in rat brain. Biol

Psychiatry 54: 703-709.

Beasley C, Cotter D, Everall I, (2002). An investigation of

the Wnt-signalling pathway in the prefrontal cortex in

schizophrenia, bipolar disorder and major depressive disorder.

Schizophr Res 58: 63-67.

Bëíque JC, de Montigny C, Blier P, Debonnel G, (1998).

Blockade of 5-hydroxytryptamine and noradrenaline uptake by

venlafaxine: a comparative study with paroxetine and

desipramine. Br J Pharmacol 125: 526-532.

Brazil DP, Hemmings BA, (2001). Ten years of protein kinase B

signalling: a hard Akt to follow. Trends Biochem Sci 26: 657-664.

Angel Pazos 34

Cadigan KM, Nusse R, (1997). Wnt signaling: a common theme in

animal development. Genes Dev 11: 3286-3305.

Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA,

(1995). Inhibition of glycogen synthase kinase-3 by insulin

mediated by protein kinase B. Nature 378: 785-789.

Czéh B, Michaelis T, Watanabe T, Frahm J, de Biurrun G, van

Kampen M et al. (2001) : Stress-induced changes in cerebral

metabolites, hippocampal volume, and cell proliferation are

prevented by antidepressant treatment with tianeptine. Proc Natl

Acad Sci USA 98: 12796-12801.

Dowlatshahi D, MacQueen GM, Wang JF, Young LT, (1998).

Increased temporal cortex CREB concentrations and

antidepressant treatment in major depression. Lancet 352: 1754-

1755.

Dranovsky A, Hen R, (2006). Hippocampal neurogenesis:

regul.ation by stress and antidepressants. Biol.Psychiatry 59:

1136-1143.

D'Sa C, Duman RS, (2002). Antidepressants and

neuroplasticity. Bipolar Disord 4: 183-194.

Angel Pazos 35

Encinas JM, Vaahtokari A, Enikopolov G, (2006). Fluoxetine

targets early progenitor cells in the adult brain. Proc Natl Acad

Sci USA 103: 8233-8238.

Frechilla D, Otano A, Del Rio J (1998). Effect of chronic

antidepressant treatment on transcription factor binding

activity in rat hippocampus and frontal cortex. Prog

Neuropsychopharmacol Biol Psychiatry 22: 787-802.

Fumagalli F, Molteni R, Calabrese F, Frasca A, Racagni G, Riva

MA, (2005). Chronic fluoxetine administration inhibits

extracellular signal-regulated kinase 1/2 phosphorylation in

rat brain. J Neurochem 93: 1551-1560.

Gould TD, Picchini AM, Einat H, Manji HK, (2006). Targeting

glycogen synthase kinase-3 in the CNS: implications for the

development of new treatments for mood disorders. Curr Drug

Targets 7: 1399-1409.

Hajszan T, MacLusky NJ, Leranth C, (2005). Short-term

treatment with the antidepressant fluoxetine triggers

pyramidal dendritic spine synapse formation in rat

hippocampus. Eur J Neurosci 21: 1299-1303.

Angel Pazos 36

He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT et

al. (1998). Identification of c-MYC as a target of the APC

pathway. Science 281: 1509-1512.

Hsiung SC, Adlersberg M, Arango V, Mann JJ, Tamir H, Liu KP,

(2003). Attenuated 5-HT1A receptor signaling in brains of

suicide victims: involvement of adenylyl cyclase,

phosphatidylinositol 3-kinase, Akt and mitogen-activated

protein kinase. J Neurochem 87: 182-194.

Kaidanovich-Beilin O, Milman A, Weizman A, Pick CG, Eldar-

Finkelman H, (2004). Rapid antidepressive-like activity of

specific glycogen synthase kinase-3 inhibitor and its effect

on β-catenin in mouse hippocampus. Biol Psychiatry 55: 781-784.

Khawaja X, Xu J, Liang JJ, Barrett JE, (2004). Proteomic

analysis of protein changes developing in rat hippocampus

after chronic antidepressant treatment: Implications for

depressive disorders and future therapies. J Neurosci Res 75: 451-

460.

Lie DC, Colamarino SA, Song HJ, Desire L, Mira H, Consiglio A

et al.(2005). Wnt signalling regulates adult hippocampal

neurogenesis. Nature 437: 1370-1375.

Angel Pazos 37

Logan CY, Nusse R, (2004). The Wnt signaling pathway in

development and disease. Annu Rev Cell Dev Biol 20: 781-810.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, (1951). Protein

measurement with the Folin phenol reagent. J Biol Chem 193: 265-

275.

Lucas JJ, Hernandez F, Avila J, (1999). Nuclear localization

of β-catenin in adult mouse thalamus correlates with low

levels of GSK-3β. Neuroreport 10: 2699-2703.

Lucas JJ, Hernandez F, Gomez-Ramos P, Moran MA, Hen R, Avila

J, (2001). Decreased nuclear β-catenin, tau

hyperphosphorylation and neurodegeneration in GSK-3β

conditional transgenic mice. EMBO J 20: 27-39.

Madsen TM, Newton SS, Eaton ME, Russell DS, Duman RS, (2003).

Chronic electroconvulsive seizure up-regulates β-catenin

expression in rat hippocampus: role in adult neurogenesis. Biol

Psychiatry 54: 1006-1014.

Madsen TM, Treschow A, Bengzon J, Bolwig TG, Lindvall O,

Tingstrom A, (2000). Increased neurogenesis in a model of

electroconvulsive therapy. Biol Psychiatry 47: 1043-1049.

Angel Pazos 38

Malberg JE, Duman RS, (2003). Cell proliferation in adult

hippocampus is decreased by inescapable stress: reversal by

fluoxetine treatment. Neuropsychopharmacology 28: 1562-1571.

Malberg JE, Eisch AJ, Nestler EJ, Duman RS, (2000). Chronic

antidepressant treatment increases neurogenesis in adult rat

hippocampus. J Neurosci 20: 9104-9110.

Manier DH, Shelton RC, Sulser F, (2002). Noradrenergic

antidepressants: does chronic treatment increase or decrease

nuclear CREB-P? J Neural Transm 109: 91-99.

Mehtonen OP, Sogaard J, Roponen P, Behnke K, (2000).

Randomized, double-blind comparison of venlafaxine and

sertraline in outpatients with major depressive disorder.

Venlafaxine 631 Study Group. J Clin Psychiatry 61: 95-100.

Nibuya M, Nestler EJ, Duman RS, (1996). Chronic

antidepressant administration increases the expression of cAMP

response element binding protein (CREB) in rat hippocampus. J

Neurosci 16: 2365-2372.

Paxinos G, Watson C (1982): The Rat Brain in Stereotaxic

Coordinates. Academic Press: Sydney.

Angel Pazos 39

Perera TD, Coplan JD, Lisanby SH, Lipira CM, Arif M, Carpio C

et al. (2007). Antidepressant-indiced neurogenesis in the

hippocampus of adult nonhuman primates. J. Neurosci. 27: 4894-4901.

Poirier MF, Boyer P, (1999). Venlafaxine and paroxetine in

treatment-resistant depression. Double-blind, randomised

comparison. Br J Psychiatry 175: 12-16.

Richelson E, (1991). Biological basis of depression and

therapeutic relevance. J Clin Psychiatry 52 Suppl: 4-10.

Russo-Neustadt AA, Alejandre H, Garcia C, Ivy AS, Chen MJ,

(2004). Hippocampal brain-derived neurotrophic factor

expression following treatment with reboxetine, citalopram,

and physical exercise. Neuropsychopharmacology 29: 2189-2199.

Sairanen M, Lucas G, Ernfors P, Castren M, Castren E, (2005).

Brain-derived neurotrophic factor and antidepressant drugs

have different but coordinated effects on neuronal turnover,

proliferation, and survival in the adult dentate gyrus. J

Neurosci 25: 1089-1094.

Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S

et al. (2003). Requirement of hippocampal neurogenesis for the

behavioral effects of antidepressants. Science 301: 805-809.

Angel Pazos 40

Sheline YI, Wang PW, Gado MH, Csernansky JG, Vannier MW,

(1996). Hippocampal atrophy in recurrent major depression.

Proc Natl Acad Sci USA 93: 3908-3913.

Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M,

Pestell R et al. (1999). The cyclin D1 gene is a target of the

β-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 96: 5522-5527.

Stahl SM, Entsuah R, Rudolph RL, (2002). Comparative efficacy

between venlafaxine and SSRIs: a pooled analysis of patients

with depression. Biol Psychiatry 52: 1166-1174.

Thome J, Sakai N, Shin K, Steffen C, Zhang YJ, Impey S et al.

(2000). cAMP response element-mediated gene transcription is

upregulated by chronic antidepressant treatment. J Neurosci 20:

4030-4036.

Tiraboschi E, Tardito D, Kasahara J, Moraschi S, Pruneri P,

Gennarelli M et al. (2004). Selective phosphorylation of nuclear

CREB by fluoxetine is linked to activation of CaM kinase IV

and MAP kinase cascades. Neuropsychopharmacology 29: 1831-1840.

Tzanakaki M, Guazzelli M, Nimatoudis I, Zissis NP, Smeraldi E,

Rizzo F, (2000). Increased remission rates with venlafaxine

Angel Pazos 41

compared with fluoxetine in hospitalized patients with major

depression and melancholia. Int Clin Psychopharmacol 15: 29-34.

van de Wetering M, Cavallo R, Dooijes D, van Beest M, van Es

J, Loureiro J et al.(1997). Armadillo coactivates transcription

driven by the product of the Drosophila segment polarity gene

dTCF. Cell 88: 789-799.

Vermetten E, Vythilingam M, Southwick SM, Charney DS, Bremner

JD, (2003). Long-term treatment with paroxetine increases

verbal declarative memory and hippocampal volume in

posttraumatic stress disorder. Biol Psychiatry 54: 693-702.

Vythilingam M, Vermetten E, Anderson GM, Luckenbaugh D,

Anderson ER, Snow J et al. (2004). Hippocampal volume, memory,

and cortisol status in major depressive disorder: effects of

treatment. Biol Psychiatry 56: 101-112.

Xu H, Chen Z, He J, Haimanot S, Li X, Dyck L, Li XM, (2006).

Synergetic effects of quetiapine and venlafaxine in preventing

the chronic restraint stress-induced decrease in cell

proliferation and BDNF expression in frat hippocampus.

Hippocampus 16: 551-559.

Angel Pazos 42

Yamada S, Yamamoto M, Ozawa H, Riederer P, Saito T, (2003).

Reduced phosphorylation of cyclic AMP-responsive element

binding protein in the postmortem orbitofrontal cortex of

patients with major depressive disorder. J Neural Transm 110:

671-680.

Yu X, Malenka RC, (2003). β-Catenin is critical for dendritic

morphogenesis. Nat Neurosci 6: 1169-1177.

Angel Pazos 43

Tables

Table 1. Relative -catenin immunoreactivity in SGZ of saline

and venlafaxine treated animals.

+ ++ +++ ++++

Saline 7 2 - -

Venlafaxine, 10

mg/kg/day

1 2 5 1

Venlafaxine, 40

mg/kg/day

- 1 2 6

-Catenin immunoreactivity is increased in the SGZ of

venlafaxine treated animals. Number of animals which -catenin

immunoreactivity was ranged as follows: +, number of positive

-catenin immunostained clusters in the SGZ < 25% of that

observed in the section with the maximal number of

immunopositive clusters; ++, ranging 25-50%; +++, ranging 50-

75%; ++++, > 75% with respect the section with the highest

density of positive immunostained clusters.

Angel Pazos 44

Titles and legends to figure

Figure 1. Chronic venlafaxine treatment increases the number

of BrdU+ cells in the SGZ of adult rat hippocampus when

administered at a high dose. Representative microphotographs

of BrdU immunohistochemistry from saline (a), 10 mg/kg/day

venlafaxine (b), and 40 mg/kg/day venlafaxine (c) chronically

treated animals. Cresyl violet counter-staining. (d) The

number of BrdU+ cells in the subgranular zone (SGZ) is

significantly increased after chronic treatment with 40

mg/kg/day venlafaxine. The results are the mean ± SEM number

of BrdU+ cells in SGZ. **p < 0.01; *p < 0.05. GCL, granule cell

layer; H, Hilus. Bar = 100 µm

Figure 2. Chronic venlafaxine treatment affects expression of

main effector proteins of Wnt and AKT/PKB signaling pathways.

Graphs represent densitometry levels of β-catenin and AKT

proteins in AD treated animals as a percentage of these

proteins in saline group animals (Mean ± SEM). (a) Venlafaxine

treatment increases β-catenin immunoreactivity in total cell

lysate (TCL), membrane-associated (M) and nuclear (N)

Angel Pazos 45

subcellular fractions of rat hippocampus in Western blot

studies. (b) Representative Western blot analyses of β-catenin

and AKT levels in total cell lysates (TCL) and subcellular

fractions, Citosol (C), membrane-associated (M) and nuclear

(N), from saline, 10 and 40 mg/kg/day venlafaxine chronically

treated animals. Densitometric measurement levels were

normalized to actin protein amounts. Purity of subcellular

fractions used in Western blot assays was assessed by analysis

of GAPDH and histone proteins expression. All samples showed

for comparison were run in the same gel. (c) Venlafaxine

treatment differentially affects AKT protein levels depending

on the dose and the subcellular fraction analyzed. *p < 0.05;

**p < 0.01; ***p < 0.001.

Figure 3. Chronic venlafaxine treatment does not affect

nuclear expression of CREB nor p-CREB. Graphs represent

densitometry levels of CREB as well as p-CREB proteins in AD

treated animals as a percentage of these proteins in saline

group animals, and the p-CREB/CREB levels ratio (all data are

expressed as mean ± SEM). Representative Western blot analyses

of CREB and p-CREB levels in nuclear subcellular fractions

Angel Pazos 46

from saline, 10 and 40 mg/kg/day venlafaxine chronically

treated animals.

Figure 4. Chronic venlafaxine treatment affects the expression

of p-ERK1 and p-ERK2 without modifying the expression of ERK1

or ERK2. Graphs represent densitometry levels of ERK1/2 and p-

ERK1/2 proteins in AD treated animals as a percentage of these

proteins in saline group animals, and the p-ERK1/ERK1 and p-

ERK2/ERK2 levels ratios (all data are expressed as mean ±

SEM). Representative Western blot analyses of ERK1 and ERK2,

and p-ERK1 and p-ERK2 in tissue cell lysates from saline, 10

and 40 mg/kg/day venlafaxine chronically treated animals. *p <

0.05; **p < 0.01; ***p < 0.001.

Figure 5. Representative microphotographs of β-catenin

immunohistochemistry from saline (a), 10 mg/kg/day venlafaxine

(b), and 40 mg/kg/day venlafaxine (c) chronically treated

animals showing increased immunoreactivity after chronic

treatment with 10 and 40 mg/kg/day venlafaxine. Arrows:

positive staining; Bar = 200 µm.

Angel Pazos 47

Figure 6. Left panel shows representative confocal

microphotographs of SGZ from animals from saline group (a) and

40 mg/kg/day venlafaxine group (b). No nuclear presence of β-

catenin could be observed in any animal from these

experimental groups, although an increased β-catenin

immunolabeling was detected in the treated animals. Right

panel shows representative microphotographs of BrdU/β-catenin

double labeling from a 40 mg/kg/day venlafaxine chronically

treated animal. No overlapping could be detected in any sample

using immunohistochemistry techniques (arrow). CGL: granule

cell layer; H: hilus; SGZ: subgranular zone; Bar = 20 µm.

Figure 7. Electron microscopy photomicrographs showing nuclei

from SGZ cells from saline (a), 10 mg/kg/day venlafaxine (b),

and 40 mg/kg/day venlafaxine (c) chronically treated animals.

β-Catenin positive labeling (arrows) is increased in

venlafaxine treated animals. Nu: nucleolus. Bar = 0.2 µm.

Figure 8. Diagram depicting the suggested effects of AD on

Wnt pathway in the SGZ. Adult hippocampal stem/progenitor

cells (AHPs), neurons and glial cells are present in SGZ and

Angel Pazos 48

adjacent GCL. AD treatment increases the extracellular levels

of 5-HT and NE, which, in neurons and glial cells, through

serotonin and adrenergic receptors, elicit a number of

reactions that lead to AKT activation. Inhibition of GSK-3 by

AKT increases β-catenin cytoplasmatic levels and promotes the

traslocation to the nucleus of this protein. In these very

differentiated cells, no expression of cell cycle induction

proteins will take place. However they express Wnt pathway

proteins, among them Wnt receptors agonists, which are

secreted to the extracellular space. There, secreted Wnt

agonists act on Frizzled receptors (FZr) the surrounding

cells, neurons and glia, where they enhance the activation of

the Wnt pathway, and AHPs, where they elicit the expression of

cell cycle related proteins, as CyclinD, promoting cell

division.

Angel Pazos 49

Angel Pazos 50