Seizure: European Journal of Epilepsy
Volume 19, Issue 1 , Pages 17-22, January 2010

Seizure susceptibility alteration through 5-HT3 receptor: Modulation by nitric oxide

  • Taha Gholipour

      Affiliations

    • 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
    • Interdisciplinary Neuroscience Research Program, Tehran University of Medical Sciences, Tehran, Iran
    • ,
  • Mehdi Ghasemi

      Affiliations

    • Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran
    • 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).
    • ,
  • Kiarash Riazi

      Affiliations

    • Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran
    • Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
    • ,
  • Majid Ghaffarpour

      Affiliations

    • Iranian Center of Neurological Research, Tehran University of Medical Sciences, Tehran, Iran
    • ,
  • Ahmad Reza Dehpour

      Affiliations

    • Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran
    • Interdisciplinary Neuroscience Research Program, Tehran University of Medical Sciences, Tehran, Iran
    • Corresponding Author InformationCorresponding 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.

Received 6 May 2009; received in revised form 28 October 2009; accepted 29 October 2009. published online 27 November 2009.

Article Outline

Abstract 

There is some evidence that epileptic seizures could be induced or increased by 5-hydroxytryptamine (5-HT) 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 (10mg/kg, i.p.) significantly increased the seizure threshold compared to control group, while high dose granisetron (10mg/kg, i.p.) proved proconvulsant. Co-administration of sub-effective doses of the 5-HT3 agonist with l-NAME (5 and 60mg/kg, i.p., respectively) exerted a significant anticonvulsive effect, while sub-effective doses of granisetron (3mg/kg) was observed to have a proconvulsive action with the addition of l-arginine (75mg/kg, i.p.). Our data demonstrate that enhancement of 5-HT3 receptor function results in as anticonvulsant effect in the PTZ-induced 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.

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

 

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1. Introduction 

Among seven known classes of receptors for serotonin (5-hydroxytryptamine: 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, 7, 8, 9 resulting in an overall inhibitory effect despite the excitatory nature of the receptor. Besides the conventional synapse formation, serotonergic axons show a paracrine release of 5-HT through varicose axonal fibers.10

According to accumulating evidence, epileptic seizures can be induced or amplified 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 Löscher,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 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-d-aspartate (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 influx 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-l-arginine 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.

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2. Materials and methods 

2.1. Animals 

Male adult Swiss mice weighting 24–30g (Razi Institute) were used in this study. Animals were housed in standard conditions, including controlled temperature (∼25°C), 12h dark/12h light cycle, and with access to food and water ad labitum. Each mouse was used only once and each treatment group consisted of 6–8 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. 85–23; 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.

2.2. Chemicals 

Granisetron was used as 1mg/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 10ml/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 0.6ml/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.30, 31, 32

2.4. Experiments 

First, animals received an intraperitoneal (i.p.) injection of different doses of SR57227 (5 or 10mg/kg), or granisetron (3 or 10mg/kg) 30min 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 l-NAME (60mg/kg; 45min prior to seizure, i.e. 15min prior to SR57227), or sub-effective doses of granisetron with l-arginine (75mg/kg; again 45min 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.). Student's t-test or One-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparisons were used, where appropriate, to analyze the data. The significance level was defined as p<0.05.

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3. Results 

3.1. Effect of different doses of granisetron on seizure threshold 

Granisetron (0 (saline), 3 or 10mg/kg, i.p.) was injected 30min prior to the seizure determination. One-way ANOVA followed by post hoc comparisons demonstrated that granisetron at 10mg/kg could lower the threshold of PTZ-induced clonic seizure (P<0.01; Fig. 1). However, a lower dose of granisetron (3mg/kg, i.p.) failed to show this proconvulsant activity, and was thus selected as a sub-effective dose.

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  • Fig. 1. 

    Effects of granisetron and SR57227 on pentylenetetrazole (PTZ)-induced seizure threshold in mice. Drugs were administered intraperitoneally 30min prior to seizure threshold determination. Data represent mean±S.E.M. of 6–8 mice. **P<0.01, and ***P<0.001 compared to control group receiving normal saline (shown as dose 0).

3.2. Effect of different doses of SR57227 on seizure threshold 

The 5-HT3 receptor agonist SR57227 (0 (saline), 5 or 10mg/kg, i.p.) was injected to groups of mice 30min before the seizure experiment. Analysis using one-way ANOVA followed by post hoc comparisons showed the anticonvulsive effect of this agent at 10mg/kg (P<0.001), but not at 5mg/kg (P>0.05; Fig. 1).

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 100mg/kg, i.p.) was administered 45min prior to seizure threshold determination. As shown in Fig. 2, a significant proconvulsive effect of l-arginine on PTZ-induced clonic seizures33 was first demonstrated at 100mg/kg (P<0.01). Therefore, a dose of 75mg/kg was selected as a sub-effective dose.

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  • Fig. 2. 

    Effects of different doses of l-arginine (50, 75, and 100mg/kg, i.p.) on pentylenetetrazole (PTZ)-induced seizure threshold in mice. l-Arginine was administered intraperitoneally 45min prior to seizure threshold determination. Data represent mean±S.E.M. of 6–8 mice. **P<0.01 compared to control group receiving normal saline (shown as dose 0).

l-NAME (10, 20, 60, and 100mg/kg, i.p.) was administered 45min prior to PTZ challenge. Post hoc comparison following one-way ANOVA revealed a highly significant anticonvulsive effect at 100mg/kg (P<0.01; not figured), but not at lower doses. l-NAME at 60mg/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 (3mg/kg, i.p.) was co-administered with l-arginine (75mg/kg, i.p.), each individually at a dose below that yielding a seizure modulating effect. As revealed by Student's t-test and demonstrated in Fig. 3, the combination significantly decreased PTZ seizure threshold (P<0.001).

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  • Fig. 3. 

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

3.5. Administration of SR57227 with l-NAME 

Sub-effective dose of the 5-HT3 receptor agonist SR57227 (i.e. 5mg/kg, i.p.) was administered together with minimally effective dose of l-NAME (60mg/kg, i.p.), and this combination yielded a very significant increase in seizure threshold (P<0.001; Fig. 4).

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  • Fig. 4. 

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

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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. 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 first 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 scientific 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 deficit in serotonergic neurotransmission has been postulated to be the etiology of seizures experienced by a certain subset of epileptic patients,36, 37, 38, 39 and 5-HT depletion appears to exacerbate paroxysms in genetic models of epilepsy and chemically induced paroxysms.40, 41, 42 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.45, 46, 47

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 3mg/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 profile 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 firing rate of hippocampal inhibitory interneurons,54 leading to an increase firing rate of pyramidal cells.

Cortical and hippocampal interneurons expressing this 5-HT3 receptor are mainly inhibitory in nature, acting on excitatory neurons.2, 55, 56, 57 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 5-HT3 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 m- chlorophenylbiguanide (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 GABAergic-mediated increase in seizure threshold.61, 62

The additive actions of sub-effective granisetron and l-arginine together to produce a significant proconvulsive effect, along with the additive anticonvulsant actions of sub-effective SR57227 and l-NAME 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 anticonvulsant66, 67, 68 or a proconvulsant69, 70 endogenous substance. However, In the PTZ-induced 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 release72, 73, 74 of inhibitory and excitatory neurotransmitters, and even modulation of their 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

Briefly, 5-HT3 activation may have two opposite effects on the inhibitory interneuron; one toward increased firing 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 5-HT3 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.

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  • 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.

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 benefit 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 specific 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.

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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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PII: S1059-1311(09)00211-8

doi:10.1016/j.seizure.2009.10.006

Seizure: European Journal of Epilepsy
Volume 19, Issue 1 , Pages 17-22, January 2010

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