Neuropharmacology 57 (2009) 415424

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Antidepressant like effect of selective serotonin reuptake inhibitors involve

modulation of imidazoline receptors by agmatine
Brijesh G. Taksande, Nandkishor R. Kotagale, Sunil J. Tripathi, Rajesh R. Ugale, Chandrabhan T. Chopde*
Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, Maharashtra 441 002, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 September 2008
Received in revised form
28 June 2009
Accepted 29 June 2009

Recent ndings demonstrated the dysregulation of imidazoline receptor binding sites in major depression and their normalization by chronic treatment with antidepressants including selective serotonin
reuptake inhibitors (SSRIs). Present study investigated the role of agmatine and imidazoline receptors in
antidepressant like effect of SSRIs and imipramine in mouse forced swimming test (FST) paradigm. The
antidepressant like effect of uoxetine or paroxetine was potentiated by imidazoline I1/I2 receptor
agonist agmatine (510 mg/kg, ip), imidazoline I1 receptor agonists, moxonidine (0.250.5 mg/kg, ip) and
clonidine (0.0150.03 mg/kg, ip), imidazoline I2 receptor agonist, 2-(2-benzofuranyl)-2-imidazoline
(510 mg/kg, ip) as well as by the drugs known to increase endogenous agmatine levels in brain viz.,
L-arginine, an agmatine biosynthetic precursor (40 mg/mouse, icv), ornithine decarboxylase inhibitor,
diuoromethyl ornithine (12.5 mg/mouse, icv), diamine oxidase inhibitor, aminoguanidine (6.5 mg/mouse,
icv) and agmatinase inhibitor, arcaine (50 mg/mouse, icv). Conversely, prior administration of I1 receptor
antagonist, efaroxan (1 mg/kg, ip), I2 receptor antagonist, idazoxan (0.25 mg/kg, ip) and arginine
decarboxylase inhibitor, D-arginine (100 mg/kg, ip) blocked the antidepressant like effect of paroxetine
(10 mg/kg, ip) and uoxetine (20 mg/kg, ip). On the other hand, antidepressant like effect of imipramine
was neither augmented nor attenuated by any of the above drugs. Mice pretreated with SSRIs but not
imipramine and exposed to FST showed higher concentration of agmatine in brain as compared to saline
control. This effect of SSRIs on agmatine levels was completely blocked by arginine decarboxylase
inhibitor D-arginine but not by imidazoline receptor antagonists, efaroxan or idazoxan. These results
demonstrate that modulation of imidazoline receptors by agmatine are implicated in the antidepressant
like effect of SSRIs and may be projected as a potential therapeutic target for the treatment of depressive
disorders.
2009 Elsevier Ltd. All rights reserved.

Keywords:
SSRI
Imidazoline receptors
Agmatine
Depression
5-HT

1. Introduction
Selective serotonin reuptake inhibitors (SSRIs) such as uoxetine, paroxetine and citalopram are widely used in the treatment of
psychiatric disorders including depression, obsessive compulsive
disorder, panic disorder and several other conditions (Eriksson
et al., 1995; Steiner et al., 1995; Su et al., 1997; Masand et al., 2005;
Duman et al., 2006). SSRIs act by inhibiting brain serotonin transporters (SERT) leading to enhanced serotonergic transmission in
the several subcortical regions (Stanford, 1996; Gourion et al.,
2004). However, the mechanism through which they exert antidepressant effect remains uncertain (Gourion et al., 2004). Indeed,
it has been argued that antidepressive efcacy of SSRIs may involve
modulation of other neurotransmitters and/or receptor systems

* Corresponding author. Tel.: 91 7109 288650; fax: 91 7109 287094.

E-mail address: chopdect@hotmail.com (C.T. Chopde).
0028-3908/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2009.06.035

along with their direct effect on serotonergic signaling (Stanford,

1996; Tunnicliff et al., 1999; Gourion et al., 2004). There is accumulative evidence that favors the involvement of imidazoline
receptor binding sites in the pathophysiology of depression
(Garcia-Sevilla et al., 1996, 1998; Piletz et al., 1996a, 2008; Zhu et al.,
1999; Halaris and Piletz, 2001; Holt, 2003) and these sites have
been proposed as an novel therapeutic targets for the treatment of
depressive disorders (Holt, 2003).
Imidazoline receptors are the unique non-adrenergic high
afnity binding sites that exist in three major subclasses (I1, I2 and I3)
based upon their ligand selectivity, subcellular distribution
and physiological functions (Michel and Insel, 1989; Michel and
Ernsberger, 1992; Parini et al., 1996; Eglen et al., 1998; Bousquet et al.,
2000; Santos et al., 2005). In human brain, I1 sites are distributed in
a regional manner with highest density in striatum, pallidum and
gyrus dentatus of hippocampus, amygdala and susbstantia nigra (De
Vos et al., 1994). The imidazoline I2 binding sites (I2A and I2B) are
allosteric, widely distributed in brain and located on monoamine

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B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

oxidase (MAO) (Tesson et al., 1991; Raddatz et al., 1997, 2000; Eglen
et al., 1998) an enzyme that causes oxidative deamination of
neurotransmitters like serotonin (5-hydroxy tryptamine, 5-HT),
noradrenaline, dopamine and exogenous amines. Several imidazoline I2 ligands have been shown to inhibit MAO activity (Carpene
et al., 1995; Raasch et al., 1999; Jones et al., 2007) and chronic
treatment with MAO inhibitors (like clorgyline and pargyline)
downregulate I2 receptor density (Alemany et al., 1995). Previous
studies have shown that major depression is associated with dysregulation of imidazoline receptors (Garcia-Sevilla et al., 1996, 1998;
Halaris and Piletz, 2001; Piletz et al., 2008, 1994; Sastre et al., 1995) in
human brain. Meanwhile, some studies in human have demonstrated lowered or normalized I1 binding sites in platelets of
depressed patients treated with either of antidepressants imipramine, desipramine, uoxetine, citalopram, clomipramine (Piletz
et al., 1996a,b; Garcia-Sevilla et al., 1996) or bupropion (Zhu et al.,
1999; Halaris et al., 2002). However, none of these antidepressants
interact directly with I1 imidazoline receptors at therapeutic
concentration (Holt, 2003). Subsequently, numerous imidazoline I1/
I2 ligands like agmatine, BU-224 (2-(4, 5-dihydroimidazole-2-yl)
quinoline hydrochloride), harmane and b-carboline have been
reported to exert antidepressant like activity in rodents (Abramets
and Dolzhenko, 1986; Adell et al., 1996; Finn et al., 2003; Farzin and
Mansouri, 2006; Zomkowski et al., 2002). Recently, agmatine a novel
neurotransmitter has gained attention in depressive disorders.
Agmatine is synthesized following decarboxylation of L-arginine by
arginine decarboxylase (ADC) in brain and other tissues. L-arginine is
also converted into ornithine and nitric oxide by enzyme arginase
and nitric oxide synthase, respectively (Reis and Regunathan, 2000).
Ornithine subsequently converted into putrescine by L-ornithine
decarboxylase. Agmatine is also metabolized to putrescine and
guanido-butanoic acid by an enzyme agmatinase and diamine
oxidase respectively (Reis and Regunathan, 2000; Lu et al., 2003;
Huang et al., 2003; Regunathan, 2006).
Besides imidazoline receptors, agmatine also binds to a2-adrenoceptors, N-methyl D-aspartate (NMDA) receptors as well as 5-HT
receptors with lower afnity and inhibit nitric oxide synthase
(NOS) in brain as well (Yang and Reis, 1999; Reis and Regunathan,
2000; Raasch et al., 2001). Exogenous administration of agmatine
have been shown to produce not only antidepressant effect (Zomkowski et al., 2002; Li et al., 2003) but also possesses antinociceptive (Onal et al., 2004), anxiolytic (Lavinsky et al., 2003),
anticonvulsant (Bence et al., 2003), antiinammatory (Satriano
et al., 2001), antiproliferative (Regunathan and Reis, 1997), neuroprotective (Olmos et al., 1999) properties and modulates morphine
tolerance or dependence (Kolesnikov et al., 1996; Aricioglu-Kartal
and Uzbay, 1997; Li et al., 1998; Wu et al., 2007). It is worth noting
that agmatine induced effects including antidepressant action are
said to be mediated through its selective interaction with imidazoline receptors (Zeidan et al., 2007).
Thus, it is a matter of investigation whether antidepressant like
effect of SSRIs involve imidazoline receptors. It is speculated that
SSRIs may modulate release of some endogenous mediators to
interact with imidazoline I1 and I2 receptors. In this study, we
determined the effect of imidazoline receptor agents and drugs that
alter the brain levels of agmatine on the antidepressant like effect
of SSRIs (uoxetine and paroxetine) and prototype typical antidepressant (imipramine) using mouse forced swimming test (FST).
2. Materials and methods
2.1. Subjects
Male Swiss albino mice (2025 g body weight) were group housed in perspex
cages (ve per cage) maintained on a 12 h light/dark cycle (lights on at 07.00 h) in
a room at controlled temperature (24  1  C) with free access to food pellets

(Hindustan Lever Ltd., Mumbai) and water. Animals were handled and acclimatized
to laboratory conditions at least 12 h prior to experiment. All experiments were
conducted between 0900 and 1500 h. The experiments were executed in strict
accordance with the ethical principles and guidelines given by Committee for the
Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forest, Govt. of India and approved by the Institutional Animal Ethical
Committee. Every possible effort was made to reduce the suffering of animals.

2.2. Drug solutions and administration

Agmatine sulfate, moxonidine hydrochloride, clonidine hydrochloride, DL-adiuoromethyl ornithine hydrochloride (DFMO), aminoguanidine hemisulfate,
arcaine sulfate, efaroxan hydrochloride, idazoxan hydrochloride, L-arginine monohydrochloride and D-arginine monohydrochloride were purchased from Sigma
Aldrich Co., USA. 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) was
purchased from Tocris Biosciences, UK. Fluoxetine, imipramine (Sun Pharmaceuticals, Vadodara, India) and paroxetine (Dr. Reddys Laboratory, Hyderabad, India)
were received as gift samples.
Agmatine, moxonidine, clonidine, 2-(2-benzofuranyl)-2-imidazoline, efaroxan,
idazoxan and D-arginine were dissolved in 0.9% saline and administered by intraperitoneal (ip) route. DFMO, arcaine, aminoguanidine and L-arginine were injected
by intracerebroventricular (icv) route to alter the levels of brain agmatine and avoid
peripheral effects. For icv administration of drugs, dilutions were made with articial cerebrospinal uid (aCSF) of following composition 0.2 M NaCl, 0.02 M
NaH2CO3, 2 mM KCl, 0.5 mM KH2PO4, 1.2 mM CaCl2, 1.8 mM MgCl2, 0.5 mM Na2SO4,
and 5.8 mM D-glucose.
Doses employed in the protocols were selected on the basis of our preliminary
experiments and available literature (Cervo and Samanin, 1991; Lu et al., 2003; Su
et al., 2003; MacInnes and Handley, 2003; Inan et al., 2004; Gentili et al., 2006;
Zeidan et al., 2007; Velloso et al., 2008).

2.3. Intracerebroventricular administration

For icv administration of drugs, mice were anaesthetized with pentobarbital
sodium (60 mg/kg, ip) and unilateral cannula was implanted stereotaxically (David
Kopf Instruments, CA, USA) as described earlier (Hirani et al., 2002; Ugale et al.,
2004). Briey, 28 gauge stainless steel guide cannula (C315 G/Spc, Plastic One Inc.,
Virginia, USA) was implanted into right lateral ventricle [coordinates: AP 
0.22 mm; ML 1 mm and DV 2.5 mm; relative to bregma, Paxinos and Franklin,
1997] and secured in place by dental cement (Dental Products of India, Mumbai)
afxed to two stainless steel screws. A stainless steel dummy cannula was used to
occlude the guide cannula when not in use. The animals were then housed
individually and allowed to recover for 1 week, before being tested in FST.
Oxytetracycline injection (25 mg/kg, im, Pzer Ltd., Chennai) was given and
neosporin ointment (Burroughs Wellcome Ltd., Mumbai) was applied topically for
3 days post surgery to avoid infection. During this period animals were habituated
to the experimental protocols to minimize nonspecic stress. Mice were then
assigned to different treatment groups (n 68) and injections (5 ml/mouse) were
made into right lateral ventricle over 1 min period with microliter syringe
(Hamilton, Nevada, USA) connected to 30 gauge internal cannula (C315 I/spc) by
polyethylene tubing. The injection cannula was left in place for further 1 min
before being slowly withdrawn to avoid back ow. At the end of all icv experiments, dilute India ink was injected by icv route and animals were euthanized by
an overdose of pentobarbital sodium. Immediately, the brain was dissected out
and the cannula placement was veried histologically by distribution of dilute
India ink in the ventricle. In some animals (<20%), guide cannula was found to be
placed incorrectly and hence excluded from the study. Data from only those
animals showing uniform distribution of ink into lateral ventricle was used for
statistical analysis.

2.4. Forced swimming test (FST)

The procedure was quite similar to that described by Porsolt et al. (1977)
except that mice were subjected to a pretest session to maintain consistency in
the basal immobility time between different groups. Briey, mice were placed
individually in plexiglass cylinders (21 cm height  12 cm internal diameter)
containing fresh water upto a height of 9 cm at 25  1  C and forced to swim for
15 min. Twenty four hours later, the animals were randomly divided into different
groups (6-8 animals/group) and treated with either a drug (test group) or vehicle
(control group). Each mouse was again forced to swim in a similar environment
for the period of 6 min in a test session and immobility time was measured by
the trained observer blind to the treatment. A mouse was judged to be immobile
when it remained oating motionless in the water, making only necessary
movements to keep its head above water. Each mouse was used only once in test
session. Reduction in the duration of immobility was considered as antidepressant like effect of the drug. Forced swimming tests were conducted 30 min after
the administration of drugs.

B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

2.5. Dose specic effect of SSRIs, imipramine and imidazoline receptor ligands
on immobility time in FST
This experiment examined the dose dependent effect of SSRIs, imipramine and
imidazoline receptor agonists/antagonists on immobility time in FST. Different
group of mice (n 6) were administered with various doses of either uoxetine
(2.520 mg/kg, ip) or paroxetine (2.520 mg/kg, ip) or imipramine (2.515 mg/kg,
ip) or saline. Thirty min later each mouse was subjected to FST for 6 min and the
duration of immobility was measured.
In separate group of experiments, mice (n 6) were treated with either moxonidine
(0.251 mg/kg, ip, I1 agonist), clonidine (0.0150.06 mg/kg, ip, I1 agonist), agmatine
(520 mg/kg, ip, an endogenous I1/I2 agonist), 2-BFI (515 mg/kg, ip, I2 agonist),
efaroxan (12 mg/kg, ip, I1 antagonist), idazoxan (0.251 mg/kg, ip, I2 antagonist) or
saline and after 30 min immobility time was measured in the test session.
2.6. Inuence of imidazoline receptor agonists on SSRIs and imipramine induced
antidepressant like effect
This experiment examined the antidepressant like effect of SSRIs and imipramine in animals pretreated with I1/I2 receptor agonists. For combination studies
submaximal or ineffective doses of drugs were used. Different groups of mice were
pretreated with either moxonidine (0.25 or 0.5 mg/kg, ip, I1 agonist), clonidine
(0.015 or 0.03 mg/kg, ip, I1 agonist), agmatine (5 or 10 mg/kg, ip, an endogenous I1/I2
agonist), 2-BFI (5 or 10 mg/kg, ip, I2 agonist) or saline. Thirty min following these
treatments, all animals received ineffective dose (2.5 mg/kg, ip) of either uoxetine,
paroxetine, imipramine or saline and the antidepressant effect was measured for
6 min test session in FST.
2.7. Inuence of imidazoline receptor antagonists on antidepressant like effect
of SSRIs and imipramine
Mice were injected with efaroxan (1 mg/kg, ip, I1 antagonist) or idazoxan
(0.25 mg/kg, ip, I2 antagonist) or saline 30 min prior to administration of maximal
effective dose of either uoxetine (20 mg/kg, ip), paroxetine (10 mg/kg, ip), imipramine (15 mg/kg, ip) or saline. Forced swimming tests were conducted as
mentioned earlier.
2.8. Inuence of imidazoline receptor antagonists on synergistic antidepressant
like effect of SSRIs and agmatine
In order to substantiate the involvement of endogenous agmatine and subsequent modulation of imidazoline receptors in the antidepressant like effect of SSRIs,
mice were pretreated with either saline, efaroxan (1 mg/kg, ip, I1 antagonist) or
idazoxan (0.25 mg/kg, ip, I2 antagonist) 30 min prior to administration of combined
doses of agmatine (5 mg/kg, ip) with uoxetine (2.5 mg/kg, ip) or paroxetine
(2.5 mg/kg, ip) or saline. Thirty min later mice were subjected to FST for 6 min and
the duration of immobility was measured. We used the doses of antidepressant that
were ineffective by themselves if administered alone but their combination resulted
into synergistic response.
2.9. Effect of agmatine modulators on antidepressant like effect of SSRIs
We examined the inuence of drugs that induces or inhibit enzymes involved in
agmatine synthesis or catabolism leading to altered brain agmatine levels. Different
group of mice were injected with agmatine precursor L-arginine (40 mg/mouse, icv),
DFMO (12.5 mg/mouse, icv), an ornithine decarboxylase inhibitor, aminoguanidine
(6.5 mg/mouse, icv), a diamine oxidase inhibitor, arcaine (50 mg/mouse, icv), an
agmatinase inhibitor or aCSF (icv) or saline (ip) 30 min before ineffective dose
(2.5 mg/kg, ip) of uoxetine, paroxetine or saline (ip). Thirty min later each mouse
was subjected to FST for 6 min and the duration of immobility was measured. DFMO
has been shown to inhibit conversion of L-arginine to ornithine and subsequently to
putrescine (Lu et al., 2003). Aminoguanidine and arcaine have been demonstrated to
inhibit metabolism of endogenous agmatine to guanido-butanoic acid and putrescine respectively (Lu et al., 2003; Regunathan, 2006).
In separate set of experiment, mice received D-arginine, an arginine decarboxylase (ADC) inhibitor (Rosenfeld and Roberts, 1976; Hao et al., 2005) or saline 30 min
prior to effective dose of uoxetine (20 mg/kg, ip), paroxetine (10 mg/kg, ip) or
saline. Thirty min later each mouse was subjected to FST for 6 min to assess the
duration of immobility.
Different group of mice were injected with D-arginine (100 mg/kg, ip) or saline
30 min prior to administration of agmatine precursor L-arginine (40 mg/mouse, icv),
DFMO (12.5 mg/mouse, icv), aminoguanidine (6.5 mg/mouse, icv), arcaine (50 mg/
mouse, icv) or aCSF (icv) before being subjected to FST.
2.10. Analysis of brain agmatine levels
As described above, mice treated with SSRIs (Experiment 2.5), imidazoline
receptor antagonists plus SSRIs (Experiment 2.7), brain agmatine enhancers alone or
D-arginine (arginine decarboxylase inhibitor) plus agmatine enhancers or SSRIs

417

(Experiment 2.9) were considered for brain agmatine analysis. Immediately after
completion of 6 min forced swimming test mice were euthanized by an overdose of
pentobarbital sodium, brain tissues (excluding cerebellum and brain stem) were
dissected out on an ice-cold petridish and agmatine levels in brain were measured by
HPLC, based on the methods reported earlier (Zhao et al., 2002; Roberts et al., 2005).
Briey, brain was homogenized with 150 ml HCl solution (0.1M) using tissue
grinder and the homogenate was sonicated for 10 min on ice and centrifuged
(6000  g for 10 min) at 4  C. The supernatant was transferred to a vial containing
30 ml of trichloroacetic acid solution (30% w/v). The solution was kept on ice for
60 min and again centrifuged at 6000  g for 10 min at 4  C (Zhao et al., 2002). The
supernatant was removed and stored at 80  C until used for further derivatization
and HPLC analysis (Roberts et al., 2005). For the precolumn derivatization of
agmatine, 50 ml of prepared supernatant was transferred to a reaction vial to which
100 ml of borate buffer (pH 9.4), 40 ml of NaCN solution (0.025 M) and 100 ml of
naphthalene dicarboxaldehyde (0.05 M in methanol) was added and set aside for
20 min at room temperature to produce highly stable and uorescent derivative of
agmatine with naphthalene dicarboxaldehyde. 10 ml of this derivatized mixture was
injected into 4.6 mm Nucleosil C8 10 mm HPLC cartridge and eluted with 80%
acetonitrile (ACN) in a phosphate buffer (pH 6.81) at a ow rate of 1.5 ml/min.
Fluorescence was recorded using an excitation wavelength of 249 nm and an
emission wavelength of 466 nm. Concentration of agmatine in brain was calculated
by using external standards.
2.11. Open eld test in mice
This test was performed to assess whether drug effect on immobility was
associated with changes in motor activity. The apparatus consisted of circular base
(84 cm diameter, 30 cm high wall) with three concentric circles of 14, 28 and 42 cm
radius, divided into 36 segments by radiating lines drawn from the center. Illumination was identical to that used for forced swim test. Animals were placed individually into the center of arena and the ambulations (partitions crossed with all
four paws) and rearings (number of times mouse stood on its hind limbs) within
5 min test period were recorded 30 min after ip/icv dosing of drugs or vehicle. The
doses and treatment schedules were identied as described earlier.
2.12. Data analysis
Data presented in Tables 1 and 2 was analyzed by one way analysis of variance
(ANOVA) followed by post hoc Dunnetts test. For multiple comparisons two way
ANOVA followed by Bonferroni test was utilized. Data was expressed as a mean  SEM
and value of P < 0.05 was considered to be statistically signicant in all the cases.

3. Results
3.1. Dose specic effect of antidepressants and imidazoline
receptor agents in FST
As shown in Table 1, SSRIs (520 mg/kg, ip) or imipramine
(515 mg/kg, ip) produced signicant dose dependent reduction in
the duration of immobility in mice exposed to FST. At higher doses
uoxetine (20 mg/kg, ip), paroxetine (10 mg/kg, ip) and imipramine

Table 1
Dose specic effect of SSRIs and imipramine on immobility time in mouse forced
swimming test.
Treatment

Dose (mg/kg, ip)

Immobility time (sec)  SEM

Saline
Fluoxetine

0
2.5
5
10
20
2.5
5
10
20
2.5
5
10
15

233.8
221.8
215.5
211.3
100.5
229.8
214.8
141.7
138.0
228.5
215.3
212.0
106.3

Paroxetine

Imipramine















4.54
3.270
2.825*
6.791$
4.403#
4.362
3.572*
2.848#
5.568#
3.905
3.756*
6.367*
4.310#

Mice were treated intraperitoneally with saline, uoxetine, paroxetine and imipramine.
Thirty min thereafter animals were subjected to test session. Each value represents mean  SEM immobility time in seconds (n 6). *p < 0.05, $p < 0.01,
#
p < 0.001 as compared with saline treated group (Dunnetts test).

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B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

Table 2
Dose specic effect of imidazoline receptor ligands on immobility time in mouse
forced swimming test.
Treatment

Dose (mg/kg, ip)

Immobility time (sec)  SEM

Saline
Agmatine

0
5
10
20
0.25
0.5
1
0.015
0.03
0.06
5
10
15
1
1.5
2
0.25
0.5
1

233.8
229.0
219.5
191.3
232.8
227.3
198.2
228.5
221.7
179.7
224.8
219.2
184.5
230.3
236.2
232.5
228.7
229.0
231.5

Moxonidine

Clonidine

2-BFI

Efaroxan

Idazoxan





















4.542
3.821
5.136
4.702#
2.676
4.088
3.449#
4.372
3.930
5.530#
4.293
5.224
6.869#
2.929
4.078
6.479
3.293
4.274
4.836

Mice were treated intraperitoneally with saline, agmatine, moxonidine, clonidine,

2-BFI, efaroxan and idazoxan and thirty min thereafter subjected to test session.
Each value represents mean  SEM immobility time in seconds (s) (n 6).
#
p < 0.001 as compared with saline treated group (Dunnetts test).

the antidepressant like effect elicited by effective dose of uoxetine

(20 mg/kg, ip) or paroxetine (10 mg/kg, ip) in FST [Two way ANOVA:
Factor antidepressants treatment F(3, 72) 519.4, p < 0.0001, Factor
imidazoline antagonist treatments F(2, 72) 250.8, p < 0.0001
and interaction antidepressant treatment  imidazoline antagonists
treatment F(6, 72) 88.25, p < 0.0001]. However, the effect of
imipramine (15 mg/kg, ip) was not altered by any of these agents
(p > 0.05). Administration of the efaroxan or idazoxan to saline treated
animals did not evoke any response at the doses used here.
3.4. Effect of imipramine, SSRIs alone and in presence of
imidazoline receptor antagonists on brain agmatine content
As shown in Fig. 2B, following uoxetine (20 mg/kg, ip) or
paroxetine (10 mg/kg, ip) administration brain agmatine concentration was signicantly increased as compared to saline treated
animals exposed to FST (p < 0.001). In contrast, imipramine (15 mg/
kg, ip) failed to produce any effect (p > 0.05). Two way ANOVA followed by Bonferroni test interestingly revealed that this effect of
SSRIs could not be attenuated by prior administration of imidazoline
receptor antagonist, efaroxan (1 mg/kg, ip) or idazoxan (0.25 mg/kg,
ip) [Two way ANOVA: Factor antidepressant treatment F(3,
60) 403.5, p < 0.0001, Factor imidazoline antagonist treatments
F(2, 60) 0.6721, p > 0.05 and interaction antidepressants
treatment  imidazoline antagonists treatments F(6, 60) 0.5085,
p > 0.05].

(15 mg/kg, ip) shortened immobility time to maximum extent as

compared to saline treated animals [one way ANOVA: F(30,
185) 65.66, p < 0.0001]. Their lower dose (2.5 mg/kg) however,
did not affect duration of immobility signicantly.
Similarly (Table 2), moxonidine (1 mg/kg, ip, I1 agonist), clonidine (0.06 mg/kg, ip, I1 agonist), 2-BFI (15 mg/kg, ip, I2 agonist) and
agmatine (20 mg/kg, ip, an endogenous I1/I2 agonist) at higher
doses also signicantly reduced the duration of immobility
[p < 0.0001] as compared to saline treated animals. However, their
effect at lower doses was not signicantly different from control
group (p > 0.05). On the other hand, imidazoline receptor antagonists efaroxan (12 mg/kg, I1 antagonist) and idazoxan
(0.251 mg/kg, I2 antagonist) failed to exhibit antiimmobility effect
at any dose level used here as compared to saline treated animals.

As depicted in Fig. 3, pretreatment with either efaroxan (1 mg/

kg, ip) or idazoxan (0.25 mg/kg, ip) blocked synergistic antidepressant like effect induced by combination of agmatine (5 mg/kg,
ip) with uoxetine (2.5 mg/kg, ip) or paroxetine (2.5 mg/kg, ip)
[Two way ANOVA: Factor SSRIs agmatine combination F(5,
90) 61.66, p < 0.0001, Factor imidazoline antagonist treatments
F(2, 90) 27.65, p < 0.0001 and interaction SSRI agmatine
combination x imidazoline antagonists treatments F(10,
90) 10.53, p < 0.0001].

3.2. Imidazoline receptor agonists potentiated

antidepressant like effect of SSRIs

3.6. Increased brain agmatine levels augmented

antidepressant like effect of SSRIs

Fig. 1 depicts the effects of a combination of ineffective doses of

SSRIs or imipramine and imidazoline I1 or I2 receptor agonists.
At the given doses combination of the moxonidine (0.25 or 0.5 mg/
kg, ip, I1 agonist), clonidine (0.015 or 0.03 mg/kg, ip, I1 agonist),
2-BFI (5 or 10 mg/kg, ip, I2 agonist) or agmatine (5 or 10 mg/kg, ip,
I1/I2 agonist) with uoxetine (2.5 mg/kg, ip) or paroxetine (2.5 mg/
kg, ip) resulted in a signicant antidepressant like effect [Two way
ANOVA: Factor antidepressant treatment F(3, 180) 517.9,
p < 0.0001, Factor imidazoline agonist treatment F(8, 180) 36.99,
p < 0.0001 and interaction antidepressant treatment  imidazoline agonists treatment F(24, 180) 10.00, p < 0.0001]. In contrast,
combination of imidazoline receptor agonists with imipramine
(2.5 mg/kg, ip) failed to reveal a signicant interaction (p > 0.05).
The administration of moxonidine, clonidine, 2-BFI, agmatine and
antidepressants at the doses used here alone did not produce
signicant effect as compared to saline treated group (p > 0.05).

As shown in Fig. 4, animals injected with drugs that augment

brain agmatine content, 30 min before ip administration of ineffective dose (2.5 mg/kg, ip) of uoxetine or paroxetine resulted in
signicant antiimmobility effect. These agents include DFMO, an
ornithine decarboxylase inhibitor (12.5 mg/mouse, icv), aminoguanidine, a diamine oxidase inhibitor, (6.5 mg/mouse, icv), arcaine,
an agmatinase inhibitor (50 mg/mouse, icv) and agmatine precursor,
L-arginine (40 mg/mouse, icv). Two way ANOVA followed by Bonferroni test revealed signicant decrease in immobility time [Two
way ANOVA: Factor SSRI treatments F(2, 165) 210.2, p < 0.0001,
Factor agmatine enhancers F(4, 165) 49.30, p < 0.0001 and
interaction SSRI treatments  agmatine enhancers F(8, 165)
14.88, p < 0.0001]. DFMO or aminoguanidine or arcaine or L-arginine per se failed to alter immobility time as compared to saline
(p > 0.05).

3.5. Imidazoline receptor antagonists attenuated synergistic

antidepressant like effect of SSRIs and agmatine

3.3. Imidazoline receptor antagonists blocked the

antidepressant like effect of SSRIs

3.7. Effect of agmatine enhancers alone and in presence

of D-arginine, an arginine decarboxylase inhibitor
on brain agmatine content

As depicted in Fig. 2A, pretreatment of mice with efaroxan (1 mg/

kg, ip, I1 antagonist) or idazoxan (0.25 mg/kg, ip, I2 antagonist) blocked

As shown in Fig. 5, pretreatment of mice with agmatine

precursor L-arginine (40 mg/mouse, icv) or an ornithine

B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

419

Fig. 1. Effect of (A) moxonidine (0.25 or 0.5 mg/kg, ip), (B) clonidine (0.015 or 0.03 mg/kg, ip), (C) 2-BFI (5 or 10 mg/kg, ip) and (D) agmatine (5 or 10 mg/kg, ip) on antidepressant
like effect of imipramine, uoxetine or paroxetine. Moxonidine, clonidine, 2-BFI, agmatine or saline were administered 30 min prior to antidepressants. Each bar represent the mean
immobility time  SEM (n 6). # *p < 0.001 when compared with their respective control group (Bonferroni multiple comparison test).

decarboxylase inhibitor, DFMO (12.5 mg/mouse, icv), or diamine

oxidase inhibitor, aminoguanidine (6.5 mg/mouse, icv) or an
agmatinase inhibitor, arcaine (50 mg/mouse, icv) considerably
enhanced the brain agmatine content as compared to saline treated
animals when exposed to FST (p < 0.001). Pretreatment with Darginine (100 mg/kg, ip) which inhibits the conversion of L-arginine
to agmatine was utilized in order to determine whether it also
blocks the above drug induced agmatine levels. The L-arginine,
DFMO, arcaine and aminoguanidine evoked increase in brain
agmatine contents were signicantly suppressed following D-arginine (100 mg/kg, ip) administration 30 min prior to these agents
(Two way ANOVA: Factor agmatine enhancer F(4, 50) 7.287,
p < 0.001, Factor D-arginine treatment F(1, 50) 96.65, p < 0.0001
and interaction agmatine enhancer  D-arginine treatment
F(4, 50) 3.753, p < 0.05).

3.9.

3.8. Agmatine synthesis inhibitor, D-arginine blocked SSRIs

antidepressant like effect

None of the above mentioned treatments (I1/I2 agonist, antagonist or antidepressant) alone or in combination at different doses
produced signicant effect on ambulations and rearings in OFT as
compared to controls. In control animals the total mean  SEM
(n 6) count of ambulations and rearings was 78.67  4.53 and
8.83  0.60 respectively (results not shown).

Pretreatment of animals with D-arginine (100 mg/kg, ip) 30 min

prior to SSRIs injection signicantly blocked the uoxetine (20 mg/
kg, ip) or paroxetine (10 mg/kg, ip) induced antiimmobility effect
[Two way ANOVA: Factor SSRI treatments F(2, 30) 103.5,
p < 0.0001, Factor D-arginine treatment F(1, 30) 324.5,
p < 0.0001 and interaction SSRI treatments  D-arginine treatment
F(2, 30) 69.06, p < 0.0001]. The administration of D-arginine
alone to saline treated animals did not show any response in FST at
the dose used here. These results are shown in Fig. 6A.

D-Arginine

blocked agmatine increase induced by SSRIs

As shown in Fig. 6B, administration of uoxetine (20 mg/kg, ip)

or paroxetine (10 mg/kg, ip) to mice 30 min prior to FST, produced
marked increase in basal brain agmatine concentration. This effect
of SSRIs was signicantly reduced by pretreatment with D-arginine
(100 mg/kg, ip) 30 min prior to uoxetine or paroxetine injection
[Factor SSRI treatments F(2, 30) 85.55, p < 0.0001, Factor
D-arginine treatment F(1, 30) 41.72, p < 0.0001 and interaction
SSRI treatments  D-arginine treatment F(2, 30) 10.41,
p < 0.0001]. D-arginine to saline treated animals did not alter basal
brain agmatine levels.
3.10. Inuence of drug treatments on ambulations
and rearings in open eld test (OFT)

4. Discussion
Although the antidepressive effect of SSRIs is explained on the
basis of their ability to enhance synaptic serotonin concentration
(Stanford, 1996; Tunnicliff et al., 1999; Gourion et al., 2004), other

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B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

Fig. 4. Effect of (A) DFMO (12.5 mg/mouse, icv), aminoguanidine (6.5 mg/mouse, icv),
arcaine (50 mg/mouse, icv) and L-arginine (40 mg/mouse, icv) on antidepressant like
effect of uoxetine (2.5 mg/kg, ip) and paroxetine (2.5 mg/kg, ip). Each bar represents
the mean immobility time recorded for 6 min in FST  SEM (n 12). * #p < 0.001 when
compared with their respective control group (Bonferroni multiple comparison test).

Fig. 2. (A) Effect of efaroxan (1 mg/kg, ip) and idazoxan (0.25 mg/kg, ip) on antidepressant like effect of imipramine, uoxetine and paroxetine. Each bar represents the
mean immobility time recorded for 6 min in FST  SEM (n 7). * # $p < 0.001 when
compared with their respective positive control group (Bonferroni multiple comparison test). (B) Effect of efaroxan (1 mg/kg, ip) or idazoxan (0.25 mg/kg, ip) on brain
agmatine contents of mice pretreated with imipramine, uoxetine or paroxetine and
subjected to FST. Each bar represents the mean agmatine levels (ng/gm wet
weight)  SEM (n 6). *p < 0.001 when compared with their respective control group
(Bonferroni multiple comparison test).

Fig. 3. Effect of efaroxan (1 mg/kg) and idazoxan (0.25 mg/kg) on the response to
combined ip administration of agmatine (5 mg/kg) and uoxetine (2.5 mg/kg) or
paroxetine (2.5 mg/kg). Each bar represents the mean immobility time recorded for
6 min in FST  SEM (n 6). # *p < 0.001 when compared with their respective control
group (Bonferroni multiple comparison test).

mechanisms may also account for their actions. The key observation
of this study is that antidepressant effect of SSRIs is derived from an
interaction of endogenous agmatine with imidazoline receptors in
brain. Clinical depression in human is associated with dysregulation
or overexpression of imidazoline receptors (Garcia-Sevilla et al.,
1996; Piletz et al., 1996b, 2000, 2008) in platelets and brain tissues
of depressed patients (Garcia-Sevilla et al., 1996; Zhu et al., 1999;
Halaris and Piletz, 2001; Holt, 2003; Piletz et al., 2008). Moreover,
chronic treatment with several antidepressants like uoxetine,
citalopram, desipramine, imipramine or bupropion normalized the
functioning of imidazoline receptors (Garcia-Sevilla et al., 1996;
Piletz et al., 1996b; Zhu et al., 1999; Halaris et al., 2002). In addition,
agmatine and other endogenous ligands of imidazoline receptors
(Harmane and bcarboline) produced antidepressant like effect in
animal models of depression (Zomkowski et al., 2002; Li et al., 2003;
Farzin and Mansouri, 2006). Thus, it is conceivable that imidazoline
receptors may be potential target for antidepressant action of
several drugs. In our study, the antiimmobility effects of uoxetine
or paroxetine were dose dependently augmented by ineffective
doses of agmatine, an endogenous ligand at imidazoline I1/I2

Fig. 5. Effect of D-arginine (100 mg/kg, ip) on brain agmatine content of mice pretreated with DFMO (12.5 mg/mouse, icv), aminoguanidine (6.5 mg/mouse, icv), arcaine
(50 mg/mouse, icv) and L-arginine (40 mg/mouse, icv) and subjected to FST. Each bar
represents the mean agmatine levels (ng/gm wet weight)  SEM (n 6). *p < 0.001 Vs
saline; # @ $ 3p<0.001 Vs respective control (Bonferroni multiple comparison test).

B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

Fig. 6. (A) Effect of D-arginine (100 mg/kg, ip) on antidepressant like effect of uoxetine (20 mg/kg, ip) and paroxetine (10 mg/kg, ip). Each bar represents the mean
immobility time (sec) recorded for 6 min in FST  SEM (n 6). (B) Effect of D-arginine
(100 mg/kg, ip) on brain agmatine content of mice pretreated with uoxetine (20 mg/
kg, ip) and paroxetine (10 mg/kg, ip) and subjected to FST. Each bar represents
the mean agmatine levels (ng/gm wet weight)  SEM (n 6). *p < 0.001 Vs saline;
# @
p < 0.001 Vs respective control (Bonferroni multiple comparison test).

receptors and moxonidine or clonidine, mixed imidazoline I1/a2

receptor agonists or 2-BFI, imidazoline I2 receptor agonist. On
the other hand, antiimmobility effect of imipramine remained
unchanged. This may be due to differential mechanism of imipramine and SSRIs. The synergism observed following coadministration of subeffective doses of agmatine and uoxetine is in
agreement with recent reports (Zomkowski et al., 2004). These
studies indicate possible interaction between imidazoline I1 and I2
receptors and SSRIs. It is worth noting that, none of the above SSRIs
possess even moderate afnity for I1 receptor or interact directly
with them (Holt, 2003). It could therefore be argued that, some
endogenous mediators of imidazoline receptors might be critically
involved in the antidepressant like effect of SSRIs.
Brain regions that regulate endocrine and affective functions
have abundant imidazoline binding sites and endogenous ligands
are colocalized at these sites (De Vos et al., 1994; Raasch et al.,
1995). There is considerable evidence that I2 imidazoline binding
sites exist on enzyme monoamine oxidase and regulate its activity
(Alemany et al., 1995; Raddatz et al., 1997; Remaury et al., 2000).
The other studies revealed that imidazoline receptor agonist or
ligands including 2-BFI and BU-224 reversibly inhibited MAO-A and
MAO-B, the enzymes that causes oxidative deamination of neurotransmitters like 5-HT, noradrenaline and dopamine (Raasch et al.,
1996; Bour et al., 2006). Thus, apparently imidazoline receptors and
its endogenous ligands might be implicated in depressive disorders. These I2 binding sites were disappeared in MAO-B but not

421

MAO-A knockout mice (Remaury et al., 2000). Several SSRIs have

been shown to inhibit MAO activity (Holt and Baker, 1996;
Mukherjee and Yang, 1999). Moreover, administration of a selective
imidazoline I2 ligand, BU-224, not only augmented the 5-HT levels
but also suppressed its turnover in frontal cortex and hypothalamus
of the rats subjected to FST (Finn et al., 2003). Since, none of the
SSRIs interact directly with imidazoline receptors (Holt, 2003), it is
reasonable to assume that SSRIs activate the release of endogenous
substance like agmatine that interact with imidazoline I1/I2
receptors to elicit antidepressant like effect.
To further characterize this interaction, antiimmobility effect of
SSRIs was investigated in presence of I1/I2 imidazoline receptor
antagonists. We found that, efaroxan (1 mg/kg, ip) and idazoxan
(0.25 mg/kg, ip) an antagonists of imidazoline I1 and I2 receptors
completely blocked the antiimmobility effect of SSRIs but not of
imipramine. These imidazoline receptor antagonists also
completely blocked the synergistic antidepressant effect observed
following combination of SSRI and agmatine. This is in agreement
with previous reports that efaroxan and idazoxan both attenuated
antidepressant like effect of agmatine (Zeidan et al., 2007). This
strengthens our assumption that antidepressant like effect of SSRIs
like uoxetine and paroxetine are mediated via endogenous
agmatine interaction with imidazoline I1/I2 receptors. Agmatine
induced antidepressant like effect was attributed to enhanced 5-HT
neurotransmission in brain (Li et al., 2003) and was attenuated by
5-HT depletion and 5-HT1 and 5-HT2 receptors antagonists
(Zomkowski et al., 2004). The MAO activity is regulated by imidazoline binding sites located on it (Tesson et al., 1991; Eglen et al.,
1998; Raddatz et al., 1997, 2000). Further, several imidazoline
receptor ligands have been shown to inhibit MAO activity (Alemany
et al., 1995) and might increase the levels of neurotransmitters
including serotonin. So, it may be implied that SSRIs produce
antidepressant like effect by involving agmatine and imidazoline
receptors.
Agmatine has been implicated in the modulation of mood
disorders (Zomkowski et al., 2002; Lavinsky et al., 2003; Gong et al.,
2006). Furthermore, Piletz et al. (1994) proposed that low brain
agmatine levels caused upregulation of imidazoline receptors in
depressive patients. Although, exogenous administration of
agmatine produced antidepressant like effect in FST and also
enhanced SSRIs effects (Zomkowski et al., 2002), it remains to be
investigated whether endogenous agmatine show similar property.
Increasing biosynthesis of endogenous agmatine and blocking its
degradation are the approaches to elevate agmatine levels.
Biosynthesis of agmatine by L-arginine decarboxylase (L-ADC)
depends upon the availability of L-arginine (Su et al., 2003). We
found that icv injections of L-arginine (40 mg/mouse, icv) enhanced
the antiimmobility effect of SSRIs. These ndings are consistent
with previous reports demonstrating potentiation of antidepressant effect of sertraline by ip administration of L-arginine (Inan
et al., 2004). Although the antidepressant potential of L-arginine is
well documented (Inan et al., 2004; Spiacci et al., 2008), we did not
observed any signicant effect of L-arginine per se at the dose
(40 mg/mouse, icv) employed here in FST. The differential effect of Larginine may depend on its dose, route of administration and
differential metabolic pathways. L-arginine is also converted in to
ornithine and nitric oxide (NO) by an enzyme arginase and nitric
oxide synthase, respectively (Reis and Regunathan, 2000). Ornithine subsequently converted into putrescine by L-ornithine
decarboxylase. Inhibition of either metabolic pathway is reported
to increase activity in the other metabolic pathways. DFMO, an
inhibitor of L-ornithine decarboxylase and arginase (Slotkin et al.,
1982; Selamnia et al., 1998) and stimulator of L-ADC (Hernandez
and Schwarcz de Tarlovsky, 1999), might increase the availability of
agmatine in brain (Lu et al., 2003). Agmatine is metabolized to

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B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

putrescine and guanido-butanoic acid by enzyme agmatinase and

diamine oxidase respectively (Reis and Regunathan, 2000) and
inhibition of these enzymes result in augmentation of endogenous
agmatine (Lu et al., 2003; Huang et al., 2003; Regunathan, 2006). In
present study we used diamine oxidase (DAO) inhibitor, aminoguanidine (Lu et al., 2003) or agmatinase inhibitor, arcaine (Huang
et al., 2003; Regunathan, 2006) and L-ornithine decarboxylase and
arginase inhibitor, DFMO (Slotkin et al., 1982; Selamnia et al., 1998)
to block the agmatine metabolic pathways leading to increase
agmatine levels in brain (Lu et al., 2003). All these manipulations
potentiated the antidepressant like effect of uoxetine and paroxetine in FST. Furthermore, it has been speculated that, inhibition of
DAO by phenelzine leads to an increase in serum and tissue
concentrations of agmatine which is found to be relevant for antidepressive efcacy (Holt and Baker, 1995). This shows that
increasing endogenous agmatine levels in brain by different means
has the same actions as exogenous agmatine on SSRIs effects in FST.
L-arginine is also a precursor for NO in brain and enzyme nitric
oxide synthase (NOS) is responsible for its production (Reis and
Regunathan, 2000). Indeed, NOS inhibitors have been shown to
produce antidepressant like effect in rodents (Yildiz et al., 2000;
Harkin et al., 2003; Sevgi et al., 2006) also augment the behavioral
effect of SSRIs in FST (Harkin et al., 2004). Wang et al. (2008)
recently demonstrated suppression of chronic stress induced
depression by aminoguanidine, the inhibitors of NOS besides DAO
(Lu et al., 2003). There are reports that agmatine by itself inhibits all
isomers of NOS in brain. Moreover, NOS inhibitors have been shown
to increase 5-HT release in brain regions like hypothalamus, raphe
nuclei, frontal cortex and hippocampus (Kaehler et al., 1999; Smith
and Whitton, 2000; Wegener et al., 2000).
Thus, enhanced endogenous agmatine levels in brain
augmented the antidepressant like effect of SSRIs. However, neither
of these drugs in the present doses could exhibit any antiimmobility effect per se. This indicates that augmented agmatine
concentration may not be adequate to induce antidepressant like
effect and perhaps desired concentration might be achieved by
coadministration of SSRIs and agmatine enhancers. These ndings
suggest that SSRIs like uoxetine and paroxetine evoke antidepressant like activity by downregulating imidazoline I1 and I2 sites
through release of some endogenous mediator like agmatine or
alternately by subsequent inhibition of NOS by agmatine. However,
it should be veried whether NOS inhibition and imidazoline
receptors down regulation are directly related.
To strengthen these behavioral ndings, we also measured the
brain agmatine levels following treatment with SSRIs, imipramine,
L-arginine, DFMO, aminoguanidine and arcaine. All these agents
except imipramine enhanced the level of agmatine in brain of mice
exposed to FST. Interestingly, imidazoline receptor antagonists,
idazoxan and efaroxan although blocked the antidepressant like
effect of SSRIs, failed to suppress their effect on agmatine content.
We have also investigated the ability of ADC inhibitor to block the
antidepressant like effect of SSRIs as well as effect on agmatine
content. D-arginine (isomer of L-arginine) is known to inhibit ADC
in plants and bacteria (Rosenfeld and Roberts, 1976; Hao et al.,
2005) and thereby blocks conversion of L-arginine to agmatine. It is
extensively used in studies on L-arginine/nitric oxide pathway in
mammals (Navarro et al., 2005). In the present study D-arginine not
only attenuated the antiimmobility effect of SSRIs in FST but also
blocked the effect of SSRIs, L-arginine, DFMO, aminoguanidine and
arcaine on augmented brain agmatine levels. However, more
studies are required to conrm the inhibition of ADC by D-arginine
in mammals.
Thus, antidepressant like effect of SSRIs in FST might be closely
intertwined with endogenous imidazoline receptor ligands like
agmatine and not their direct interaction with these receptors.

Therefore, it is postulated that administration of SSRIs like uoxetine or paroxetine may elicit release of endogenous ligands like
agmatine in the brain and interacts with both I1 and I2 imidazoline
receptors leading to their down regulation. This interaction directly
or through MAO inhibition may facilitate 5-HT neurotransmission
and produce antidepressant like effect. The antidepressant-like
effect of uoxetine, paroxetine and imipramine alone or in
combination with imidazoline receptor agonists and agmatine
metabolic inhibitors is not due to any locomotor component since
activity in open eld remained unaffected.
In view of extension of the present study into clinical aspects
several studies have already conrmed the existence of ADC and
formation of agmatine in mammalian tissues like brain, liver, kidney
etc. (Li et al., 1994, 1995; Lortie et al., 1996; Regunathan and Reis,
2000; Zhu et al., 2007, 2008). Indeed, immunohistochemistry and
HPLC studies support the wide distribution of agmatine not only in
the cerebral cortex, the lower brain stem, the midbrain, frontal
brain, thalamus and the hypothalamus in rat brain (Otake et al.,
1998; Wang et al., 1995) but also in many other organs (Raasch et al.,
1995). Moreover, the increase in agmatine content in rat brain
regions by ADC is found to be down-regulated by specic siRNA (Iyo
et al., 2006). In addition, human complementary DNA (cDNA)
sequence of mammalian ADC has been identied and this human
cDNA is found to produce agmatine after transfection in COS-7 cells
(Zhu et al., 2004). These results further strengthen the importance of
present study in clinical context. Nevertheless, more clinical studies
are awaited conrming the distribution of agmatine and ADC in
mammalian tissues. On the contrary, a study by Coleman et al.
(2004) has created doubts for synthesis of agmatine in mammalian
tissues. This study did not nd the presence of ADC in liver and
kidney extracts of rodents. Conversely, Horyn et al. (2005) strongly
criticized these nding and provided compelling evidence for the
presence of mitochondrial ADC in rat liver as demonstrated by
others (Regunathan and Reis, 2000; Lortie et al., 1996; Li et al., 1994;
Grillo and Colombatto, 2004; Nissim et al., 2002). Thus, the existence of ADC in mammalian brain and tissue can not be denied. The
results of present study also support these reports since exogenous
administration of several pharmacological agents known to increase
endogenous agmatine have substantially augmented its level in
mouse brain. However, this issue needs to be addressed carefully in
clinical context due to lack of very precise and specic tools used
here for determining agmatine formation. Also, it is essential to have
selective pharmacological agents to target imidazoline receptors.
Further, it is important to have specic mammalian ADC inhibitor
since there is signicant difference between mammalian and
non-mammalian ADC (Reis and Regunathan, 2000).
To summarize, present study demonstrated that antidepressant
like effect of SSRIs in FST is potentiated by exogenous agmatine or
by increasing brain agmatine levels by different means or by
imidazoline receptor agonists. These effects were prevented by
imidazoline antagonists and arginine decarboxylase inhibitor.
Furthermore, brain agmatine levels were signicantly elevated by
SSRIs. Taken together these results suggest that antidepressant like
effects of SSRIs atleast in part may depend on release of some
endogenous mediator like agmatine which then interact with
imidazoline I1 and I2 receptors. However, apart from imidazoline
receptors, the role of other biological targets of agmatine like nitric
oxide synthase, NMDA and a2-adrenoreceptors in observed antidepressant like effect of SSRI can not be ruled out. These ndings
may be of latent implication to explore the unrevealed mechanisms
of SSRIs and suggest imidazoline receptors as a therapeutic target
for the development of novel antidepressants. Further studies using
other selective or highly specic analytical or pharmacological tools
are necessary to dene more reasonably the role of imidazoline
receptors in antidepressant effect of SSRIs.

B.G. Taksande et al. / Neuropharmacology 57 (2009) 415424

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