Seizure 19 (2010) 1722

Contents lists available at ScienceDirect

Seizure
journal homepage: www.elsevier.com/locate/yseiz

Seizure susceptibility alteration through 5-HT3 receptor: Modulation by

nitric oxide
Taha Gholipour a,b,c, Mehdi Ghasemi a,1, Kiarash Riazi a,d, Majid Ghaffarpour b, Ahmad Reza Dehpour a,c,*
a

Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran
Iranian Center of Neurological Research, Tehran University of Medical Sciences, Tehran, Iran
c
Interdisciplinary Neuroscience Research Program, Tehran University of Medical Sciences, Tehran, Iran
d
Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 6 May 2009
Received in revised form 28 October 2009
Accepted 29 October 2009

There is some evidence that epileptic seizures could be induced or increased by 5-hydroxytryptamine (5HT) attenuation, while augmentation of serotonin functions within the brain (e.g. by SSRIs) has been
reported to be anticonvulsant. This study was performed to determine the effect of selective 5-HT3
channel/receptor antagonist granisetron and agonist SR57227 hydrochloride on the pentylenetetrazole
(PTZ)-induced seizure threshold in mice. The possible interaction of this effect with nitrergic system was
also examined using the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME)
and the NO precursor L-arginine. SR57227 (10 mg/kg, i.p.) signicantly increased the seizure threshold
compared to control group, while high dose granisetron (10 mg/kg, i.p.) proved proconvulsant. Coadministration of sub-effective doses of the 5-HT3 agonist with L-NAME (5 and 60 mg/kg, i.p.,
respectively) exerted a signicant anticonvulsive effect, while sub-effective doses of granisetron (3 mg/
kg) was observed to have a proconvulsive action with the addition of L-arginine (75 mg/kg, i.p.). Our data
demonstrate that enhancement of 5-HT3 receptor function results in as anticonvulsant effect in the PTZinduced seizure model, and that selective antagonism at the 5-HT3 receptor yields proconvulsive effects.
Furthermore, the NO system may play a role in 5-HT3 receptor function.
2009 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Keywords:
5-HT3 receptor
Nitric oxide (NO)
Clonic seizure threshold
Pentylenetetrazole
Mice

1. Introduction
Among seven known classes of receptors for serotonin (5hydroxytryptamine: 5-HT), the 5-HT3 receptor is unique as being a
ligand-gated ion channel.1 It is structurally similar to other
neurotransmitter-dependent ion channels which also includes the
nicotinic acetylcholine channel/receptor.2,3 Unlike other 5-HT
receptors, 5-HT3 receptor causes an early depolarization effect in
postsynaptic membranes by directly conducting Na+, K+ and Ca2+
ions.1,4 As shown in animal brains, serotonergic brainstem
projections have excitatory effects on target neurons expressing
5-HT3 receptors.5 These targets are generally a subset of inhibitory
interneurons in cerebral neocortex6 and hippocampus,5,79 resulting in an overall inhibitory effect despite the excitatory nature of

* Corresponding author at: Department of Pharmacology, School of Medicine,

Tehran University of Medical Sciences, P. O. Box: 13145-784, Tehran, Iran.
Tel.: +98 21 6611 2802; fax: +98 21 6640 2569.
E-mail address: dehpour@yahoo.com (A.R. Dehpour).
1
Current address: Institute for Cell Engineering, Departments of Neurology and
Neurosciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205,
USA; mghasem2@jhmi.edu, m82_ghasemi@yahoo.com (M. Ghasemi).

the receptor. Besides the conventional synapse formation,

serotonergic axons show a paracrine release of 5-HT through
varicose axonal bers.10
According to accumulating evidence, epileptic seizures can be
induced or amplied by experimental manipulations that attenuate serotonergic neurotransmission.11 In contrast, augmentation of
serotonin activity in the brain, either by dietary manipulation,12
administration of 5-hydroxytryptophan13 or selective serotonin
reuptake inhibitors (SSRIs),14,15 or via dorsal raphe nucleus
stimulation16 has been reported to be anticonvulsant. In a recent
review by Loscher,17 the apparent biphasic effect of antidepressants
on seizure threshold was documented: low but clinically relevant
doses of antidepressants appear to exert an anticonvulsant effect in a
variety of animal seizure models (e.g. the i.v. PTZ seizure threshold)
in mice and rats via an increase in noradrenaline or 5-HT synaptic
levels, while proconvulsant activity may be seen at higher or
supratherapeutic doses of PTZ or other proconvulsant and other
models. Furthermore, increase in extracellular 5-HT concentration
has been suggested to be an underlying mechanism for the action of
some well-known antiepileptic drugs.18,19
Nitric oxide (NO) is synthesized from L-arginine by different
types of NO synthase20 in many tissues. In the brain, NO is

1059-1311/$ see front matter 2009 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.seizure.2009.10.006

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T. Gholipour et al. / Seizure 19 (2010) 1722

considered as both neurotransmitter and neuromodulator,21,22

acting mainly through increasing levels of cyclic GMP (cGMP).23,24
Neuronal NOS (nNOS) activity is well known to be regulated by
membrane channel/receptors, including glutamate N-methyl-Daspartate (NMDA) receptor and gamma amino butyric acid
(GABA)A receptor, as well as intracellular signaling mediators
such as Ca2+.25,26 Since 5-HT3 receptor activation could increase
the calcium inux directly through its ion channel component
activation or indirectly by affecting other calcium regulating
mechanisms,27 one may hypothesize an NO-mediated intracellular
signaling for 5-HT3 receptor activation. To date, no one has
reported such interaction in seizure paradigms.
A number of selective 5-HT3 receptor agonist and antagonists
have been synthesized and pharmacologically characterized. None
of these drugs has not been investigated in the seizure treatment.
In this study, we determined effects of selective 5-HT3 receptor
agonist and antagonist on an experimental model of clonic seizure
threshold induced by intravenous pentylenetetrazole (PTZ).28 We
also examined the possible involvement of the nitrergic system
using the NOS substrate L-arginine and inhibitor NG-nitro-Larginine methyl ester (L-NAME) in the seizure propensity outcomes of these agents. This research could provide new insight into
the role of the 5-HT3 receptor and its relative pharmacological
agents in modulating seizures, which could in turn divulge this
branch in antiepileptic drug studies.
2. Materials and methods
2.1. Animals
Male adult Swiss mice weighting 2430 g (Razi Institute) were
used in this study. Animals were housed in standard conditions,
including controlled temperature (25 8C), 12 h dark/12 h light
cycle, and with access to food and water ad labitum. Each mouse
was used only once and each treatment group consisted of 68
animals. All experiments were conducted between 10:00 and
14:00. The study was conducted in accordance with the Guide for
the Care and Use of Laboratory Animals published by National
Institutes of Health (NIH publication no. 8523; revised 1985) and
with the recommendations and approval of the Ethics Committee
on Animal Experiments of the Tehran University/Medical Sciences.
Each tested animal was immediately euthanized after seizure
threshold determination.

2.4. Experiments
First, animals received an intraperitoneal (i.p.) injection of
different doses of SR57227 (5 or 10 mg/kg), or granisetron (3 or
10 mg/kg) 30 min prior to seizure threshold measurement. In the
control group, mice received identical volumes of isotonic saline.
Next, we investigated the effects of NO on interaction between
serotonin and seizures. Sub-effective doses of SR57227 with LNAME (60 mg/kg; 45 min prior to seizure, i.e. 15 min prior to
SR57227), or sub-effective doses of granisetron with L-arginine
(75 mg/kg; again 45 min prior to seizure) were co-administered,
seeking for any probable additive effect.
2.5. Data analysis
Data are presented as mean  standard error of the mean
(S.E.M.). Students t-test or One-way analysis of variance (ANOVA)
followed by the TukeyKramer multiple comparisons were used,
where appropriate, to analyze the data. The signicance level was
dened as p < 0.05.
3. Results
3.1. Effect of different doses of granisetron on seizure threshold
Granisetron (0 (saline), 3 or 10 mg/kg, i.p.) was injected 30 min
prior to the seizure determination. One-way ANOVA followed by
post hoc comparisons demonstrated that granisetron at 10 mg/kg
could lower the threshold of PTZ-induced clonic seizure (P < 0.01;
Fig. 1). However, a lower dose of granisetron (3 mg/kg, i.p.) failed to
show this proconvulsant activity, and was thus selected as a subeffective dose.
3.2. Effect of different doses of SR57227 on seizure threshold
The 5-HT3 receptor agonist SR57227 (0 (saline), 5 or
10 mg/kg, i.p.) was injected to groups of mice 30 min before
the seizure experiment. Analysis using one-way ANOVA
followed by post hoc comparisons showed the anticonvulsive
effect of this agent at 10 mg/kg (P < 0.001), but not at 5 mg/kg
(P > 0.05; Fig. 1).

2.2. Chemicals
Granisetron was used as 1 mg/ml injectable solution (manufactured and marketed by Roche, Switzerland) and was diluted,
when needed, by saline solution. SR57227 (1-(6-chloro-2-pyridinyl)-4-piperidinamine) hydrochloride was purchased from Tocris
(UK). Pentylenetetrazole (PTZ), L-NAME, and L-arginine were
purchased from Sigma (UK). Drugs were all dissolved in sterile
physiological saline. All injections were done in the volume of
10 ml/kg of the bodyweight of the mice. PTZ was prepared in
physiological saline as 1% solution.
2.3. Seizure threshold determination
The threshold of PTZ-induced seizures was measured by an
infusion of PTZ into the tail vein of freely moving mice at a constant
rate of 0.6 ml/min via a 30-gauge needle, connected by a
polyethylene tube to a Hamilton microsyringe.29 Minimal dose
of PTZ (mg/kg) required to induce general clonus was recorded. The
general clonus was characterized by forelimb clonus followed by
whole body clonus. As such, seizure threshold is dependent on PTZ
dose administered and time-related.3032

Fig. 1. Effects of granisetron and SR57227 on pentylenetetrazole (PTZ)-induced

seizure threshold in mice. Drugs were administered intraperitoneally 30 min prior
to seizure threshold determination. Data represent mean  S.E.M. of 68 mice.
**P < 0.01, and ***P < 0.001 compared to control group receiving normal saline (shown
as dose 0).

T. Gholipour et al. / Seizure 19 (2010) 1722

Fig. 2. Effects of different doses of L-arginine (50, 75, and 100 mg/kg, i.p.) on
pentylenetetrazole (PTZ)-induced seizure threshold in mice. L-Arginine was
administered intraperitoneally 45 min prior to seizure threshold determination.
Data represent mean  S.E.M. of 68 mice. **P < 0.01 compared to control group
receiving normal saline (shown as dose 0).

19

Fig. 3. Additive effect of granisetron and L-arginine when co-administered in subeffective doses (3 and 75 mg/kg, i.p., respectively). Both drugs were administered
intraperitoneally; L-arginine 45 min and granisetron 30 min prior to seizure
threshold determination. In appropriate groups, normal saline was injected as
control. ***P < 0.001 compared to control group receiving no drug.

3.3. Determination of the sub-effective doses of L-arginine

and L-NAME
The NO precursor L-arginine and the NOS inhibitor L-NAME
were used in subsequent steps and dose response studies for these
agents were performed. L-Arginine (50, 75, and 100 mg/kg, i.p.) was
administered 45 min prior to seizure threshold determination. As
shown in Fig. 2, a signicant proconvulsive effect of L-arginine on
PTZ-induced clonic seizures33 was rst demonstrated at 100 mg/
kg (P < 0.01). Therefore, a dose of 75 mg/kg was selected as a subeffective dose.
L-NAME (10, 20, 60, and 100 mg/kg, i.p.) was administered
45 min prior to PTZ challenge. Post hoc comparison following oneway ANOVA revealed a highly signicant anticonvulsive effect at
100 mg/kg (P < 0.01; not gured), but not at lower doses. L-NAME
at 60 mg/kg (P < 0.05 compared to saline treated mice) was
therefore selected as the minimally effective dose.
3.4. Co-administration of granisetron and L-arginine
Granisetron (3 mg/kg, i.p.) was co-administered with L-arginine
(75 mg/kg, i.p.), each individually at a dose below that yielding a
seizure modulating effect. As revealed by Students t-test and
demonstrated in Fig. 3, the combination signicantly decreased
PTZ seizure threshold (P < 0.001).
3.5. Administration of SR57227 with L-NAME
Sub-effective dose of the 5-HT3 receptor agonist SR57227 (i.e.
5 mg/kg, i.p.) was administered together with minimally effective
dose of L-NAME (60 mg/kg, i.p.), and this combination yielded a
very signicant increase in seizure threshold (P < 0.001; Fig. 4).
4. Discussion
In the present study, we demonstrated that 5-HT3 receptor
agonists and antagonists alter the clonic seizure threshold induced
by PTZ in mice, suggesting a role for 5-HT3 receptors in brain
excitability and expression of convulsive discharges. Using a
selective 5-HT3 receptor agonist and antagonist, we clearly found
that higher 5-HT3 receptor stimulation was accompanied with
higher thresholds of PTZ-induced clonic seizures in mouse.

Fig. 4. Additive effect of SR57227 and L-NAME when co-administered in subeffective doses (5 and 60 mg/kg, i.p., respectively). Both drugs were administered
intraperitoneally; L-NAME 45 min and SR57227 30 min prior to seizure threshold
determination. In appropriate groups, normal saline was injected as control.
***P < 0.001 compared to control group receiving no drug.

Selective antagonism of baseline 5-HT3 activity by granisetron

decreased the PTZ-induced clonic seizure threshold. In contrast,
selective augmentation of 5-HT3 activity increased seizure threshold, suggesting a possible therapeutic role of serotonin agonists
against seizures. Furthermore, this study demonstrated for the rst
time that the NO system may modulate the action of 5-HT3
receptors during brain excitability.
The role of serotonin as an important neurotransmitter in
seizure development and epileptogenesis is increasingly addressed
in the scientic literature.11 Although soma of serotonergic
neurons in the central nervous system are limited to a series of
midline brainstem nuclei the raphe formations,10 neuronal
projections spread throughout most of brain, and particularly high
concentrations of this receptor are distributed the cortical and
limbic areas.2,34,35 A decit in serotonergic neurotransmission has
been postulated to be the etiology of seizures experienced by a
certain subset of epileptic patients,3639 and 5-HT depletion
appears to exacerbate paroxysms in genetic models of epilepsy

20

T. Gholipour et al. / Seizure 19 (2010) 1722

and chemically induced paroxysms.4042 Concerning SSRIs, they

have been shown to exert anticonvulsive properties in epileptic
patients17,43 and different animal models of epilepsy,14,15,17,44 and
they also have been demonstrated to enhance the effect of
antiepileptic drugs in experimental settings.4547
Most of 5-HT receptors are metabotropic, and found to be
genetically related; but 5-HT3 is considered dissimilar to other
classes particularly in being a ligand-gated ion channel.3 Clinically,
5-HT3 antagonists (ondansetron, granisetron, and tropisetron) are
typically used as potent antiemetics, due to the important central
and peripheral role of 5-HT3 receptors in the nausea/emesis
centers. Some case reports have described seizures in patients
provoked by ondansetron.48,49 Previous experimental studies on
the effects of 5-HT3 receptor agonists and antagonists on seizure
propensity, however, have been limited.
Watanabe et al.50 have studied electrically induced focal
hippocampal seizures in rats and found no effect of the 5-HT3
receptor agonist SR57227 (up to 3 mg/kg; i.p.),50 and only a
decrease in the primary after-discharge duration and the latency of
secondary after-discharge measures following granisetron administration.51 Wada et al. have suggested a facilitator role for 5-HT3
in development of amygdala-kindled rats using intra-cerebroventricular injection of a 5-HT3 agonist.52 Balakrishnan and colleagues
even reported an anticonvulsant prole for ondansetron in
maximal electroshock-induced seizures in rats.53 Contrarily, this
5-HT3 antagonist was also shown to increase hippocampal theta
rhythm5 and to decrease the ring rate of hippocampal inhibitory
interneurons,54 leading to an increase ring rate of pyramidal cells.
Cortical and hippocampal interneurons expressing this 5-HT3
receptor are mainly inhibitory in nature, acting on excitatory
neurons.2,5557 Thus, stimulation of these interneurons increase
the seizure threshold. Ropert and Guy had shown that hippocampal GABAergic interneurons are directly excited via 5-HT3
transmission.58 A postulated mechanism for this anticonvulsive
effect of 5-HT3 agonism may be activation of the 5-HT3 receptor as
an inward Na+, Ca2+, outward K+ conducting ion channel, resulting
in early depolarization of inhibitory interneurons.1,4 This may
explain the anticonvulsive properties reported for SSRIs (which
generally augment synaptic serotonin) as well our data presented
here. Similarly, blocking the baseline endogenous stimulation of 5HT3 receptors by granisetron induces proconvulsive properties.
GABAergic interneurons also express presynaptic 5-HT3 receptors on their axonal terminals,59,60 and the 5-HT3 agonist mchlorophenylbiguanide (mCPBG) has been shown to facilitate
GABA release in a synaptic bouton preparation of hippocampal CA1
pyramidal neurons.60 It is plausible that increased Ca2+ conductance at the 5-HT3 channel/receptor could modulate a GABAergicmediated increase in seizure threshold.61,62
The additive actions of sub-effective granisetron and L-arginine
together to produce a signicant proconvulsive effect, along with
the additive anticonvulsant actions of sub-effective SR57227 and LNAME suggest that the NO system modulates 5-HT3 receptors
function during seizure expression.
Nitric oxide synthase (NOS) enzyme has been shown to have
close connection with other Ca2+ conducting neuronal channels,
especially the glutamate NMDA receptor/channel, and to also take
part in NMDA modulation of seizure paradigms.63 In general, NOS
is considered as an enzyme highly dependent to Ca2+ and calcium
binding proteins (especially calmodulin) in terms of activity64 and
even destruction.65 Depending on the studied seizure model, NO
has been suggested to be either an anticonvulsant6668 or a
proconvulsant69,70 endogenous substance. However, In the PTZinduced clonic seizure paradigm, NOS overactivity is shown to be
proconvulsive.70 This is explained by the consequences of the NO/
cGMP pathway in metabolism71 and release7274 of inhibitory and
excitatory neurotransmitters, and even modulation of their

Fig. 5. Proposed mechanism for the interaction of 5-HT3 and NO system in

modulation of seizure threshold in GABAergic interneurons. 5-HT3 R, 5-HT3
receptor; NMDA R, NMDA receptor; nNOS, neuronal NO synthase; ", increase.

receptors.75 In rat dorsal spinal cord neurons, Inoue et al.

described NO-mediated release of substance P via 5-HT3.76 5-HT3
activation is reported to increase intracellular cGMP levels,77
which rises the question of how an ion channel could link with this
system. Reiser has suggested cytosolic Ca2+ and NO as possible
mechanisms.78 5-HT3 receptor activation could directly affect NOS
by changing cytosolic Ca2+ levels. Again, by contributing to NMDA
facilitation due to the resulting membrane depolarization, more
prolonged effects on the Ca2+ concentration through 5-HT3
activation could be postulated. Furthermore, it is well known
that NMDA directly enhances nNOS activity.79,80
Briey, 5-HT3 activation may have two opposite effects on the
inhibitory interneuron; one toward increased ring and subsequent GABA release, which is anticonvulsive, and another towards
increased NOS activity and proconvulsive consequences. Hence,
one could suggest a masked anticonvulsant effect from pure 5HT3 agonists, at least at lower doses. Concomitant presence of a
NOS inhibitor agent, as we performed with the addition of L-NAME,
could unmask and potentiate the anticonvulsive pathway
(Fig. 5). This effect might also take part in other downstream
neuronal circuits where NO may again oppose decreased seizure
propensity.
One limitation for immediate progression to clinical application
is the potential central and peripheral side effects of 5-HT3
agonists. Using low doses of these agents with NOS inhibitors, or
development of mixed 5-HT3 agonist/NOS inhibitor agents could
be solutions to benet their anticonvulsant properties. In addition,
thanks to human genome research, 5-HT3 receptor subtypes are
widely researched. Identifying the potential differences in human
expression of 5-HT3 which could guide pharmaceutical research
towards development of more specic antiepileptic drugs.
In conclusion, this study demonstrates an anticonvulsant role
for a selective 5-HT3 receptor agonist. Furthermore, it demonstrates that NO system is involved in the effects of 5-HT3 receptor
signaling on seizure propensity.
Acknowledgements
Authors wish to thank Dr. Hamed Shafaroodi and Dr. Ali
Mojtahed for their kind helps during the study. This study was
supported by Tehran University of Medical Sciences research grant
(85-02-54-3981) to A.R. Dehpour.
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