The antidepressant-like action of metabotropic glutamate 7 receptor agonist N,N'-bis(diphenylmethyl)-1,2-ethanediamine (AMN082) is serotonin-dependent.

Abstract

Behavioral studies show that modulation of the glutamatergic system might be an efficient way to achieve antidepressant activity. Among the group III metabotropic glutamate (mGlu) receptors, the mGlu7 receptor subtype seems to be the most promising target for potential antidepressants. It has been shown that a selective, allosteric mGlu7 receptor agonist, N,N'-bis (diphenylmethyl)-1,2-ethanediamine (AMN082), induced antidepressant-like action in behavioral tests in mice, although the mechanisms responsible for this action remained unknown. Here, we decided to investigate the possible role of the serotonergic system in the antidepressant-like activity of AMN082 in both the forced swim test (FST) in rats and the tail suspension test (TST) in mice. We found that AMN082 (1-10 mg/kg i.p.) induced a dose-dependent reduction in the immobility of rats and an increase in their swimming behavior, whereas there were not any changes in climbing behavior in the FST in rats. In the TST in mice we found that AMN082 (3 mg/kg i.p.) did not induce an antidepressant-like effect after depletion of serotonin (5-HT) with para-chlorophenylalanine. Moreover, we revealed that citalopram, but not reboxetine, when combined with AMN082 (all compounds used at low subeffective doses), induced a significant antidepressant-like effect in the TST. We also discovered that the 5-HT1A receptor antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohexane-carboxamide (WAY100635) (0.1 mg/kg s.c.), but not the 5-HT2A/2C receptor antagonist ritanserin (0.5 mg/kg i.p.), blocked the antidepressant-like action of AMN082. Altogether, the results of our studies show that the antidepressant-like action of the mGlu7 receptor-positive modulator AMN082 depends on the activation of the serotonergic system.

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The Antidepressant-Like Action of Metabotropic Glutamate 7
Receptor Agonist N,N?-Bis(Diphenylmethyl)-1,2-Ethanediamine
(AMN082) Is Serotonin-Dependent
Agnieszka Pałucha-Poniewiera, Piotr Bran ´ski, Tomasz Lenda, and Andrzej Pilc
Institute of Pharmacology, Polish Academy of Sciences, Krako ´w, Poland (A.P.-P., P.B., T.L., A.P.); and Faculty of Health
Sciences, Jagiellonian University Medical College, Krako ´w, Poland (A.P.)
Received April 27, 2010; accepted June 17, 2010
ABSTRACT
Behavioral studies show that modulation of the glutamatergic
system might be an efficient way to achieve antidepressant activ-
ity. Among the group III metabotropic glutamate (mGlu) receptors,
the mGlu7 receptor subtype seems to be the most promising
target for potential antidepressants. It has been shown that a
selective, allosteric mGlu7 receptor agonist, N,N?-bis (diphenyl-
methyl)-1,2-ethanediamine (AMN082), induced antidepressant-
like action in behavioral tests in mice, although the mechanisms
responsible for this action remained unknown. Here, we decided
to investigate the possible role of the serotonergic system in the
antidepressant-like activity of AMN082 in both the forced swim
test (FST) in rats and the tail suspension test (TST) in mice. We
found that AMN082 (1–10 mg/kg i.p.) induced a dose-dependent
reduction in the immobility of rats and an increase in their swim-
ming behavior, whereas there were not any changes in climbing
behavior in the FST in rats. In the TST in mice we found that
AMN082(3mg/kgi.p.)didnotinduceanantidepressant-likeeffect
after depletion of serotonin (5-HT) with para-chlorophenylalanine.
Moreover, we revealed that citalopram, but not reboxetine, when
combined with AMN082 (all compounds used at low subeffective
doses), induced a significant antidepressant-like effect in the TST.
We also discovered that the 5-HT1A receptor antagonist N-{2-[4-
(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohex-
ane-carboxamide (WAY100635) (0.1 mg/kg s.c.), but not the
5-HT2A/2C receptor antagonist ritanserin (0.5 mg/kg i.p.), blocked
the antidepressant-like action of AMN082. Altogether, the results
of our studies show that the antidepressant-like action of the
mGlu7 receptor-positive modulator AMN082 depends on the ac-
tivation of the serotonergic system.
Introduction
The glutamatergic system constitutes the main excitatory
neurotransmitter system in the mammalian central nervous
system (CNS). Two main classes of glutamate receptors have
been identified: the ionotropic receptors, i.e., ion channels
responsible for fast synaptic transmission, and the metabo-
tropic glutamate (mGlu) receptors, which couple to G pro-
teins to modulate slow synaptic transmission through intra-
cellular second messengers (Nakanishi et al., 1998). mGlu
receptors are a family of eight G protein-coupled receptors,
which are classified into three groups according to their
sequence homology, effector coupling, and pharmacology.
Group I mGlu receptors (mGlu1 and mGlu5) are positively
coupled to phospholipase C; group II mGlu receptors (mGlu2
and mGlu3), and group III mGlu receptors (mGlu4, mGlu6,
mGlu7, and mGlu8) are negatively coupled to adenylyl cy-
clase (Pin and Duvoisin, 1995).
mGlu receptors are implicated in many diverse functions of
the CNS, including regulation of selective and nonselective
ion channels, regulation of neurotransmitter release, induc-
tion of long-term potentiation and long-term depression, and
regulation of the function of the ionotropic receptors (for
review see Conn and Pin, 1997; Ferraguti and Shigemoto,
2006). Moreover, mGlu receptors are known to be involved in
various CNS diseases, including those that are neuropsychi-
atric, such as depression. Numerous behavioral studies have
shown that action on the mGlu receptors might be an effi-
cient way to achieve antidepressant activity (Pałucha and
This work was supported by Funds for Statutory Activity of the Institute of
Pharmacology, Polish Academy of Sciences and the Ministry of Science and
Higher Education [Grant N405 055737] (to A.P.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.169730.
ABBREVIATIONS: mGlu, metabotropic glutamate; AMN082, N,N?-bis(diphenylmethyl)-1,2-ethanediamine; AD, antidepressant drug; CNS, central
nervous system; FST, forced swim test; PCPA, para-chlorophenylalanine; SSRI, selective serotonin reuptake inhibitor; TST, tail suspension test;
WAY100635, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohexane-carboxamide; 5-HT, serotonin; ANOVA, analysis of
variance; KO, knockout; MMPIP, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one; ACPT-I, (1S,3R,4S)-1-amino-
cyclopentane-1,3,4-tricarboxylic acid.
0022-3565/10/3343-1066–1074$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET 334:1066–1074, 2010
Vol. 334, No. 3
169730/3618312
Printed in U.S.A.
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Pilc, 2002, 2005; Pałucha, 2006; Pilc et al., 2008). Preclinical
studies collected from the last decade have shown that
among all mGlu receptor ligands the mGlu5 receptor antag-
onists and group II mGlu receptor antagonists seem to be the
most promising antidepressants (for review see Pałucha and
Pilc, 2007). However, group III mGlu receptor ligands have
been studied less, because of a lack of high-affinity, highly
selective, brain-penetrating agents (Lavreysen and Dautzen-
berg, 2008). Among group III mGlu receptors, the mGlu7
receptor subtype seems to be the most promising target for
potential antidepressants. First, mGlu7 receptor KO mice
displayed antidepressant-like behavior in both the FST and
TST (Cryan et al., 2003). Second, the mGlu7 receptor is
known to be widely distributed in the brain areas related to
mood disorders (Kinoshita et al., 1998). Finally, our recent
study showed that the novel, selective, allosteric mGlu7 re-
ceptor agonist N,N?-bis (diphenylmethyl)-1,2-ethanediamine
(AMN082) (Mitsukawa et al., 2005) induced a potential an-
tidepressant-like action in both the FST and TST in mice
(Pałucha et al., 2007), although the mechanisms responsible
for the behavioral effects of AMN082 remains unknown. Be-
cause activation of the mGlu7 receptor is known to be respon-
sible for the regulation of glutamate and GABA release (Cart-
mell and Schoepp, 2000), it can be supposed that the
mechanism of the antidepressant-like effect of AMN082
might be related to the regulation of glutamatergic or
GABAergic neurotransmission. However, activation of mGlu
receptors may also be related to the regulation of other neu-
rotransmitters, including those engaged in the mechanism of
action of classic antidepressant drugs (ADs), such as 5-HT
(Lee and Croucher, 2003; Kawashima et al., 2005; Stachow-
icz et al., 2007b, 2009). Furthermore, several studies have
shown the structural and functional interaction between the
serotonergic and glutamatergic systems and its implication
in psychiatric diseases such as psychosis and anxiety (Marek
et al., 2000; Klodzinska et al., 2002; Stachowicz et al., 2007a,
2007b, 2009; Gonza ´lez-Maeso et al., 2008). As such, we de-
cided to investigate the possible role of the serotonergic sys-
tem in the antidepressant-like activity of the mGlu7 receptor
agonist AMN082.
Materials and Methods
Animals and Housing. Male Sprague Dawley rats (Charles
River Laboratories, Inc., Wilmington, MA) (220–230 g) were used to
assess the antidepressant-like activity of AMN082 in the FST and
locomotor activity test. Male C57BL/6J mice (23–25 g) were used in
the TST and locomotor activity test. The animals were kept under
standard laboratory conditions of lighting (light phase 7:00 AM to
7:00 PM) and temperature (19–21°C). Food and water were freely
available. Each experimental group consisted of seven to nine ani-
mals. All of the subjects were experimentally naive and used only
once in each test. Behavioral experiments were performed during the
light period (9:00 AM to 2:00 PM) by an observer unaware of the
treatment. All procedures were conducted according to the guide-
lines of the National Institutes of Health Animal Care and Use
Committee and were approved by the Ethics Committee of the In-
stitute of Pharmacology, Polish Academy of Sciences in Krako ´w.
Drug Administration. AMN082 (Ascent Scientific, Bristol, UK),
ritanserin (Tocris Bioscience, Bristol, UK), and para-chloropheny-
lalanine (PCPA) (Sigma-Aldrich, St. Louis, MO) were dispersed in a
suspension of 0.5% methylcellulose, which was used as vehicle. N-{2-
[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohex-
ane-carboxamide (WAY100635) (Sigma-Aldrich), reboxetine (Phar-
macia and Upjohn, Kalamazoo, MI), and citalopram (Sigma-Aldrich)
were soluble in sterile water. In the rats, AMN082 was injected
intraperitoneally, at a constant volume of 2 ml/kg, 24, 5, and 1 h
before the behavioral test. In the mice, AMN082 and ritanserin were
administered intraperitoneally 60 min before the experiment,
WAY100635 was given subcutaneously 45 min before the test, and
citalopram and reboxetine were administered intraperitoneally 30
min before the experiment. The drugs were injected in the mice at a
constant volume of 10 ml/kg. Drugs were chosen based on previous
experiments (Stachowicz et al., 2007a,b, 2009). Because lower doses
of AMN082 are required in mice than rats to reach equivalent brain
levels (Fendt et al.,2008), the doses were adjusted accordingly.
5-HT Depletion. To deplete 5-HT, the mice were pretreated with
the tryptophan hydroxylase inhibitor PCPA (300 mg/kg i.p.). Injec-
tions were made twice daily (at 9:00 AM and 9:00 PM), for 3 consec-
utive days. Experiments were done on the fourth day and started at
10:00 AM. The dose and schedule of PCPA treatment were adopted
from O’Leary et al. (2007).
Forced Swim Test. The experiments were performed according
to the procedure of Porsolt et al. (1977). In brief, the rats were placed
individually in glass cylinders (40 cm high, 18 cm in diameter)
containing 25 cm of water maintained at 25°C. The water column
was deep enough so that the rats could not support themselves by
placing their paws on the base of the cylinder. After 15 min, they
were removed to a drying room (30°C) for 30 min. They were placed
again in the cylinder 24 h later, and the total duration of immobility
was measured during a 5-min test. Behavioral scoring was per-
formed according to Detke et al. (1995). Three different behaviors
were rated: 1) immobility, rats were judged to be immobile when they
remained floating passively in the water; 2) swimming, rats were
judged to be swimming if they were making active swimming mo-
tions, more than necessary to solely maintain their head above
water; and 3) climbing, rats were judged to be climbing when they
were making active movements in and out of the water with their
forepaws, usually directed against the walls. The time of immobility,
swimming, and climbing was measured. AMN082 (1, 5, or 10 mg/kg)
was administered as a series of three intraperitoneal injections at 23,
5, and 1 h before the 5-min test on the second day. The first injection
was given at the end of a drying period (i.e., 30 min after removal
from the water).
Tail Suspension Test. Immobility was induced by tail suspen-
sion according to the procedure of Steru et al. (1985). C57BL/6J mice
were individually suspended by their tails by a plastic string posi-
tioned horizontally 75 cm above the tabletop by using adhesive tape
placed approximately 1 cm from the tip of the tail. The immobility
duration was recorded for 6 min. The mice were considered immobile
only when they were both hanging down passively and completely
motionless.
Locomotor Activity Test in Rats. Spontaneous locomotor ac-
tivity was recorded individually for each animal in Opto-Varimex
cages (Columbus Instruments, Columbus, OH) linked on-line to a
compatible IBM (White Plains, NY) computer. Each cage (43 ? 44 ?
25 cm) was surrounded with a 15 ? 15 array of photocell beams
located 3 cm from the floor surface. Rats were injected in their home
cages with a series of three injections of AMN082 given 23, 5, and 1 h
before the locomotor activity test. One hour after the last dose of
AMN082, the rats were placed individually into locomotor activity
chambers. Immediately afterward, locomotor activity was measured
for 30 min and recorded every 5 min. Interruptions of photobeams
resulted in horizontal activity defined as distance traveled (in cen-
timeters). The data were analyzed with Auto-Track software (Colum-
bus Instruments).
Locomotor Activity Test in Mice. The locomotor activity of
mice was measured in Plexiglas locomotor activity chambers (40 ?
20 ? 15 cm) in a 20-station photobeam activity system (Opto-M3
Activity Meter; Columbus Instruments). The animals were placed
there individually, and the total number of ambulations was re-
corded for 30 min and recorded every 5 min.
Antidepressant-Like Action of AMN082 Is Serotonin-Dependent
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Determination of 5-HT Concentration. Thirty minutes after
the TST, the mice were sacrificed by decapitation, and their frontal
cortices were dissected on an ice-cold plate, then immediately frozen
and stored at ?80°C for the tissue analysis. 5-HT was assayed in
cortical homogenates by high-performance liquid chromatography
coupled with coulometric detection. Tissue samples were weighed
and homogenized in 0.5 ml of ice-cold 0.1 M perchloric acid contain-
ing 0.05 mM ascorbic acid. After centrifugation (10,000g, 10 min),
the supernatants were filtered through 0.2-?m cellulose filters (All-
tech Associates, Deerfield, IL). Ten-microliter samples were injected
into a high-performance liquid chromatography system (Dionex Inc.,
Sunnyvale, CA) equipped with a Hypersil Gold C18 analytical col-
umn (Thermo Fisher Scientific, Waltham, MA) and the Coulochem
III detector (ESA Inc., Chelmsford, MA). The mobile phase consisted
of 50 mM citrate-phosphate buffer (pH 4.2), 0.25 mM EDTA, 0.25
mM sodium octyl sulfonate, 2.4% methanol, and 1.3% acetonitrile.
The flow rate was maintained at 0.8 ml/min. Data were collected and
chromatograms were integrated with Chromeleon 6.8 SP3 software
(Dionex Inc.). 5-HT was quantified by peak area comparisons with
standards, run on the day of analysis. The results are presented as a
nanogram of the analyzed compound per gram of weight of the brain
tissue.
Statistical Analysis. The data are presented as means ? S.E.M.
and were evaluated by one-way ANOVA followed by Dunnett’s post
hoc test (FST in rats), two-way ANOVA followed by Bonferroni’s post
hoc test (TST in mice), or the repeated-measures ANOVA followed by
Dunnett’s test (locomotor activity). Two-way ANOVA was used to
analyze 5-HT concentrations in the frontal cortex of the mice. Prism
version 4.00 for Windows 2000 (GraphPad Software Inc., San Diego,
CA) was used to analyze the data.
Results
Effect of AMN082 on the Behavior of Rats in the FST.
One-way ANOVA showed the effects of AMN082 on immo-
bility time [F(3,24) ? 8.755; p ? 0.0004] and swimming time
[F(3,24) ? 4.444; p ? 0.0128] but no effect on climbing dura-
tion [F(3,24) ? 1.257; p ? 0.3115]. Post hoc tests showed that
AMN082, given at a dose of 5 mg/kg, significantly decreased
the immobility time of rats (p ? 0.05), whereas at the dose of
10 mg/kg, AMN082 not only decreased the duration of the
immobility (p ? 0.01), but it increased swimming time (p ?
0.01; Fig. 1).
Effect of AMN082 on the Locomotor Activity of Rats.
In rats, AMN082 at a dose of 5 mg/kg did not induce changes
in locomotor activity [F(1,65) ? 0.008385; p ? 0.9284], al-
though a significant effect of time on the parameter was
observed [F(5,65) ? 40.54; p ? 0.0001] (Fig. 2). AMN082 at a
dose of 10 mg/kg significantly decreased the locomotor activ-
ity of rats (the curves were significantly different) [F(1,65) ?
25.06; p ? 0.0002]. Furthermore, there was a significant
influence of time on locomotor activity [F(5,65) ? 58.97, p ?
0.0001] and time-dose interaction [F(5,65) ? 28.50; p ?
0.0001]. Post hoc analysis revealed that AMN082 at a dose of
10 mg/kg decreased locomotor activity at 5 min (p ? 0.001)
and 10 min (p ? 0.05) (Fig. 2).
Effect of 5-HT Depletion on the Antidepressant-Like
Activity of AMN082 in the TST. AMN082, given at a dose
of 3 mg/kg, significantly decreased the immobility time of
vehicle-treated mice in the TST [F(1,26) ? 14.88; p ? 0.0007].
Two-way ANOVA also revealed significant interaction be-
tween PCPA and AMN082 [F(1,26) ? 4.373; p ? 0.0464],
showing that AMN082 was not active in the TST in 5-HT-
depleted mice (Fig. 3).
Effect of AMN082 on the Locomotor Activity of 5-HT-
Depleted and Control Mice. Two-way ANOVA showed
that PCPA-pretreated mice were less active in the locomotor
activity test than vehicle-treated animals (i.e., the curves
were significantly different) [F(1,70) ? 10.34; p ? 0.0062]. No
significant differences between groups treated with vehicle/
AMN082 (3 mg/kg) and PCPA/AMN082 (3 mg/kg) were ob-
served [F(1,70) ? 1.642; p ? 0.2209]. Statistical analysis of
the behavior of mice at 5 min revealed that AMN082 de-
creased basal locomotor activity (p ? 0.0327) (Fig. 4, inset),
Fig. 1. The effect of AMN082 (AMN; 1, 5, and 10 mg/kg i.p.) on
theimmobilitytime,climbing,andswimmingofratsintheFST.
Rats were administrated AMN082 23, 5, and 1 h before the
behavioral test. Values expressed as means ? S.E.M. were an-
alyzed by one-way ANOVA. ?, p ? 0.05; ??, p ? 0.01 versus
respective control. Veh, vehicle.
Fig. 2. The effect of AMN082 (AMN; 5 and 10 mg/kg i.p.) on the locomotor
activity of rats. Rats were administrated AMN082 23, 5, and 1 h before
the behavioral test. Values expressed as the means ? S.E.M. were eval-
uated by repeated-measures ANOVA. ?, p ? 0.05; ???, p ? 0.001 versus
respective control.
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Pałucha-Poniewiera et al.

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suggesting the decreased exploratory activity of mice. PCPA,
which attenuated the locomotor activity of mice when given
by itself [F(1,28) ? 12.98; p ? 0.0012], did not significantly
influence AMN082-induced changes in the locomotor activity
of mice at the first 5 min of the locomotor activity test
[F(1,28) ? 4.097; p ? 0.05] (Fig. 4, inset).
Verification of 5-HT Depletion after PCPA Pretreat-
ment. Two-way ANOVA showed a significant influence of
PCPA on the concentration of 5-HT in the frontal cortex of
the mice [F(1,26) ? 929.8; p ? 0.0001]. Statistical analysis
did not reveal any influence of AMN082 (3 mg/kg) on the
concentration of 5-HT in the frontal cortex (Table 1). PCPA
treatment did not cause significant changes in the level of
dopamine or noradrenaline (results not shown).
Effects of Citalopram or Reboxetine and AMN082 on
the Immobility Time of Mice in the TST. Two-way
ANOVA showed that citalopram, used in the TST at a non-
active dose of 5 mg/kg, coadministered with a nonactive dose
of AMN082 (0.3 mg/kg), induced a significant decrease in the
immobility time of mice [F(1,28) ? 19.33; p ? 0.0001] in the
TST (Fig. 5A). However, reboxetine used in the TST at a
nonactive dose of 2 mg/kg, coadministered with a nonactive
dose of AMN082 (0.3 mg/kg), did not change the behavior of
the mice in this test [F(1,27) ? 0.09886; p ? 0.7556] (Fig. 5B).
Subeffective doses of citalopram, reboxetine, and AMN082
were defined on the basis of dose-response curves, performed
in the preceding set of experiments (data not shown).
Effects of Citalopram or Reboxetine and AMN082 on
the Locomotor Activity of Mice. AMN082, at a dose of 0.3
mg/kg, did not induce changes in the locomotor activity of
mice [F(1,60) ? 0.4494; p ? 0.5153]. Citalopram, used at a
dose of 5 mg/kg, also did not influence this parameter
[F(1,60) ? 0.006506; p ? 0.9370]. The locomotor activity of
the mice injected with AMN082 (0.3 mg/kg) and citalopram (5
mg/kg) was comparable with that of the control animals (the
curves were not significantly different) [F(1,60) ? 2.254; p ?
0.1591]. No changes in the locomotor activity between all of
the groups of mice at the first time point of 5 min (p ? 0.05)
were revealed, suggesting no influence of used compounds on
the exploratory activity (Fig. 6A).
Likewise, reboxetine (2 mg/kg) [F(1,60) ? 0.6506; p ?
0.6621] or reboxetine (2 mg/kg), coadministered with
AMN082 (0.3 mg/kg) [F(1,60) ? 0.3830; p ? 0.8585], did not
change the locomotor activity of mice. Moreover, statistical
analysis of all of the groups of animals did not reveal any
changes at the first time point of 5 min (p ? 0.05) (Fig. 6B).
Effects of Antagonists of 5-HT Receptors on the An-
tidepressant-Like Activity of AMN082 in the TST. Two-
way ANOVA showed that AMN082, administered at a dose of
3 mg/kg, significantly decreased the immobility time of the
C57BL/6J mice in the TST [F(1,28) ? 19.58; p ? 0.0001]. An
antagonist of the 5-HT1A receptor, WAY100635 (0.1 mg/kg),
when given by itself, did not change the behavior of the
animals in this test, although it antagonized a AMN082 (3
mg/kg)-induced decrease in the immobility time of mice
[F(1,28) ? 6.781; p ? 0.0146] (Fig. 7A).
The 5-HT2A/2C receptor antagonist ritanserin (0.5 mg/kg)
was not active in the TST when given by itself and did not
influence the AMN082 (3 mg/kg)-induced attenuation of the
antidepressant-like activity of mice in the TST [F(1,28) ?
4.016; p ? 0.0548] (Fig. 7B).
AMN082 was not active in the TST at a dose of 0.3 mg/kg.
Fig. 3. The effect of 5-HT depletion on the antidepressant-like activity of
AMN082 (AMN) in the TST in mice. PCPA (300 mg/kg i.p.) was given
twice daily for 3 consecutive days. AMN082 (3 mg/kg) was administered
intraperitoneally 60 min before the behavioral test. Values expressed as
the means ? S.E.M. were analyzed by two-way ANOVA. ???, p ? 0.001
versus control; #, p ? 0.05 versus AMN082-treated group.
Fig. 4. The effects of AMN082 (AMN; 3 mg/kg i.p.) on the
locomotor activity of 5-HT-depleted and control mice.
PCPA (300 mg/kg i.p.) was given twice daily for 3 consec-
utive days. AMN082 (3 mg/kg) was administered 60 min
before the behavioral test. Values expressed as the
means ? S.E.M. were evaluated by repeated-measures
ANOVA. ?, p ? 0.05; ??, p ? 0.01 versus respective control
(Cont) group. veh, vehicle.
Antidepressant-Like Action of AMN082 Is Serotonin-Dependent
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Two-way ANOVA revealed that WAY100635 (0.1 mg/kg)
showed the tendency to enhance the nonactive dose of
AMN082 (0.3 mg/kg), although the effect was not statistically
significant [F(1,28) ? 3.056; p ? 0.0914] (Fig. 8).
EffectsofAntagonistsof5-HTReceptorsandAMN082on
the Locomotor Activity of Mice. AMN082, at a dose of 3
mg/kg, decreased the locomotor activity of mice [F(1,60) ?
32.81, p ? 0.0001], and a significant effect of time on the
parameter [F(5,60) ? 9.920, p ? 0.0001] was observed.
WAY100635, at a dose of 0.1 mg/kg, did not influence the
basal locomotor activity of mice [F(1,60) ? 4.722, p ?
0.0505]. Mice injected with AMN082 coadministered with
WAY100635 were statistically less active in the locomotor
activity test than the control animals (the curves were sig-
nificantly different) [F(1,60) ? 16.52, p ? 0.0016], although
their activity did not change compared with the group of mice
injected with AMN082 (the curves were not significantly differ-
ent) [F(1,60) ? 2.533, p ? 0.1374] (Fig. 9).
Statistical analysis also revealed that AMN082 decreased
basal locomotor activity at the first time point of 5 min (p ?
0.0209) (Fig. 9, inset), suggesting decreased exploratory activity
of the mice. Two-way ANOVA showed that WAY100635 did not
significantly influence AMN082-induced changes in the locomo-
toractivityofthemiceinthefirst5minofthelocomotoractivity
test [F(1,24) ? 3.329, p ? 0.0805] (Fig. 9, inset).
Similar effects were observed in the group of animals ad-
Fig. 5. The effects of citalopram (Cit) or reboxetine (Reb) and AMN082
(AMN) on the immobility time of mice in the TST. AMN082 (0.3 mg/kg
i.p.) was administered 60 min before the behavioral test. Citalopram
(5 mg/kg i.p.) and reboxetine (2 mg/kg i.p.) were given 30 min before the
test. Values expressed as means ? S.E.M. were analyzed by two-way
ANOVA. ???, p ? 0.0001 versus control.
TABLE 1
The effect of PCPA pretreatment on 5-HT content in the frontal cortex
of mice
Values are expressed as the means ? S.E.M. PCPA was administered twice daily for
3 days (300 mg/kg i.p.). On the fourth day, the TST was performed. Thirty minutes
after the test the mice were sacrificed and the frontal cortices were dissected and
frozen for tissue analysis by high-performance liquid chromatography.
Treatment
5-HT Content
Change
Vehicle PCPA
ng/g tissue%
Vehicle
AMN082
592.3 ? 18.44
563.0 ? 14.82
135.3 ? 10.90
138.2 ? 12.69
?77.0
?75.4
Fig. 6. The effects of citalopram (Cit; 5 mg/kg) (A) or reboxetine (Reb; 2
mg/kg) (B) and AMN082 (AMN; 0.3 mg/kg) on the locomotor activity of
mice. AMN082 was administered intraperitoneally 60 min before the
behavioral test, and citalopram and reboxetine were given intraperitone-
ally 30 min before the test. Values expressed as the means ? S.E.M. were
evaluated by repeated-measures ANOVA.
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Pałucha-Poniewiera et al.

Page 6

ministered AMN082 and ritanserin. Locomotor activity of
mice coadministered ritanserin and AMN082 was not signif-
icantly changed, compared with the AMN082 group of mice
[F(1,60) ? 3.057; p ? 0.1059] (Fig. 10). Moreover, ritanserin
did not influence AMN082-induced changes in the explor-
atory locomotor activity measured at the first time point of 5
min (p ? 0.7778) (Fig. 10, inset).
Discussion
The FST in rats is one of the most widely used animal
models for estimating antidepressant-like activity of new
potential ADs (Porsolt et al., 1977; Cryan et al., 2002). Here,
we used the modified version of the FST, originally intro-
duced by Detke et al. (1995), in which three specific types of
behavior, i.e., immobility, climbing, and swimming, were
measured. It has been proposed that climbing is sensitive to
catecholamine-based ADs, whereas swimming is modified by
ADs, acting via modulation of the serotonergic system, in-
cluding SSRIs (Detke et al., 1995; Page et al., 1999). In this
study, conducted for the first time on rats, it was demon-
strated that AMN082 induced a dose-dependent reduction in
their immobility and an increase in their swimming behav-
ior, whereas there were no changes in their climbing behav-
ior. This profile of action may suggest that the mechanism of
the antidepressant-like activity of AMN082 is related to the
modulation of the serotonergic system.
Our experiments showing antidepressant-like effects of
AMN082 yield paradoxical results to the data describing
the antidepressant-like phenotype of mGlu7 receptor KO
mice (Cryan et al., 2003). However, recently reported pro-
found changes in GABAergic system in mGlu7 KO mice
(Wieron ´ska et al., 2010) may explain the antidepressant-
like phenotype of mGlu7 knockouts and resolve that dis-
crepancy. Moreover, the recent data demonstrating that
pharmacological blockade of mGlu7 receptors by 6-(4-
methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-
c]pyridin-4(5H)-one (MMPIP) did not cause antidepressant
effects (Hikichi et al., 2010) support the view that it is the
stimulation of mGlu7 receptors that brings about antide-
pressant-like activity. The possibility that AMN082 may
act via additional mechanisms not related to the stimula-
tion of mGlu7 receptors also has to be considered. AMN082
causes internalization of mGlu7 receptors (Pelkey et al.,
2007) and is active in mGlu7 KO mice, producing a decrease in
locomotor activity (Pałucha et al., 2007). However, its antide-
pressant-like action is absent in mGlu7 KO mice, showing that
it is related to mGlu7 receptor stimulation.
Compounds that increase locomotor activity can often pro-
vide false positive results in the FST. We observed that
AMN082 at a dose of 5 mg/kg had no effect on the locomotor
activity of rats and there was a significant decrease of this
parameter after an injection of AMN082 at a dose of 10
mg/kg, adding validity to the antidepressant-like activity of
the drug in the FST. These results confirm our previously
published data showing AMN082-induced decreases in the
locomotor activity of mice at doses active in the FST (Pałucha
et al., 2007).
To further investigate the potential serotonergic-depen-
dent mechanism of the antidepressant-like activity of
Fig. 7. A, the effect of WAY100635 (WAY; 0.1 mg/kg s.c.) on the antide-
pressant-like activity of AMN082 (AMN; 3 mg/kg i.p.) in the TST in mice.
Mice were administered AMN082 and WAY100635 60 and 45 min, re-
spectively, before the test. Values expressed as the means ? S.E.M. were
analyzed by two-way ANOVA, ???, p ? 0.001 versus control group; #, p ?
0.05 versus AMN082-treated group. B, the effect of ritanserin (Rit; 0.5
mg/kg i.p.) on the antidepressant-like activity of AMN082 (3 mg/kg i.p.) in
the TST in mice. Mice were administered AMN082 and ritanserin 60 min
before the test. Values expressed as the means ? S.E.M. were analyzed
by two-way ANOVA.
Fig. 8. The effect of WAY100635 (WAY; 0.1 mg/kg s.c.) and AMN082
(AMN; 0.3 mg/kg i.p.) on the immobility time of mice in the TST. Mice
were administered AMN082 and WAY100635 60 and 45 min, respec-
tively, before the test. Values expressed as the means ? S.E.M. were
analyzed by two-way ANOVA.
Antidepressant-Like Action of AMN082 Is Serotonin-Dependent
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AMN082, we used the TST in the C57BL/6J mice. In this
test, mice hung by the tail develop an immobile posture and
acutely given ADs decrease the time of immobility. The TST
detects the antidepressant-like activity of not only typical
ADs drugs but also atypical and new potential ADs, such as
the mGlu receptor ligands (Cryan et al., 2005), including
mGlu7 receptor agonist (Pałucha et al., 2007).
To investigate the role of the serotonergic system in the
antidepressant-like activity of AMN082 in the TST, we used
mice after the pharmacological depletion of 5-HT by a selec-
tive inhibitor of tryptophan hydroxylase, PCPA (Koe and
Weissman, 1966). We adopted a schedule of treatment with
PCPA from O’Leary et al. (2007) obtaining identical 77%
depletion of 5-HT levels in frontal cortices. The antidepres-
sant-like effect of AMN082 was abolished by PCPA adminis-
tration, suggesting that a regular level of 5-HT in the brain is
essential to the antidepressant-like action of AMN082 in the
TST. The lack of antidepressant-like efficacy of AMN082 in
serotonin-depleted rats resembles the results of O’Leary et
al. (2007), who demonstrated that SSRIs were not active in
the TST in the PCPA-pretreated mice, and may suggest
similar mechanisms of the antidepressant efficacy of both
groups of compounds.
Locomotor activity studies showed that PCPA did not sig-
nificantly influence AMN082-induced changes in motility,
suggesting that the results of the TST studies did not result
from possible hypolocomotion of mice treated with PCPA and
AMN082.
Our experiments have shown that a selective inhibitor of
the 5-HT transporter, citalopram, combined with AMN082
(both used at low, subeffective doses) induced a significant
antidepressant-like effect in the TST. However, a selective
inhibitor of the noradrenaline transporter, reboxetine, given
at a nonactive dose, together with a nonactive dose of
AMN082, did not induce any changes in the immobility time
of mice. These results suggest a synergistic action of the SSRI
and the mGlu7 receptor agonist, indicating that both agents
act at different targets, stimulation of which leads to the
same final results. This further indicates that the serotoner-
gic system is engaged in the mechanism of action of AMN082,
whereas the noradrenergic system is not.
Locomotor activity studies showed that citalopram, rebox-
Fig. 9. The effect of WAY100635 (WAY; 0.1 mg/kg s.c.) and
AMN082 (AMN; 3 mg/kg i.p.) on the locomotor activity of
mice. AMN082 and WAY100635 were administered 60 and
45 min, respectively, before the test. Values expressed as
the means ? S.E.M. were evaluated by repeated-measures
ANOVA. ?, p ? 0.05; ??, p ? 0.01; ???, p ? 0.001 versus
respective control group.
Fig. 10. The effect of ritanserin (Rit; 0.5 mg/kg i.p.) and
AMN082 (AMN; 3 mg/kg i.p.) on the locomotor activity of
mice. AMN082 and ritanserin were administered 60 min
before the test. Values expressed as the means ? S.E.M.
were evaluated by repeated-measures ANOVA. ???, p ?
0.001 versus respective control group.
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etine, and AMN082, given alone or in combination at doses
nonactive in the TST, did not change the locomotion of mice,
ensuring the specificity of antidepressant-like effect of com-
bination of AMN082 with citalopram.
To further investigate the role of the serotonergic system
in the mechanism of the antidepressant-like activity of
AMN082, we studied the engagement of specific 5-HT re-
ceptors in the action of this drug in the TST. 5-HT1A and
5-HT2A receptors have been shown to play a crucial role in
the regulation of serotonergic neurotransmission and the
mechanism of action of several ADs, including SSRIs,
monoamine oxidase inhibitors, and tricyclics (Middlemiss
et al., 2002; Hensler, 2006). Furthermore, pretreatment
with antagonists of 5-HT1A and 5-HT2A receptors has
been shown to antagonize the action of the SSRIs in the
TST in mice (Miyata et al., 2004). Thus, we examined the
influence of the pretreatment of mice with the 5-HT1A
receptor antagonist WAY100635 (Forster et al., 1995) and
the 5-HT2A/2C receptor antagonist ritanserin (Hoyer et
al., 1994) by using TST. The action of AMN082 was inhib-
ited by WAY100635, but not by ritanserin, indicating the
involvement of 5-HT1A but not the 5-HT2/2C receptors in
the antidepressant-like action of AMN082. Direct influ-
ence of AMN082 on the serotonergic receptors can be ex-
cluded, because no significant binding interaction of 1 ?M
AMN082 with the serotonergic receptors was observed
(Mitsukawa et al., 2005).
Locomotor activity studies showed that WAY100635 or
ritanserin, given by itself, did not change the locomotion of
mice and did not significantly change AMN082-induced de-
creases in this parameter, confirming the specificity of the
observed effects.
5-HT1A receptors are localized presynaptically on seroto-
nergic neurons in the raphe, where they play the role of
autoreceptors and postsynaptically mainly on nonserotoner-
gic neurons, including glutamatergic ones (Santana et al.,
2004). Generally, postsynaptic 5-HT1A receptors exist in lim-
bic structures, including the hippocampus, frontal cortex,
entorhinal cortex, and amygdaloid complex (Hensler, 2006).
Numerous data show, that 5-HT1A receptor antagonists,
used at subeffective doses, enhance the antidepressant-like
activity of several ADs (Artigas et al., 1996). It is supposed
that such effects resulted probably from a blockade of
5-HT1A autoreceptors and a subsequent increase in 5-HT
concentration in the serotonergic synapse in the dorsal raphe
nucleus. Thus, the antagonists of 5-HT1A receptors synergis-
tically augment the 5-HT level, increased by ADs acting as
inhibitors of 5-HT transporters (Romero et al., 1996). In the
present study, we found that the 5-HT1A receptor antago-
nist, WAY100635, and the mGlu7 receptor agonist, AMN082,
both used in the TST at subactive doses, did not induce an
antidepressant-like effect, although a nonsignificant ten-
dency was observed. These data may suggest that postsyn-
aptic 5-HT1A receptors, rather then presynaptic ones, are
engaged in the mechanism of the antidepressant-like activity
of AMN082.
A large amount of data shows that an increased function of
the glutamatergic system is observed in depression (see
Pałucha and Pilc 2007; Sanacora et al., 2008; Wieron ´ska and
Pilc, 2009). Limbic structures, including the prefrontal cor-
tex, are believed to be related to emotional processes and
depressive states. Thus, it may be supposed that the activa-
tion of glutamatergic cerebral pyramidal neurons might play
an important role in depression. Therefore, AMN082, act-
ing at presynaptic mGlu7 receptors, may induce its anti-
depressant-like effect by inhibition of glutamate release.
On the other hand, prefrontal pyramidal neurons have
been shown to be inhibited by 5-HT via activation of the
inhibitory 5-HT1A receptors (Amargo ´s-Bosch et al., 2004).
Therefore, a selective blockade of 5-HT1A receptors may
antagonize the inhibitory effects of 5-HT on pyramidal
neurons, thus inducing an increased activity of these cells.
This mechanism may account for a WAY100635-induced
blockade of the antidepressant-like effect of AMN087 in
the TST. Similar data were reported earlier, when an
anxiolytic effect of ACPT-I [(1S,3R,4S)-1-aminocyclopen-
tane-1,3,4-tricarboxylic acid], a group III mGlu receptor
agonist, was antagonized by WAY100635 (Stachowicz et
al., 2009). This indicates that an interaction between
group III mGlu receptors and 5-HT1A receptors might be a
general phenomenon involved not only in depression but
also anxiety processes.
Altogether, the results of our studies show that the anti-
depressant-like action of the mGlu7 receptor-positive modu-
lator AMN082 depends on the serotonergic system activa-
tion. There is a functional interaction between mGlu7
receptor and serotonergic receptors (possibly 5-HT1A recep-
tor), which may account for the behavioral effects observed in
our experiments.
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Address correspondence to: Dr. Agnieszka Pałucha-Poniewiera, Depart-
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