Folic acid administration produces an antidepressant-like effect

in mice: Evidence for the involvement of the serotonergic

and noradrenergic systems
Patr cia S. Brocardo
a
, Josiane Budni
a
, Manuella P. Kaster
a
, Adair R.S. Santos
b
,
Ana Lucia S. Rodrigues
a,
*
a
Departamento de Bioqu mica, Centro de Ciencias Biolo gicas, Universidade Federal de Santa Catarina, Campus Universitario, Trindade, 88040-900
Floriano polis, SC, Brazil
b
Departamento de Ciencias Fisiologicas, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Campus Universitario,
Trindade, 88040-900 Floriano polis, SC, Brazil
Received 16 May 2007; received in revised form 21 September 2007; accepted 22 October 2007
Abstract
Clinical studies have shown that folic acid plays a role in the pathophysiology of depression. However, very few studies have investigated its
effect in behavioral models of depression. Hence, this study tested its effect in the forced swimming test (FST) and the tail suspension test (TST),
two models predictive of antidepressant activity, in mice. Folic acid administered by oral route (p.o.) produced a reduction in the immobility time
in the FST (50e100 mg/kg) and in the TST (10e50 mg/kg). The administration of folic acid by i.c.v. route also reduced the immobility time in
the FST (10 nmol/site) and in the TST (1e10 nmol/site). Both folic acid administered by oral and i.c.v. route produced no psychostimulant
effect, which indicates that its antidepressant-like effect is specic. Pretreatment of mice with p-chlorophenylalanine methyl ester (PCPA;
100 mg/kg, i.p., an inhibitor of serotonin (5-HT) synthesis, for 4 consecutive days), ketanserin (5 mg/kg, i.p., a 5-HT
2A/2C
receptor antagonist),
prazosin (1 mg/kg, i.p., an a
1
-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p., an a
2
-adrenoceptor antagonist) prevented the anti-immobility
effect of folic acid (50 mg/kg, p.o.) in the FST. Moreover, the pretreatment of mice with WAY100635 (0.1 mg/kg, s.c., a selective 5-HT
1A
receptor
antagonist) blocked the decrease in immobility time in the FSTelicited by folic acid (50 mg/kg, p.o.), but produced a synergistic effect with a sub-
effective dose of folic acid (10 mg/kg, p.o.). In addition, a subeffective dose of folic acid (10 mg/kg, p.o.) produced a synergistic antidepressant-
like effect with uoxetine (10 mg/kg, p.o.) in the FST. Overall, the results rstly indicate that folic acid produced an antidepressant-like effect in
FST and in TST and that this effect appears to be mediated by an interaction with the serotonergic (5-HT
1A
and 5-HT
2A/2C
receptors) and nor-
adrenergic (a
1
- and a
2
-adrenoceptors) systems.
2007 Elsevier Ltd. All rights reserved.
Keywords: Folic acid; Forced swimming test; Tail suspension test; Serotonin; Noradrenaline; Antidepressant
1. Introduction
Folic acid (folate) is a water-soluble B-vitamin whose
biologically active form is tetrahydrofolic acid (THF), which
participates in the transfer of 1-carbon units (such as methyl,
methylene, and formyl groups) to the essential substrates
involved in the synthesis of DNA, RNA, and proteins (Bailey
and Gregory, 1999).
Ingested folic acid can be converted to its physiological
forms. This process is initiated by dihydrofolate reductase in
a two-step reaction; the rst step, conversion to dihydrofolate
(DHF) is a slow and rate-limiting step (Wagner, 1995). In the
second, more rapid, step dihydrofolate is further reduced to
THF. THF can then be converted into additional physiological
folates including 5-methyl-THF, the form that is normally
* Corresponding author. Tel.: 55 48 3721 5043; fax: 55 48 3721 9672.
E-mail addresses: analucia@mbox1.ufsc.br, alsrodri@terra.com.br (A.L.S.
Rodrigues).
0028-3908/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2007.10.016
Available online at www.sciencedirect.com
Neuropharmacology 54 (2008) 464e473
www.elsevier.com/locate/neuropharm
found in the circulation and in tissues (Bailey and Gregory,
1999; Ramaekers, 2004). 5-Methyl-THF is also replenished
by the conversion of folinic acid (5-formyltetrahydrofolate),
an active metabolite of folic acid. Because de novo folate syn-
thesis is not present in the CNS, it depends on adequate folate
transport across the blood-brain barrier. Within neurons, part
of the folate pool will be catabolized by oxidation to dihydro-
folates and folic acid, which can be reconverted to THF by
dihydrofolate reductase (Ramaekers, 2004).
Folic acid plays a role in the methylation of homocysteine
providing the methyl group for the conversion of methionine
to S-adenosylmethionine (SAM), which itself has been shown
to inuence 5-HT metabolism. It also has an important role in
the synthesis of tetrahydrobiopterin, an essential co-factor for
the hydroxylation of phenylalanine and tryptophan, rate-
limiting steps in the synthesis of dopamine (DA), noradrena-
line (NA) and 5-HT (Mattson and Shea, 2003; Taylor et al.,
2004).
Several clinical studies have shown the association between
depressive disorder and low folic acid levels (Reynolds, 2002;
Paul et al., 2004; Abou-Saleh and Coppen, 2006). Additionally,
a deciency of folate causes elevated homocysteine concentra-
tions, which may contribute to the pathogenesis of major depres-
sion (Bottiglieri, 2005; Coppen and Bolander-Gouaille, 2005).
Moreover, patients diagnosed with major depressive disorder
tend to have lower concentrations of serum or red cell folate
than healthy control subjects (Reynolds, 2002; Morris et al.,
2003; Coppen and Bolander-Gouaille, 2005) and antidepressant
therapy is shown to be accompanied by an increase of folate in
the erythrocytes (Levitt et al., 1998). Moreover, it has been
shown that supplementing antidepressant medication with folic
acid enhances the therapeutic effect (Coppen and Bailey, 2000;
Abou-Saleh and Coppen, 2006).
According to the monoaminergic hypothesis of depression,
the major neurochemical process in depression is the impair-
ment of monoaminergic neurotransmission and the conco-
mitant decrease of extracellular concentration of NA and/or
5-HT (Schildkraut, 1965). The theory was originally based
on the observations that most antidepressants were inhibitors
of transporters of 5-HT and NA in the presynaptic nerve end-
ing, therefore, enhancing the monoaminergic neurotransmis-
sion (Glowinski and Axelrod, 1964; Ross and Renyi, 1969).
This action by antidepressants is one of the cornerstones of
the monoaminergic hypothesis of depression (Maas, 1975).
The serotonergic system is closely implicated in the patho-
genesis of depression and in the mechanism of action of the
antidepressant. Most of the prescribed antidepressants directly
affect 5-HT turnover in the brain (Kreiss and Lucki, 1995), in-
hibit 5-HT reuptake and also interact with 5-HT
1A
and 5-HT
2
receptors (Cryan et al., 2005). Newer antidepressants (selective
serotonin reuptake inhibitors; SSRIs) are generally more selec-
tive and more potent than the older compounds at blocking
transport of 5-HT over NA (Richelson, 2001).
Evidence for a role of NA in depression is well established,
since most of the antidepressants until the 1980s were shown
to be more potent at blocking uptake of NA than at blocking
uptake of 5-HT (Richelson and Pfenning, 1984). Many
antidepressants, such as doxepin, amitriptyline and nefazo-
done, have signicant a
1
-afnity and the antidepressant mirta-
zapine enhances neurotransmission by directly blocking
presynaptic a
2
-adrenoceptors (Richelson, 2001).
Given that depression is associated with low levels of folic
acid, which is involved in the biosynthesis of monoamines
that, in turn, exerts a signicant role in the pathogenesis of
depression, it is reasonable to hypothesize that folic acid
administration produces antidepressant-like effects which are
dependent on the serotonergic and noradrenergic systems.
Considering that lack of preclinical studies dealing with the
antidepressant-like effects of folic acid, this study therefore
sought to investigate the effect of this vitamin in the forced
swimming test (FST) and the tail suspension test (TST) in
mice and the involvement of the serotonergic and noradrener-
gic systems in its antidepressant-like effect.
2. Materials and methods
2.1. Animals
Swiss mice of either sex, weighing 30e40 g, were maintained at constant
room temperature (22e25

C) with free access to water and food, under
a 12:12 h light/dark cycle (lights on at 07:00 h) in cages separated by sex.
Animals (male and female mice were homogeneously distributed among
groups) were acclimatized to the laboratory for at least 1 h before testing
and were used only once throughout the experiments. All manipulations
were conducted in the light phase, between 09:00 and 16:00 h. The procedures
in this study were performed in accordance with the NIH Guide for the Care
and Use of Laboratory Animals. The protocol of the study was approved by
the Ethics Committee of the Institution and all efforts were made to minimize
animal suffering and to reduce the number of animals used in the experiments.
2.2. Drugs and treatment
The following drugs were used: folic acid, folinic acid, ketanserin tartrate,
uoxetine, p-chlorophenylalanine methyl ester (PCPA), N-{2-[4-(2-methoxy
phenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohexanecarboxamide (WAY
100635), prazosin hydrochloride and yohimbine hydrochloride (all from
Sigma Chemical Co., St. Louis, MO, USA). Folic acid was administered by
oral route (p.o.) by gavage, whereas PCPA, ketanserin, sulpiride, prazosin
and yohimbine were administered by intraperitoneal (i.p.) route; WAY
100635 was administered by subcutaneous (s.c.) route. Alternatively, folic
acid was administered by intracerebroventricular (i.c.v.) route in a volume
of 5 ml per mouse, as described previously (Kaster et al., 2007). The i.p.,
s.c. and p.o. administrations were given in a constant volume of 10 ml/kg
body weight. All drugs were dissolved in saline, except that folic acid and
uoxetine administered by p.o. route were dissolved in water. Appropriate
vehicle-treated groups were assessed simultaneously.
2.3. Experimental design
In order to investigate the antidepressant-like effect produced by an oral
administration of folic acid, it was administered at three doses: 10, 50 and
100 mg/kg, 1 h before the FST and 1, 10 and 50 mg/kg 1 h before the TST.
The locomotor activity test was performed in an independent group of animals
1 h after the administration of folic acid at a dose that produced an antidepres-
sant-like effect in the FST or TST. Alternatively, in order to conrm that the
effect produced by the systemic administration of folic acid is due to its enter-
ing into the central nervous system, this compound was also directly adminis-
tered through i.c.v. injection (dose range: 0.1e10 nmol/site) 15 min before the
FST or TST. The locomotor activity test was performed in an independent
465 P.S. Brocardo et al. / Neuropharmacology 54 (2008) 464e473
group of animals 15 min after the i.c.v. administration of folic acid at a dose
that produced an antidepressant-like effect in the FST or TST.
In another experiment, folinic acid, a metabolite of folic acid, was admin-
istered by i.c.v. route (dose range: 1e10 nmol/site) 15 min before the FST. The
locomotor activity test was performed in an independent group of animals
15 min after the i.c.v. administration of folinic acid at a dose that produced
an antidepressant-like effect in the FST.
In order to investigate a possible contribution of the 5-HT system to the
effect of folic acid in reducing the immobility time in the FST, animals
were pretreated with PCPA (100 mg/kg, an inhibitor of 5-HT synthesis) or
saline, once a day, for 4 consecutive days. Then, 24 h after the last PCPA or
saline injection, animals were treated with folic acid (50 mg/kg, p.o.), uoxe-
tine (20 mg/kg, p.o., positive control) or vehicle (water) and were tested in the
FST 60 min later.
We also investigated the ability of folic acid to potentiate the antidepres-
sant-like effect of uoxetine. To this end, mice received folic acid (10 mg/
kg, p.o.), uoxetine (10 mg/kg, p.o.) or vehicle by p.o. route (gavage). After
60 min, they were tested in the FST.
In a separate series of experiments, the involvement of the 5-HT receptor
subtypes in the effect of folic acid in the FST was studied. In order to inves-
tigate the possible involvement of the 5-HT
1A
receptor subtype in the antide-
pressant-like effect of folic acid, mice were pretreated with WAY100635
(0.1 mg/kg, s.c., a selective 5-HT
1A
receptor antagonist) or saline and after
30 min they received folic acid (50 mg/kg, p.o.) or vehicle by p.o. route before
being tested in the FST 60 min later. In another experiment, mice were
pretreated with WAY100635 (0.1 mg/kg, s.c.) or saline and after 30 min
they received a subeffective dose of folic acid (10 mg/kg, p.o.) or vehicle by
p.o. route before being tested in the FST 60 min later.
In a separate series of experiments, in order to investigate the involvement
of the 5-HT
2
receptor subtype in the effect of folic acid in the FST, mice were
pretreated with ketanserin (5 mg/kg, a 5-HT
2A/2C
receptor antagonist) or
saline, and after 30 min, they received folic acid (50 mg/kg, p.o.) or vehicle
by p.o. route before being tested in the FST 60 min later.
To assess the possible involvement of the noradrenergic system in the
antidepressant-like effect of the folic acid in the FST, animals were pretreated
with prazosin (1 mg/kg, i.p., an a
1
-adrenoceptor antagonist) or yohimbine
(1 mg/kg, i.p., an a
2
-adrenoceptor antagonist), and after 30 min they received
folic acid (50 mg/kg, p.o.) or vehicle (p.o.) and were tested in the FST 60 min
later.
The doses of the drugs used were selected on the basis of literature and are
previously reported not to increase locomotor activity (Redrobe et al., 1996;
Redrobe and Bourin, 1997; ONeill and Conway, 2001; Kaster et al., 2005;
Machado et al., 2007).
2.4. Behavioral tests
2.4.1. Forced swimming test (FST)
The test was conducted using the method of Porsolt et al. (1977) with
minor modications. Briey, mice were individually forced to swim in an
open cylindrical container (diameter 10 cm, height 25 cm), with a depth of
19 cm of water at 25 1

C. The immobility time, dened as the absence
of escape-oriented behaviors, such as swimming, was scored during 6 min,
as described previously (Eckeli et al., 2000; Zomkowski et al., 2004; Kaster
et al., 2005, 2007). Each mouse was judged immobile when it ceased strug-
gling and remained oating motionless in the water, making only those move-
ments necessary to keep its head above water.
2.4.2. Tail suspension test (TST)
The total duration of immobility induced by tail suspension was measured
according to the method described by Steru et al. (1985). Mice both acousti-
cally and visually isolated were suspended 50 cm above the oor by adhesive
tape placed approximately 1 cm from the tip of the tail. Immobility time was
recorded during a 6-min period.
2.4.3. Locomotor activity
In order to rule out any unspecic locomotor effect of folic acid/folinic acid
on the antidepressant-like effect these compounds, mice were administered
with the same doses that produce anti-immobility effects in the FST or TST
to have their locomotor activity evaluated. Animals were habituated to the
test chamber for 2 h and then were injected with the test compounds or saline.
Fifteen minutes post injections, locomotor activity was measured for 15 min.
The chamber consists of a cage of wood (40 14 20 cm) with steel grid
oors and equipped with three parallel horizontal infrared beans positioned
2 cm above the oor and spaced evenly along the longitudinal axis. The activity
cages were linked to a digital counter that recorded photocell bean interruptions
(Gevaerd and Takahashi, 1999).
2.5. Statistical analysis
Comparisons between experimental and control groups were performed by
one-way (dose-response curve of folic acid in the FST, TST and open-eld
test) or two-way ANOVA (experiments dealing with the involvement of the
monoaminergic system in the effect of folic acid in the FST) followed by
Tukeys HSD test when appropriate. P < 0.05 was considered signicant.
3. Results
3.1. Effect of treatment with folic acid in the mouse FST,
TST and locomotor activity
The results depicted in Fig. 1A show that the administration
of folic acid by oral route decreased the immobility time in the
FST, indicating that the systemic administration of folic acid is
effective in producing an antidepressant-like effect in this
behavioral model. One-way ANOVA revealed a signicant ef-
fect of folic acid (F
3,24
11.21, P < 0.01). Post hoc analyses
indicated a signicant decrease in the immobility time elicited
by the administration of folic acid at the doses of 50 and
100 mg/kg. Fig. 1B shows that the administration of folic
acid by p.o. route also produced a signicant effect in the
immobility time in the TST (F
3,20
5.71, P < 0.01).
Fig. 1C shows that when administered at these active doses
in the FST/TST, folic acid produced no psychostimulant effect
in mice habituated to the novel environment (F
3,19
0.81,
P 0.50).
Fig. 2A shows that folic acid administered by i.c.v. route
decreased the immobility time of mice in the FST. One-way
ANOVA revealed a signicant effect of folic acid
(F
3,22
12.31, P < 0.01). Post hoc analyses indicated a signif-
icant decrease in the immobility time elicited by folic acid
(10 nmol/site) in the FST. Fig. 2B shows that folic acid, also
decreased the immobility time of mice in the TST. One-way
ANOVA revealed a signicant effect of folic acid
(F
3,22
5.50, P < 0.01). Post hoc analyses indicated that folic
acid administered at doses of 1 and 10 nmol/site produced a
decrease in the immobility time as compared to the control
group.
In order to exclude a possibility that the reduction in the
immobility time elicited by folic acid was due to a psychosti-
mulant effect, we tested the effect of folic acid in the locomo-
tor activity. Fig. 2C shows that folic acid caused no effect on
locomotor activity in mice habituated to the novel environ-
ment when compared to the control group (F
2,18
0.032,
P 0.96).
The results of Fig. 3A show the effect of the i.c.v. adminis-
tration of folinic acid (5-formyltetrahydrofolate), an active
466 P.S. Brocardo et al. / Neuropharmacology 54 (2008) 464e473
metabolite of folic acid not susceptible to oxidative degrada-
tion, which represents a stable form of folate. One-way AN
OVA revealed a signicant effect of the treatment with folinic
acid (F
2,19
7.24, P < 0.01). Post hoc analyses revealed that
folinic acid administered at the dose of 10 nmol/site signi-
cantly reduced the immobility time in the FST as compared
to the control group. Fig. 3B shows that when administered
at these active doses in the FST, folinic acid produced no psy-
chostimulant effect in mice habituated to the novel environ-
ment (F
1,10
1.42, P 0.26).
3.2. Effect of pretreatment with PCPA on the
anti-immobility effect of folic acid and uoxetine in the
mouse FST
The effect of pretreatment of mice with PCPA (100 mg/kg,
an inhibitor of serotonin synthesis) or saline, once a day for
four consecutive days, on the antidepressant effect of folic
acid and uoxetine (positive control) is shown in Fig. 4A.
The two-way ANOVA showed signicant differences of
pretreatment (F
1,32
47.92, P < 0.01), treatment (F
2,32

37.55, P < 0.01) and of pretreatment treatment interaction

(F
2,32
28.90, P < 0.01). Post hoc analyses indicated that
the pretreatment of mice with PCPA blocked the decrease in
immobility time elicited by folic acid (50 mg/kg, p.o.) and u-
oxetine (20 mg/kg, p.o.).
3.3. Interaction of folic acid with uoxetine in the mouse
FST
Fig. 4B shows the effect of co-administration of mice with
a subeffective dose of folic acid (10 mg/kg, p.o.) and subeffec-
tive dose of uoxetine (10 mg/kg, p.o.) on the immobility time
in the FST. A two-way ANOVA revealed signicant differ-
ences for the folic acid treatment (F
1,24
13.93, P < 0.01),
uoxetine treatment (F
1,24
5.73, P < 0.05), and folic
acid uoxetine interaction (F
1,24
4.86, P < 0.05). Post
hoc analyses indicated that the co-administration of a subeffec-
tive uoxetine produced a synergistic effect with a subeffective
dose of folic acid.
3.4. Interaction of folic acid with 5-HT
1A
receptors in the
mouse FST
Fig. 5A shows the inuence of pretreatment of mice with
WAY100635 (0.1 mg/kg, s.c., a selective 5-HT
1A
receptor
C 10 50 100
0
50
100
150
200
250
300
Folic acid (mg/kg, p.o.)
**
**
A
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
0
5
10
15
20
C
N
u
m
b
e
r

o
f

c
r
o
s
s
i
n
g
s
C 1 10 50
0
50
100
150
200
250
**
*
Folic acid (mg/kg, p.o.)
B
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
C 10 50 100
Folic acid (mg/kg, p.o.)
Fig. 1. Effect of the administration of folic acid by oral route in the FST (A), in the TST (B) and in the locomotor activity (C). Values are expressed as mean
S.E.M. (n 5e7). *P < 0.05 and **P < 0.01 as compared with the vehicle-treated control.
467 P.S. Brocardo et al. / Neuropharmacology 54 (2008) 464e473
antagonist) on the reduction in immobility time elicited by
folic acid (50 mg/kg, p.o.) in the FST. A two-way ANOVA
revealed a main effect of pretreatment (F
1,20
7.15,
P 0.05, treatment (F
1,20
18.66, P < 0.01) and of pretreat-
ment treatment interaction (F
1,20
29.88, P < 0.01) on
immobility time in the FST. Post hoc analyses indicated that
the pretreatment of mice with WAY100635 signicantly
blocked the decrease in immobility time in the FST elicited
by folic acid. The results depicted in Fig. 5B show the effect
of pretreatment of mice with WAY100635 (0.1 mg/kg, s.c.)
C 1 10
0
5
10
15
20
Folic acid (nmol/site, i.c.v.)
C
N
u
m
b
e
r

o
f

c
r
o
s
s
i
n
g
s
C 0.1 1 10
0
50
100
150
200
250
300
**
Folic acid (nmol/site, i.c.v.)
A
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
0
50
100
150
200
250
300
**
*
B
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
C 0.1 1 10
Folic acid (nmol/site, i.c.v.)
Fig. 2. Effect of the intracerebroventricular administration of folic acid in the FST (A) and TST (B) and in the locomotor activity (C) in mice. Values are expressed
as mean S.E.M. (n 6e8). *P < 0.05 and **P < 0.01 as compared with the vehicle-treated control.
C 1 10
0
50
100
150
200
250
300
**
Folinic acid (nmol/site, i.c.v)
A
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
C 10
0
5
10
15
Folinic acid (nmol/site, i.c.v.)
B
N
u
m
b
e
r

o
f

c
r
o
s
s
i
n
g
s
Fig. 3. Effect of the intracerebroventricular administration of folinic acid in the FST (A) and in the locomotor activity (B) in mice. Values are expressed as mean S.E.M.
(n 6e8). *P < 0.05 and **P < 0.01 as compared with the vehicle-treated control.
468 P.S. Brocardo et al. / Neuropharmacology 54 (2008) 464e473
in the effect of subeffective dose of folic acid (10 mg/kg, p.o.)
in the FST. A two-way ANOVA revealed a main effect of pre-
treatment (F
1,23
7.63, P < 0.05) and of treatment (F
1,23

6.21, P < 0.05) and of pretreatment treatment interaction

(F
1,23
5.24, P < 0.05). Post hoc analyses indicated that the
pretreatment with WAY100635 produced a synergistic effect
with a subeffective dose of folic acid (10 mg/kg, p.o.).
3.5. Interaction of folic acid with 5-HT
2
receptors in the
mouse FST
Fig. 6 shows the effect of pretreatment of mice with ketan-
serin (5 mg/kg, a 5-HT
2A/2C
antagonist) on the reduction in im-
mobility time elicited by folic acid (50 mg/kg, p.o.). Atwo-way
ANOVA revealed a main effect of pretreatment (F
1,20
27.55,
P < 0.01), treatment (F
1,20
33.73, P < 0.01) and of pretreat-
ment treatment interaction (F
1,20
53.06, P < 0.01). Post
hoc analyses indicated that the pretreatment with ketanserin
blocked the decrease in immobility time in the FST elicited
by folic acid.
3.6. Involvement of the noradrenergic system
The results depicted in Fig. 7A show that pretreatment of
mice with prazosin (1 mg/kg, i.p) was able to reverse the
antidepressant-like effect the folic acid (50 mg/kg, p.o.) in
the FST. The two-way ANOVA revealed a main effect of the
pretreatment (F
1,20
17.92, P < 0.01), treatment (F
1,20

50.56, P < 0.01), and of treatment pretreatment interaction

(F
1,20
52.58, P < 0.01). Fig. 7B shows that the pretreatment
of mice with yohimbine (1 mg/kg, i.p.) was also able to pre-
vent the anti-immobility effect the folic acid (50 mg/kg,
p.o.) in the FST. The two-way ANOVA revealed a main effect
of the pretreatment (F
1,20
28.26, P < 0.01), treatment
(F
1,20
27.37, P < 0.01), and of treatment pretreatment in-
teraction (F
1,20
48.65, P < 0.01).
0
50
100
150
200
250
300
350
Control Folic acid Fluoxetine
**
#
#
**
A
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
Saline
PCPA
0
50
100
150
200
250
300
350
Control Folic acid
**
B
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
Saline
Fluoxetine
Fig. 4. Effect of pretreatment with the 5-HT synthesis inhibitor, PCPA (100 mg/kg, 4 consecutive days, an inhibitor of serotonin synthesis), on the folic acid
(50 mg/kg, p.o.) and uoxetine (20 mg/kg, p.o.)-induced reduction in the immobility time in the FST (A). Effects of co-administration of mice with folic acid
(10 mg/kg, p.o.) and uoxetine (10 mg/kg, p.o.) in the immobility time in the FST (B). Values are expressed as mean S.E.M. (n 6e7). **P < 0.01 as compared
with the vehicle-treated control;
#
P < 0.01 as compared with the same group pretreated with vehicle and folic acid.
0
50
100
150
200
250
300
350
Saline
WAY 100635
A
**
#
Control Folic acid
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
0
50
100
150
200
250
300
350
*
Control Folic acid
B
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
Fig. 5. (A) Inhibition the anti-immobility effect of folic acid (50 mg/kg, p.o.) in the FST by pretreatment of mice with WAY100635 (0.1 mg/kg, s.c., a selective 5-
HT
1A
receptor antagonist). Values are expressed as mean S.E.M. (n 6e7). **P < 0.01 as compared with the control group (vehicle);
#
P < 0.01 as compared
with the same group pretreated with vehicle and folic acid. (B) Inuence of pretreatment of mice with WAY100635 (0.1 mg/kg, s.c.) on the effect of a subeffective
dose of folic acid (10 mg/kg, p.o.) in the FST. Values are expressed as mean S.E.M. (n 6e7). **P < 0.01 as compared with the control group (vehicle) or with
the vehicle/folic acid group.
469 P.S. Brocardo et al. / Neuropharmacology 54 (2008) 464e473
4. Discussion
Our results show, to our knowledge for the rst time, that
folic acid given either systemically (p.o. route) or centrally
(i.c.v. route) is effective in reducing the immobility time in
the FST and in the TST, consistent with an antidepressant-
like effect in these two animal models predictive of antidepres-
sant action (Porsolt et al., 1977; Steru et al., 1985). Both tests
are widely used to assess the antidepressant properties of new
drugs, as they are sensitive to all major classes of antidepres-
sant drugs, including tricyclics, serotonin-specic reuptake in-
hibitors, monoamine oxidase inhibitors, and atypicals (Porsolt
et al, 1977; Steru et al, 1985) and are important tools to study
neurobiological mechanisms involved in antidepressant
responses. Effective antidepressant treatments decrease immo-
bility time in both tests (Porsolt et al., 1977; Steru et al., 1985).
However, these tests have some drawbacks represented by the
possibility of obtaining some false positives or negatives.
Drugs enhancing motor activity may give a false positive
effect in these tests, whereas drugs decreasing locomotion
may give a false negative result (Borsini and Meli, 1988).
The reduction in the immobility time elicited by folic acid
cannot be attributable to a psychostimulant action of this
compound. This conclusion derives from the fact that in our
study folic acid administered either by p.o. or i.c.v. route at
doses that produced a signicant decrease in the immobility
time in the FST or TST did not alter the locomotor activity
in animals previously habituated to the novel environment,
as compared to control animals. The antidepressant-like
behavior of folic acid in the FST and in the TST are somewhat
in accordance with clinical studies that show that folic acid
exerts a signicant role in the pathophysiology of depression,
since it has long been recognized that its deciency is associ-
ated with symptoms of depression (Fava et al., 1997; Ramos
et al., 2004; Bottiglieri, 2005). In spite of this, there is
a lack of studies investigating the effect of folic acid in animal
models of depression. Indeed, a reduction in the immobility
time in the FST by folic acid was previously shown (Ali
et al., 2003), but the mechanism underlying its effect was
not addressed.
Our results also show that folinic acid, an active form of
folate, administered centrally also produced an antidepres-
sant-like effect in the FST. This result together with the one
obtained with folic acid administered centrally, suggests that
folic acid undergoes conversion to DHF and THF to exerts
its behavioral effects in the FST, since these forms can be
generated both from folic acid and folinic acid (Ramaekers,
2004). However, future experiments are needed to better
clarify this issue.
Several lines of evidence indicate that serotonergic and
noradrenergic neurotransmissions are involved in the expres-
sion of an antidepressant-like effect in the behavioral despair
models of depression (Elhwuegi, 2004). In this study we inves-
tigated the possible involvement of these systems in the
antidepressant-like effect of folic acid administered orally.
Folic acid may be directly involved in the regulation of
the serotonergic function in depression (Bottiglieri, 2005).
Alterations in the serotonergic function by folate deciency
have been reported to be associated with impaired 5-HT me-
tabolism (Botez et al., 1982; Gospe et al., 1995; Alpert et al.,
2002; Bottiglieri, 2005). In the present study, the involve-
ment of the 5-HT system the antidepressant-like effect of
folic acid in the FST was investigated by inhibiting 5-HT
synthesis with the tryptophan hydroxylase inhibitor PCPA
and by using 5-HT
1A
and 5-HT
2A/2C
receptor agonists or an-
tagonists to examine the behavioral responses to folic acid in
the FST.
The reversal of the antidepressant-like effect of folic acid
by the pretreatment of mice with PCPA suggests that its effect
in the FST may be dependent on the availability of 5-HT in the
synaptic cleft. Similar to previous ndings, PCPA by its own
did not alter the immobility time of control animals, but signif-
icantly blocked the anti-immobility effect of uoxetine, a
5-HT reuptake inhibitor (Page et al., 1999; Eckeli et al.,
2000; Zomkowski et al., 2004; Kaster et al., 2005). Thus,
our ndings provide convincing behavioral evidence that
PCPA treatment was effective in depleting 5-HT stores. Al-
though we did not measure the concentration of 5-HT to verify
its successful depletion following the administration of PCPA,
our behavioral data together with studies from literature (Eck-
eli et al., 2000; Zomkowski et al., 2004; Kaster et al., 2005)
suggest that it was effective in depleting the 5-HT stores. In-
deed, the administration of PCPA for four consecutive days
depletes the endogenous stores of 5-HT by about 60% in
mice, while noradrenaline and dopamine levels are not af-
fected (Redrobe et al., 1998). Considering that PCPA, which
is thought to act presynaptically (Luscombe et al., 1993), pre-
vented the antidepressant-like effect of folic acid in the FST, it
is likely that the expression of the antidepressant-like effect of
folic acid requires an intact presynaptic 5-HT system.
This study also showed that the combination of subeffective
doses of folic acid and uoxetine administered by p.o. route
produced antidepressant-like effects in the FST. This result
0
50
100
150
200
250
300
350
Saline
Ketanserin
**
#
Control Folic acid
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
Fig. 6. Effect of pretreatment of mice with ketanserin (5 mg/kg, i.p., a 5-HT
2A/2C
receptor antagonist) on the folic acid (50 mg/kg, p.o.)-induced reduction in the
immobility time in the FST. Values are expressed as mean S.E.M. (n 6e7).
**P < 0.01 as compared with the control group (vehicle);
#
P < 0.01 as com-
pared with the same group pretreated with vehicle and folic acid.
470 P.S. Brocardo et al. / Neuropharmacology 54 (2008) 464e473
is in line with a clinical study in which folic acid was shown to
augment the antidepressant action of uoxetine (Coppen and
Bailey, 2000). Moreover, folate deciency is associated with
poorer response to SSRIs in major depressive disorder (Alpert
et al., 2002). Recently, low serum folate levels were found to
be associated with a delayed onset of clinical improvement
during treatment with uoxetine in major depressive disorder
patients (Papakostas et al., 2005). One possibility to account
for the synergistic effect between folic acid and uoxetine in
the FST is that folic acid, similarly to uoxetine, increases
5-HT levels at the synaptic cleft.
Several studies have demonstrated the involvement of
5-HT
1A
receptors in the mechanism of action of many classes
of antidepressant drugs, including tricyclics, SSRIs and
MAOi (Hensler, 2002). The combined effect of folic acid
and WAY100635, a competitive antagonist at the 5-HT
1A
re-
ceptors (Zuideveld et al., 2002), in the FST depends on the
dose of folic acid. When combined with a low dose of folic
acid (10 mg/kg, p.o.), which had no effect in the FST by its
own, WAY100635, administered at the dose of 0.1 mg/kg,
s.c., decreased the immobility time, consistent with an antide-
pressant-like effect. When combined with higher doses of folic
acid (50 mg/kg, p.o.) which decreased immobility time,
WAY100635 (0.1 mg/kg, s.c.) blocked the effects of folic
acid. WAY100635 alone had no effect in the FST, as previ-
ously shown (Kaster et al., 2005). One possibility that could
account for the potentiation of the effect of a low dose of folic
acid in the FST is that this compound, similarly to uoxetine,
may increase the 5-HT levels in the synaptic cleft by inhibiting
the 5-HT reuptake, as suggested by the synergistic effect be-
tween folic acid and uoxetine shown in this study. Therefore
under this condition, WAY100635, which is supposed to have
a preferential effect at presynaptic 5-HT
1A
receptors (Ago
et al., 2003), blocks 5-HT
1A
autoreceptors, consequently
increasing 5-HT levels, resulting in an antidepressant-like
effect. It is noteworthy that the blockade of 5-HT
1A
receptors
by WAY100635 synergistically potentiates the increments of
extracellular 5-HT produced by serotonergic antidepressants
(Romero et al., 1996). The reversal of the antidepressant-like
effect of the higher dose of folic acid (50 mg/kg, p.o.) by
the same dose of WAY100635 (0.1 mg;kg, s.c.), instead of
a neutral or synergistic effect with folic acid is a nding dif-
cult to explain. However, it is possible that WAY100635 dis-
rupted that anti-immobility effect of a higher dose of folic
acid in the FST by producing excessive high synaptic levels
of 5-HT, which could reverse the anti-immobility effect of
folic acid. Although this hypothesis needs to be conrmed in
further studies, a similar hypothesis was also raised by
Cousins and Seiden (2000) to explain the reason by which, us-
ing the differential reinforcement of low rates 72-s schedule in
rats, the behaviorally inactive dose of uoxetine produced an
antidepressant-like effect when given in combination with
WAY100635, whereas the effects of the active dose of uoxe-
tine were blocked by pretreatment with WAY100635. More-
over, our nding is similar to those reported in previous
studies with agmatine (Zomkowski et al., 2004) and adenosine
(Kaster et al., 2005).
The antidepressant-like effect of folic acid in the FST was
also prevented by the pretreatment with ketanserin, a 5-
HT
2A/2C
receptor antagonist. There are studies showing that
5-HT
2A/2C
antagonism has a signicant role in the mechanism
underlying the antidepressant-like effect of conventional anti-
depressants in the FST (Redrobe and Bourin, 1997; Cryan and
Lucki, 2000; Elhwuegi, 2004). The reversal of the anti-immo-
bility effect of folic acid with ketanserin suggests that its effect
in the FST is mediated through an interaction with 5-HT
2A
or
5-HT
2C
receptors.
Preclinical and clinical studies have shown that stimulation
of the serotonergic system leads to noradrenergic effects and
vice versa, conrming that 5-HT and NA are intimately con-
nected in the central nervous system (Gorman and Sullivan,
2000). In our study prazosin, an a
1
-adrenoceptor antagonist,
and yohimbine, an a
2
-adrenoceptor antagonist, were able to
reverse the antidepressant-like effect of the folic acid. This re-
sult indicates that the folic acid may exert its effect in the FST
by interacting with either a
1
- or a
2
-adrenoceptors. Our result
0
50
100
150
200
250
300
350
**
#
Control Folic acid
A
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
0
50
100
150
200
250
300
350
B
Control Folic acid
**
#
I
m
m
o
b
i
l
i
t
y

t
i
m
e

(
s
)
Saline
Prazosin
Saline
Yohimbine
Fig. 7. Effect of pretreatment of mice with prazosin (1 mg/kg, i.p., panel A) or with yohimbine (1 mg/kg, i.p., panel B) on the folic acid (50 mg/kg, p.o.)-induced
reduction in immobility time in the FST. Values are expressed as mean S.E.M. (n 6). **P < 0.01 as compared with the control group (vehicle);
#
P < 0.01 as
compared with the same group pretreated with vehicle and folic acid.
471 P.S. Brocardo et al. / Neuropharmacology 54 (2008) 464e473
is in line with the fact that a
1
- and a
2
-adrenoceptors are impli-
cated in the actions of antidepressant agents (Richelson, 2001;
Millan, 2004).
In conclusion, our results rstly show that oral and central
administration of folic acid is able to produce an antidepres-
sant-like effect in FSTand TST, which is not due to a psychos-
timulant effect. Moreover, we provide convincing evidence
that this effect is dependent on its interaction with the seroto-
ninergic and noradrenergic systems. It is noteworthy that the
same prole of effects with the antagonists of serotonergic
and noradrenergic systems regarding the effects of folic acid
in the FST was also obtained when it was administered
centrally by i.c.v. route (data not shown). Taken together,
our ndings are somewhat in accordance with clinical results
(Coppen and Bailey, 2000; Abou-Saleh and Coppen, 2006),
further suggesting that folic acid may exert a role in the mod-
ulation of depression. However, we should take into account
that the FST is not a model of depression per se, and that
the results obtained with this model should be considered
with caution. Thus, further studies are necessary to conrm
its antidepressant effect in other animal behavioral models,
which are thought to more closely mimic depression such as
chronic stress paradigms.
Acknowledgments
This study was supported by CNPq and CAPES (Brazil).
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