:: Journal of Epilepsy Research

Gholizadeh, Eslami, Dejban, Ghasemi, Rahimi, and Dehpour: Additive Anticonvulsive Effects of Sumatriptan and Morphine on Pentylenetetrazole-Induced Clonic Seizures in Mice

Original Article

Journal of Epilepsy Research 2024; 14(1): 9-16.

Published online: June 30, 2024

DOI: https://doi.org/10.14581/jer.24002

Additive Anticonvulsive Effects of Sumatriptan and Morphine on Pentylenetetrazole-Induced Clonic Seizures in Mice

Ramtin Gholizadeh, DVM^1,^2,³, Faezeh Eslami, PharmD^2,^4,⁵, Pegah Dejban, MD^2,^5,⁶, Mehdi Ghasemi, MD, MPH⁷, Nastaran Rahimi, MS^2,⁵, Ahmad Reza Dehpour, PharmD, PhD^2,⁵

¹Department of Pharmaceutical Sciences, College of Pharmacy University of Kentucky, Lexington, KY, USA

²Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran

³Department of Pharmacology, College of Veterinary Medicine, Islamic Azad University, Karaj, Iran

⁴Department of Neurology and Rehabilitation, University of Illinois Chicago, Chicago, IL, USA

⁵Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

⁶Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, USA

⁷Department of Neurology, Lahey Hospital & Medical Center, Burlington, MA, USA

Corresponding author: Ahmad Reza Dehpour, PharmD, PhD, Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran Province, Tehran, 1416753955, Iran, Tel. +98-21-8897-3652, Fax. +98-21-6640-2569, E-mail; dehpour@yahoo.com

Received November 10, 2023 Revised December 6, 2023 Accepted January 3, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background and Purpose

Sumatriptan protects the brain from damage and enhance the anti-seizure effect of morphine. There is evidence that nitric oxide (NO) may mediate these effects of both drugs. In the present study, we investigated the effects of sumatriptan (0.1–20 mg/kg, intraperitoneal [i.p.]) and morphine (0.1–20 mg/kg, i.p.) alone or in combination on seizure thresholds in an in vivo model of seizure in mice. Using various NO synthase inhibitors as well as the NO precursor, we assessed possible involvement of NO signaling in these effects.

Methods

Clonic seizures were induced in male Naval Medical Research Institute mice by intravenous administration of pentylenetetrazol (PTZ).

Results

Acute sumatriptan administration exerted anti-convulsive effects at 0.5 (p<0.01) and 1 mg/kg (p<0.05), but pro-convulsive effects at 20 mg/kg (p<0.05). Morphine had anti-convulsive effects at 0.5 (p<0.05) and 1 mg/kg (p<0.001), but exerted pro-convulsive effect at 20 mg/kg (p<0.05). Combination treatment with sub-effective doses of sumatriptan (0.1 mg/kg) and morphine (0.1 mg/kg) significantly (p<0.05) exerted an anti-convulsive effect. Co-administration of the NO precursor L-arginine (60 mg/kg) with sub-effective doses of sumatriptan and morphine significantly (p<0.05) increased seizure threshold compared with sumatriptan alone, but not sumatriptan+morphine group. While concomitant administration of either the non-selective NO synthase (NOS) inhibitor L-N^G-nitroarginine methyl ester (5 mg/kg) or the selective inducible NOS inhibitor aminoguanidine (50 mg/kg) with combined sub-effective doses of morphine and sumatriptan produced significant anticonvulsive effects, concomitant administration with the selective neuronal NOS inhibitor 7-nitroindazole (30 mg/kg) inhibited this effect.

Conclusions

Our data suggest a possible role for the NO signaling in the anticonvulsive effects of combined sumatriptan and morphine on the PTZ-induced clonic seizures in mice.

Key words: Sumatriptan, Morphine, Seizure, Pentylenetetrazole, Nitic oxide, Mice

Introduction

Sumatriptan is a drug that activates 5-hydroxytryptamine (5-HT)_1B/1D receptors, which are found on serotonergic nerve terminals that release serotonin (5-HT).1 The levels of serotonin in the brain can have an impact on seizure activity.2,3 As a result, sumatriptan has been found to have both anti-convulsive and pro-convulsive effects, depending on the dosage, when it comes to seizures triggered by pentylenetetrazole (PTZ).4 Additionally, sumatriptan has other effects on the nervous system. For instance, it can protect against nerve damage induced by vincristine in rats,5 and it provides relief from pain during migraine attacks.6

Opioids have been used by humans for pleasure and for treating various conditions, particularly pain, for almost 6 millennia.7 One area of investigation that is not well-known is the impact of opioids on seizure susceptibility. Previous studies have demonstrated that opioids can have different effects on convulsive events. There is a growing body of evidence that indicates opioids have both anti-convulsive and pro-convulsive properties in different experimental models of seizures.8,9 Specifically, low doses of morphine, an opioid-receptor agonist, have an anti-convulsive effect, while higher doses decrease the seizure thresholds induced by agents like picrotoxin, bicuculline, and PTZ.10

Nitric oxide (NO) is one of the most important neurotransmitters in both central (CNS) and peripheral nervous system. It is synthesized by three NO synthase (NOS) enzyme isoforms (neuronal NOS [nNOS], endothelial NOS [eNOS], and inducible NOS [iNOS]) from the amino acid L-arginine.11 Compelling evidence suggest that NO modulates the susceptibility to seizure in a bimodal direction.12,13 Additionally, studies reported that low doses of sumatriptan exert anti-convulsive effects on the PTZ-induced seizure in mice via mediating the NO pathway.4 In addition, the involvement of NO in the biphasic effects of morphine on the PTZ-induced seizure susceptibility has been reported.10,14 In the present study, we aimed to investigate the effects of combining sumatriptan and morphine on the seizure threshold and explore the possible role of the NO pathway.

Methods

Animals

All animal procedures were performed under the guidelines for the care and use of laboratory animals, Tehran University of Medical Sciences as well as the National Institutes of Health (NIH publication NO. 85-23; revised 1985). Male adult Naval Medical Research Institute mice, weighting 23–30 g and aged 6–8-week, were provided from the Department of Pharmacology, Tehran University of Medical Sciences. Animals were housed in standard polycarbonate cages at an ambient temperature of 24ºC and a cycle of 12 horus-light/12 horus-dark. They also had free access to food and tap water. All behavioral trials were carried out at the same time of the day between 8:00 and 11:00 AM to minimize diurnal variations. Each mouse was used only once, and each group consisted of four to eight animals.

Chemicals

Sumatriptan, morphine sulfate, PTZ, L-arginine (a NO precursor), L-N^G-nitroarginine methyl ester (L-NAME, a non-specific NOS inhibitor), aminoguanidine (a specific iNOS inhibitor), and 7-nitroindazole (a specific nNOS inhibitor) were purchased from Sigma (St. Louis, MO, USA). All drugs were dissolved in normal saline (0.9%) except 7-nitroindazole, which was suspended in an aqueous solution of Tween^® (Sigma) 80 (1%). All drug solutions and suspensions were prepared freshly on the day of the experiment. PTZ was injected intravenously in the tail vein (0.5%). Sumatriptan, morphine sulfate, L-NAME, 7-nitroindazole, and aminoguanidine were administered intraperitoneally (i.p.). The dosage selections, route of drug administration, and injection time of different compounds were based on previously published data, preliminary experiments, and pharmacokinetic considerations.4,10,15,16

Experimental groups

In experiment 1, animals received different doses of sumatriptan (0.1, 0.5, 1, 10, and 20 mg/kg, i.p.) or its vehicle (normal saline, as control group) 30 minutes before PTZ injection. In this step, the sub-effective dose of sumatriptan was selected for subsequent experiments.

In experiment 2, animals received different doses of morphine (0.1, 0.5, 1, 10, and, 20 mg/kg, i.p.) or its vehicle (normal saline, as control group) 30 minutes before PTZ injection. In this step, the sub-effective dose of morphine was selected for subsequent experiments.

In experiment 3, a sub-effective dose of sumatriptan (0.1 mg/kg, i.p.) was concurrently administered with a sub-effective dose of morphine (0.1 mg/kg, i.p.). The seizure was induced by PTZ injection 30 minutes after the drugs injection.

In experiments 4 through 7, the role of NO and opioids in the anticonvulsant effects of sumatriptan injection was assess. The non-effective doses of the NO donor L-arginine (60 mg/kg, i.p.), the non-selective NOS inhibitor L-NAME (5 mg/kg, i.p.), the selective iNOS inhibitor aminoguanidine (50 mg/kg, i.p.), and the selective nNOS inhibitor 7-nitroidazole (30 mg/kg, i.p.) were administered 15 minutes before saline, sumatriptan (0.1 mg/kg, i.p.), morphine (0.1 mg/kg, i.p.), and combined sumatriptan (0.1 mg/kg, i.p.)+morphine (0.1 mg/kg, i.p.) injection in separate groups. The seizure was induced by PTZ injection 30 minutes after the drugs injection.

Evaluation of seizure threshold

To assess the clonic seizure thresholds, a 30-gauge butterfly needle was inserted into the tail vein and fixed by a piece of adhesive tape while the animal was placed in a mouse restrainer. The infusion pump was adjusted to deliver PTZ (0.5%) at a constant rate of 1 mL/minutes in all the experiments, and animals were allowed to move freely. All these steps take less than 10 seconds and normally no mortality was seen. The infusion of PTZ was immediately discontinued when forelimb clonus was observed which is normally followed by full clonus of the body.16 The minimal dose of PTZ (mg/kg of mice weight) needed to induce general clonus was recorded as an index of clonic seizure threshold.15,16

Statistical analysis

The data were analyzed using the Graph pad Prism data analysis program (Graph pad Software; San Diego, CA, USA) and presented as the mean±standard error of mean. One-way ANOVA (Graph pad Software) followed by Tukey’s multiple comparisons of variances was used to analyze data where appropriate. In all experimental groups, differences were considered statistically significant in case the probability of type I error (p-value) was less than 0.05.

Results

Effect of sumatriptan and morphine on the PTZ-induced seizure threshold

Fig. 1A illustrates the biphasic effects of sumatriptan (0.1, 0.5, 1, 10, and 20 mg/kg, i.p.) on PTZ-induced seizure thresholds (F_5,33=15.01; p<0.001). Sumatriptan at doses of 0.5 mg/kg (p<0.01) and 1 mg/kg (p<0.05) exerted anti-convulsive effects, whereas sumatriptan at 20 mg/kg decreased the seizure threshold compared with the control (saline-treated) group (p<0.05). Notably, sumatriptan at 0.1 mg/kg did not exert any significant anti-convulsive effects on the seizure threshold compared to the control group and was selected as a sub-effective dose for anti-convulsive activity in our experiments.

Fig. 1B shows the effects of different doses of morphine (0.1, 0.5, 1, 10, and 20 mg/kg, i.p.) on the PTZ-induced seizure thresholds (F_5,30=12.19; p<0.001). Morphine at 0.5 mg/kg (p <0.05) and 1 mg/kg (p<0.001) increased seizure thresholds compared to the saline-treated control group. However, morphine at the highest dose (20 mg/kg, i.p.) exerted a significant (p<0.05) pro-convulsive effect compared to the control group. Notably, morphine at 0.1 mg/kg did not exert any anti-convulsive effects and was considered as a sub-effective dose.

Effect of acute co-administration of morphine and sumatriptan on the PTZ-induced seizure threshold

Fig. 2 demonstrates the effects of co-administration of the sub-effective doses of morphine (0.1 mg/kg, i.p.) and sumatriptan (0.1 mg/kg, i.p.) on the PTZ-induced seizure threshold (F_3,26=8.771; p<0.001). This combined administration significantly increased the seizure thresholds in comparison with sumatriptan (p<0.05) or morphine (p<0.05) alone groups.

Effects of L-arginine on the anti-convulsive effects of sumatriptan, morphine, or their combination on the PTZ-induced seizure threshold

As shown in Fig. 3, L-arginine at 60 mg/kg did not have any significant effect on the seizure threshold compared with the saline-treated control group. Moreover, L-arginine (60 mg/kg, i.p.) plus sub-effective dose of sumatriptan (0.1 mg/kg, i.p.) or L-arginine (60 mg/kg, i.p.) plus sub-effective dose of morphine (0.1 mg/kg, i.p.) did not exert any significant effect on the PTZ-induced seizure threshold compared with sumatriptan (0.1 mg/kg, i.p.) or morphine (0.1 mg/kg, i.p.) alone group. Notably, co-administration of L-arginine (60 mg/kg, i.p.), sumatriptan (0.1 mg/kg, i.p.), and morphine (0.1 mg/kg, i.p.) significantly (p<0.05) increased seizure threshold compared with sumatriptan (0.1 mg/kg) alone group. However, this combined three-agent treatment did not significantly alter the seizure threshold (p>0.05) in comparison with sumatriptan (0.1 mg/kg, i.p.) plus morphine (0.1 mg/kg, i.p.) group (F_7,43=4.766; p<0.001).

Effects of NOS inhibitors on the anti-convulsive effects of sumatriptan, morphine, or their combination on the PTZ-induced seizure threshold

Fig. 4A shows that L-NAME at 5 mg/kg (i.p.) alone did not have any significant effect on the PTZ-induced seizure threshold compared with the saline-treated control group. However, co-treatment of L-NAME (5 mg/kg, i.p.) with the sub-effective dose of sumatriptan (0.1 mg/kg, i.p.) significantly increased the seizure threshold in comparison with sumatriptan (0.1 mg/kg, i.p.) alone (p<0.001). Moreover, co-administration of L-NAME (5 mg/kg, i.p.) with a sub-effective dose of morphine (0.1 mg/kg, i.p.) did not alter the seizure threshold in comparison with the morphine (0.1 mg/kg) alone. Notably, concurrent administration of L-NAME (5 mg/kg, i.p.), sumatriptan (0.1 mg/kg, i.p.), and morphine (0.1 mg/kg, i.p.) significantly increased the PTZ-induced seizure threshold compared with sumatriptan alone (p<0.001), morphine alone (p<0.001), or morphine plus sumatriptan (p<0.05) groups (F_7,42=13.18; p<0.001).

Fig. 4B shows that aminoguanidine at 50 mg/kg (i.p.) alone did not exert any significant effect on the PTZ-induced seizure threshold compared with the saline-treated control group. Moreover, co-administration of aminoguanidine (50 mg/kg, i.p.) with either the sub-effective dose of sumatriptan (0.1 mg/kg, i.p.) or morphine (0.1 mg/kg, i.p.) did not exert any significant effect on the PTZ-induced seizure thresholds in comparison with either sumatriptan or morphine group, respectively. Notably, the administration of aminoguanidine (50 mg/kg, i.p.) along with sumatriptan (0.1 mg/kg, i.p.) and morphine (0.1 mg/kg, i.p.) significantly increased seizure threshold compared with sumatriptan (p<0.001) and morphine (p<0.01) groups (F_7,44=8.771; p<0.001). However, this combined three-agent treatment did not alter the seizure threshold compared to sumatriptan plus morphine group (p>0.05).

Fig. 4C shows that 7-nitroindazole at 30 mg/kg (i.p.) did not have any significant effect on the seizure threshold compared with the saline-treated control group. Moreover, co-treatment of 7-nitroindazole (30 mg/kg, i.p.) with the sub-effective dose of either sumatriptan (0.1 mg/kg, i.p.) or morphine (0.1 mg/kg, i.p.) did not exert any significant effect on the PTZ-induced seizure threshold compared with corresponding sumatriptan or morphine group, respectively. Notably, co-administration of 7-nitroindazole (30 mg/kg, i.p.) with combined sumatriptan (0.1 mg/kg, i.p.) and morphine (0.1 mg/kg, i.p.) significantly reduced seizure activity induced by PTZ in comparison with morphine (p<0.001) or sumatriptan plus morphine (p<0.001), and not with sumatriptan alone group (F_7,47=5.154; p<0.001).

Discussion

Seizure induction by PTZ is a recognized and widely used experimental model for studying clonic seizures in a clinical setting.17,18 PTZ specifically binds to the picrotoxin site of the γ-aminobutyric acid (GABA) receptor complex, disrupting GABA-mediated inhibition and potentially activating the N-methyl-D-aspartate receptor. This in turn increases activity in key epileptogenic centers of the forebrain such as the amygdala and piriform cortex.19 These neurochemical processes contribute to the onset and spread of PTZ-induced seizures.20

While it was previously believed that sumatriptan only had an effect on the peripheral nervous system, recent findings have shown that it also affects the CNS.4,21–23 An increase in sumatriptan concentration in the spinal fluid after oral administration and high levels of sumatriptan in the brain after intravenous injection confirm this.23 Sumatriptan is commonly used to treat migraines and works by targeting 5-HT_1B/1D receptors. There is a connection between migraines and epilepsy, known as migralepsy,24 suggesting that seizures can be triggered by migraines and followed by headaches and more migraines. This raises questions about how sumatriptan affects seizures. It has been found that lower dose of sumatriptan (1 mg/kg) can increase the threshold for clonic seizures induced by PTZ via involvement of the nNOS/NO pathway in the hippocampus and temporal cortex, whereas higher dose (20 mg/kg) can actually lower the seizure threshold via the activation of iNOS/NO pathway in the brain.4 A study in mice showed anti-convulsive effects of sumatriptan, 22 but another study in rats reported pro-convulsive effects on the PTZ-induced seizures.21 Our present study found that low doses of sumatriptan (0.5 and 1 mg/kg) showed anti-convulsive effects, but a higher dose (20 mg/kg) had a pro-convulsive impact on the PTZ-induced seizures in mice.

Opioids, such as morphine, have a biphasic effect on seizure threshold depending on the dosage and models used.8,10,25–27 Lower doses of morphine typically have an anti-convulsive effect against seizures induced by substances that block GABA transmission, such as PTZ, picrotoxin, bicuculline, and isoniazid, in animal studies.28 Conversely, higher doses of morphine actually increase the susceptibility of animals to seizures.25 Moreover, endogenous opioids have the potential to protect against seizures induced by electroconvulsive shocks29 and can play an anti-convulsive role in acute stress.30 However, at very high doses, morphine can induce seizures and heighten susceptibility to them.27 The biphasic impact of morphine on seizures suggests that it activates both excitatory and inhibitory mechanisms.31 Moreover, opioids can modulate the activity of stimulatory guanine nucleotide-binding (Gs)32 and inhibitory guanine nucleotide-binding (Gi) proteins,33 which are involved in signal transduction pathways.34 Low doses of opioids have been shown to activate Gs proteins,32 which stimulate adenylate cyclase and increase cyclic adenosine monophosphate (AMP) levels. High doses of opioids, on the other hand, have been shown to activate Gi proteins,33 which inhibit adenylate cyclase and decrease cyclic AMP levels. Confirming earlier research, we also observed that administering low doses of morphine (0.5 and 1 mg/kg) increased the seizure threshold. On the other hand, higher doses (20 mg/kg) decreased the threshold and intensified susceptibility to seizures induced by PTZ in mice.

The NO pathway has been shown to influence seizure threshold in different ways depending on the study.35–37 Some researchers have suggested that the alterations in the activity of three enzymes that produce NO in a biological system are responsible for the seizures induced by PTZ in animals.38,39 Kirkby et al.38 reported that the effects of NOS inhibitors on seizures, whether pro or anti-convulsive, depend on genetic factors, the models employed in the seizures, and the dosage administered. Moreover, there is evidence that sumatriptan reduces levels of NO.39 Additional studies have suggested that sumatriptan aids in alleviating migraine headaches by decreasing the production of NO.40 In our recent study,41 we observed an increase in NO levels following the induction of status epilepticus (SE) in rats, but treatment with sumatriptan resulted in a significant decrease in NO levels in the rats’ brain tissues post-seizure. On the other hand, a more substantial reduction in NO levels could potentially lead to more brain damage. This was evident in our experiment when we combined sumatriptan with NOS inhibitors (L-NAME and 7-nitroindazole), which resulted in more brain injury. Moreover, there is evidence that the NO pathway mediates both the anti- and pro-convulsive effects of morphine in seizures.10,29,42 For instance, Homayoun et al.10 demonstrated that the non-selective NOS inhibitor L-NAME was able to completely reverse the anticonvulsive effects of morphine on the PTZ-induced clonic seizures in mice, whereas the pro-convulsive impact was only partially neutralized by L-NAME. Other study also found that pre-treatment with different NOS inhibitors, such as L-NAME (10 mg/kg, i.p.), 7-nitroindazole (30 mg/kg, i.p.), and aminoguanidine (50 mg/kg, i.p.), significantly reversed the inhibitory effects of morphine on the lithium chloride/pilocarpine-induced SE.43 Another study also demonstrated that constitutional NOS isoforms (i.e., eNOS and nNOS) may play an important role in the modulation of anticonvulsive effects of morphine on electroshock-induced seizure in mice.29

In a study conducted in 2013, the researchers investigated the potential role of the NO pathway in the effects of combining morphine with agmatine (a compound derived from the chemical arginine) on seizures induced by PTZ in mice.42 The results showed that when L-NAME (1 and 5 mg/kg, i.p.) or 7-nitroindazole (15 and 30 mg/kg, i.p.) were co-administered with a combination of morphine (0.1 mg/kg) and agmatine (1 mg/kg), a significant anticonvulsive effect was observed. Additionally, the administration of the NO precursor L-arginine (30 and 60 mg/kg, i.p.) inhibited the anticonvulsive effects of co-administering agmatine (3 mg/kg) and morphine (0.5 mg/kg), suggesting the involvement of the NO pathway.42 These findings are consistent with our present data that showed that different NOS inhibitors could influence the effects of combined sumatriptan and morphine administration on PTZ-induced seizures in mice. The anti-convulsive effect of morphine is connected to an increase in GABAergic transmission within the CNS.26,44 Interestingly, several studies have found a correlation between NOS activation and an increase in GABAergic activity in the brain.45,46 Therefore, it is likely that NO plays a role in mediating the anticonvulsive effect of morphine by enhancing GABAergic function. However, further investigation is needed to clarify this point.

Conflicts of Interest

The authors report no conflicts of interest related to the present study.

Figure 1

Effects of different doses of (A) sumatriptan (0.1, 0.5, 1, 10, and 20 mg/kg, i.p.) or its solvent (saline, i.p., as control group) and (B) morphine (0.1, 0.5, 1, 10, and 20 mg/kg, i.p.) or its solvent (saline, i.p., as control group) on the pentylenetetrazole (PTZ)-induced clonic seizure threshold in mice. Sumatriptan or morphine were administered 30 minutes before determination of PTZ-induced clonic seizure threshold. Data are expressed as the mean±standard deviation of seizure threshold in each group. Each group consisted of 5 to 8 mice. i.p., intraperitoneal. *p <0.05; **p <0.01; and ***p <0.001 compared with corresponding saline-treated control group.

Figure 2

Effect of co-administration of sub-effective doses of morphine (0.1 mg/kg, i.p.) and sumatriptan (0.1 mg/kg, i.p.) on the pentylenetetrazole (PTZ)-induced clonic seizure threshold in mice. Morphine and sumatriptan were administered concurrently 30 minutes before PTZ. Data are expressed as mean±standard deviation. The number of mice in each group is indicated below the corresponding bar. i.p., intraperitoneal. *p <0.05 compared with corresponding group as shown by lines connecting each of the two comparison groups.

Figure 3

Effect of pretreatment with L-arginine (60 mg/kg, i.p.) on the effects of sumatriptan (0.1 mg/kg, i.p.) and morphine (0.1 mg/kg, i.p.), alone or in combination, on the pentylenetetrazole (PTZ)-induced seizure threshold in mice. L-arginine was administered 15 minutes before sumatriptan or morphine, or their co-administration, and 45 minutes before PTZ. Data are expressed as mean±standard deviation. The number of mice in each group is indicated below the corresponding bar. i.p., intraperitoneal. *p <0.05 and **p <0.01 compared with corresponding group as shown by lines connecting each of the two comparison groups.

Figure 4

Effect of pretreatment with (A) L-NAME (5 mg/kg, i.p.), (B) aminoguanidine (AG, 50 mg/kg, i.p.), and (C) 7-NI (30 mg/kg, i.p.) on the effects of combined morphine (0.1 mg/kg, i.p.) and sumatriptan (0.1 mg/kg, i.p.) on the PTZ-induced seizure threshold in mice. L-NAME, 7-NI, or AG were administered 15 minutes before sumatriptan or morphine, or their co-administration, and 45 minutes before PTZ. Data are expressed as mean± standard deviation. The number of mice in each group is indicated below the corresponding bar. PTZ, pentylenetetrazole; L-NAME, L-N^G-nitroarginine methyl ester; 7-NI, 7-nitroindazole; i.p., intraperitoneal. *p <0.05; **p <0.01; and ***p <0.001 compared with corresponding group as shown by lines connecting each of the two comparison groups.

References

1. Peroutka SJ, McCarthy BG. Sumatriptan (GR 43175) interacts selectively with 5-HT1B and 5-HT1D binding sites. Eur J Pharmacol. 1989;163:133–6.

2. Prendiville S, Gale K. Anticonvulsant effect of fluoxetine on focally evoked limbic motor seizures in rats. Epilepsia. 1993;34:381–4.

3. Lin WH, Huang HP, Lin MX, et al. Seizure-induced 5-HT release and chronic impairment of serotonergic function in rats. Neurosci Lett. 2013;534:1–6.

4. Gooshe M, Ghasemi K, Rohani MM, et al. Biphasic effect of sumatriptan on PTZ-induced seizures in mice: modulation by 5-HT1B/D receptors and NOS/NO pathway. Eur J Pharmacol. 2018;824:140–7.

5. Khalilzadeh M, Panahi G, Rashidian A, et al. The protective effects of sumatriptan on vincristine - induced peripheral neuropathy in a rat model. Neurotoxicology. 2018;67:279–86.

6. Goadsby PJ. The pharmacology of headache. Prog Neurobiol. 2000;62:509–25.

7. Snyder S. The opiates, opioid peptides (endorphins), and the opiate receptor. Tex Med. 1979;75:41–5.

8. Homayoun H, Khavandgar S, Dehpour AR. The role of alpha2-adrenoceptors in the modulatory effects of morphine on seizure susceptibility in mice. Epilepsia. 2002;43:797–804.

9. Lauretti GR, Ahmad I, Pleuvry BJ. The activity of opioid analgesics in seizure models utilizing N-methyl-DL-aspartic acid, kainic acid, bicuculline and pentylenetetrazole. Neuropharmacology. 1994;33:155–60.

10. Homayoun H, Khavandgar S, Namiranian K, Gaskari SA, Dehpour AR. The role of nitric oxide in anticonvulsant and proconvulsant effects of morphine in mice. Epilepsy Res. 2002;48:33–41.

11. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J. 2001;357:593–615.

12. Bahramnjead E, Kazemi Roodsari S, Rahimi N, Etemadi P, Aghaei I, Dehpour AR. Effects of modafinil on clonic seizure threshold induced by pentylenetetrazole in mice: involvement of glutamate, nitric oxide, GABA, and serotonin pathways. Neurochem Res. 2018;43:2025–37.

13. Kazemi Roodsari S, Bahramnejad E, Rahimi N, Aghaei I, Dehpour AR. Methadone’s effects on pentylenetetrazole-induced seizure threshold in mice: NMDA/opioid receptors and nitric oxide signaling. Ann N Y Acad Sci. 2019;1449:25–35.

14. Tsuda M, Suzuki T, Misawa M. Aggravation of DMCM-induced seizure by nitric oxide synthase inhibitors in mice. Life Sci. 1997;60:PL339–43.

15. Dafe EA, Rahimi N, Javadian N, et al. Effect of lenalidomide on pentylenetetrazole-induced clonic seizure threshold in mice: a role for N-Methyl-D-aspartic acid receptor/nitric oxide pathway. J Epilepsy Res. 2021;11:6–13.

16. Gholizadeh R, Abdolmaleki Z, Bahremand T, Ghasemi M, Gharghabi M, Dehpour AR. Involvement of N-Methyl-D-aspartate receptors in the anticonvulsive effects of licofelone on pentylenetetrazole-induced clonic seizure in mice. J Epilepsy Res. 2021;11:14–21.

17. Swinyard EA, Kupferberg HJ. Antiepileptic drugs: detection, quantification, and evaluation. Fed Proc. 1985;44:2629–33.

18. Löscher W, Hönack D, Fassbender CP, Nolting B. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. III. Pentylenetetrazole seizure models. Epilepsy Res. 1991;8:171–89.

19. Gale K. Subcortical structures and pathways involved in convulsive seizure generation. J Clin Neurophysiol. 1992;9:264–77.

20. Kaputlu I, Uzbay T. L-NAME inhibits pentylenetetrazole and strychnine-induced seizures in mice. Brain Res. 1997;753:98–101.

21. Chavan VR, Mudium R, Sayana SB. Abolition of seizure provoking effect of sumatriptan by fluoxetine in pentylenetetrazol induced seizures in rats. Asian J Pharm Clin Res. 2014;7:107–9.

22. Jand A, Palizvan MR. Sumatriptan, an antimigraine drug, inhibits pentylenetetrazol- induced seizures in NMRI mice. Drug Res (Stuttg). 2017;67:179–82.

23. Johnson DE, Rollema H, Schmidt AW, McHarg AD. Serotonergic effects and extracellular brain levels of eletriptan, zolmitriptan and sumatriptan in rat brain. Eur J Pharmacol. 2001;425:203–10.

24. Vercueil L. Migralepsy, what it is and what it is not. Rev Neurol (Paris). 2022;178:654–8.

25. Foote F, Gale K. Proconvulsant effect of morphine on seizures induced by pentylenetetrazol in the rat. Eur J Pharmacol. 1984;105:179–84.

26. Kuriyama K, Yoneda Y. Morphine induced alterations of gamma-aminobutyric acid and taurine contents and L-glutamate decarboxylase activity in rat spinal cord and thalamus: possible correlates with analgesic action of morphine. Brain Res. 1978;148:163–79.

27. Wikler A, Altschul S. Effects of methadone and morphine of the electroencephalogram of the dog. J Pharmacol Exp Ther. 1950;98:437–46.

28. Shafaroodi H, Asadi S, Sadeghipour H, et al. Role of ATP-sensitive potassium channels in the biphasic effects of morphine on pentylenetetrazole- induced seizure threshold in mice. Epilepsy Res. 2007;75:63–9.

29. Khavandgar S, Homayoun H, Dehpour AR. Mediation of nitric oxide in inhibitory effect of morphine against electroshock-induced convulsions in mice. Pharmacol Biochem Behav. 2003;74:795–801.

30. Pavone F, Castellano C, Oliverio A. Strain-dependent effects of shock-induced release of opioids: dissociation between analgesia and behavioral seizures. Brain Res. 1986;366:326–8.

31. Lutfy K, Woodward RM, Keana JF, Weber E. Inhibition of clonic seizure- like excitatory effects induced by intrathecal morphine using two NMDA receptor antagonists: MK-801 and ACEA-1011. Eur J Pharmacol. 1994;252:261–6.

32. Crain SM, Shen KF. Opioids can evoke direct receptor-mediated excitatory effects on sensory neurons. Trends Pharmacol Sci. 1990;11:77–81.

33. Childers SR. Opioid receptor-coupled second messenger systems. Life Sci. 1991;48:1991–2003.

34. Wang D, Zeng J, Li Q, Huang J, Couture R, Hong Y. Contribution of adrenomedullin to the switch of G protein-coupled μ-opioid receptors from Gi to Gs in the spinal dorsal horn following chronic morphine exposure in rats. Br J Pharmacol. 2016;173:1196–207.

35. Ghasemi M, Shafaroodi H, Nazarbeiki S, et al. Inhibition of NMDA receptor/ NO signaling blocked tolerance to the anticonvulsant effect of morphine on pentylenetetrazole-induced seizures in mice. Epilepsy Res. 2010;91:39–48.

36. Bahremand A, Ziai P, Khodadad TK, et al. Agmatine enhances the anti-convulsant effect of lithium chloride on pentylenetetrazole-induced seizures in mice: involvement of L-arginine/nitric oxide pathway. Epilepsy Behav. 2010;18:186–92.

37. Pourshadi N, Rahimi N, Ghasemi M, Faghir-Ghanesefat H, Sharifzadeh M, Dehpour AR. Anticonvulsant effects of thalidomide on pentylenetetrazole-induced seizure in mice: a role for opioidergic and nitrergic transmissions. Epilepsy Res. 2020;164:106362

38. Kirkby RD, Carroll DM, Grossman AB, Subramaniam S. Factors determining proconvulsant and anticonvulsant effects of inhibitors of nitric oxide synthase in rodents. Epilepsy Res. 1996;24:91–100.

39. Ikeda Y, Jimbo H, Shimazu M, Satoh K. Sumatriptan scavenges superoxide, hydroxyl, and nitric oxide radicals: in vitro electron spin resonance study. Headache. 2002;42:888–92.

40. Suwattanasophon C, Phansuwan-Pujito P, Srikiatkhachorn A. 5-HT(1B/1D) serotonin receptor agonist attenuates nitroglycerin-evoked nitric oxide synthase expression in trigeminal pathway. Cephalalgia. 2003;23:825–32.

41. Eslami F, Rahimi N, Ostovaneh A, et al. Sumatriptan reduces severity of status epilepticus induced by lithium-pilocarpine through nitrergic transmission and 5-HT1B/D receptors in rats: a pharmacological-based evidence. Fundam Clin Pharmacol. 2021;35:131–40.

42. Payandemehr B, Rahimian R, Bahremand A, et al. Role of nitric oxide in additive anticonvulsant effects of agmatine and morphine. Physiol Behav. 2013;118:52–7.

43. Meskinimood S, Rahimi N, Faghir-Ghanesefat H, Gholami M, Sharifzadeh M, Dehpour AR. Modulatory effect of opioid ligands on status epilepticus and the role of nitric oxide pathway. Epilepsy Behav. 2019;101:106563

44. Frenk H. Pro- and anticonvulsant actions of morphine and the endogenous opioids: involvement and interactions of multiple opiate and non-opiate systems. Brain Res. 1983;287:197–210.

45. Jayakumar AR, Sujatha R, Paul V, Asokan C, Govindasamy S, Jayakumar R. Role of nitric oxide on GABA, glutamic acid, activities of GABA-T and GAD in rat brain cerebral cortex. Brain Res. 1999;837:229–35.

46. Paul V, Jayakumar AR. A role of nitric oxide as an inhibitor of gamma-aminobutyric acid transaminase in rat brain. Brain Res Bull. 2000;51:43–6.