Epilepsy & Behavior 20 (2011) 602606

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Epilepsy & Behavior

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Morphine sensitization in the pentylenetetrazole-induced clonic seizure threshold in mice: Role of nitric oxide and receptors
Hamed Shafaroodi a, Nazanin Baradaran b, Leila Moezi c, Siavash Dehpour b, Tina Kabiri b, Ahmad R. Dehpour d,
a

Department of Pharmacology and Toxicology, Pharmaceutical Sciences Branch and Pharmaceutical Sciences Research Centre, Islamic Azad University, Tehran, Iran Department of Pharmacology, Tehran Medical Branch, Isalmic Azad University, Tehran, Iran Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran d Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
b c

a r t i c l e

i n f o

a b s t r a c t
Behavioral sensitization occurs after repeated administration of -opioid receptor agonists following a drugfree period. It seems that the changes in dopaminergic systems induced by -opioid receptor agonists play a crucial role in behavioral sensitization to opioids. Nitric oxide also plays a role in some behavioral effects of morphine, including sensitization to the locomotor-stimulating effect. This study investigated whether morphine sensitization appears in seizure threshold and the possible role of -opioid receptor and nitric oxide in this sensitization. Sensitization was produced by daily injections of morphine (0.1, 0.5, 1, 5, 15, or 30 mg/kg), followed by a 10-day washout period. Then the challenge test was performed using morphine (0.1, 0.5, 1, 5, 15, or 30 mg/kg) in different groups. To assess clonic seizure threshold, pentylenetetrazole (PTZ) was administered intravenously. Subcutaneous administration of morphine (0.1 and 0.5 mg/kg) induced sensitization in PTZ-induced clonic seizures in mice. Intraperitoneal administration of L-NAME (20 mg/kg), a nonselective inhibitor of nitric oxide synthase, or naltrexone (10 mg/kg), an opioid receptor antagonist, along with morphine inhibited morphine-induced sensitization in PTZ-induced seizure threshold. In conclusion, at low doses, morphine induces sensitization in PTZ-induced clonic seizures in mice probably as a result of the interaction with -receptors and nitric oxide. 2010 Elsevier Inc. All rights reserved.

Article history: Received 23 May 2010 Revised 20 September 2010 Accepted 15 December 2010 Available online 21 March 2011 Keywords: Morphine Sensitization Nitric oxide Clonic seizure Mice Addiction

1. Introduction Continuous exposure to morphine in rodents leads to the development of tolerance to most of its pharmacological effects, whereas intermittent exposure produces a subsequent augmentation of the motor-stimulating effects of this drug termed behavioral sensitization. Morphine sensitization has been reported using various pretreatment protocols and various doses [13], and it has been suggested that the temporal pattern of the drug treatment and posttreatment periods may be critical to the development of long-lasting behavioral sensitization and associated neuronal adaptations [3,4]. Sensitization is characterized by a progressive augmentation of behavioral effects elicited by the repeated administration of drugs. Although repeated administration of morphine at intervals shorter than 12 hours may induce tolerance, repeated administration at interdose intervals of 24 hours elicited sensitization to the locomotor-stimulating effect [5]. In experimental studies, sensitization was observed after
Corresponding author. Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran. Fax: + 98 21 664 02 569. E-mail address: dehpour@yahoo.com (A.R. Dehpour). 1525-5050/$ see front matter 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2010.12.020

repeated administration of opioids [6], cocaine [7], amphetamine [8], ethanol [9], and nicotine [10]. Sensitization can last weeks to months after cessation of drug treatment [11], and it might contribute to a rapid relapse of drug abuse on reexposure to a drug. Behavioral sensitization is considered to be linked to central aspects of the development of drug addiction, such as drug craving and the persistence of compulsive drugseeking behavior [12]. It has been argued that behavioral sensitization may be responsible for the development of addiction and especially for the high rate of relapse among drug addicts even after very long periods of abstinence [13,14]. Therefore, investigation of morphine behavioral sensitization may help to better understand the pathophysiology of opioid-associated disorders, and a study of intervention in its behavioral sensitization may provide new strategies for the treatment of drug addiction. Although it has been assumed that expression of opiate sensitization is related to the ability of the opiate to change dopamine responsiveness in the nucleus accumbens shell and core and in the dorsal caudate putamen [15,16], it cannot be ruled out that neurochemical adaptations present in morphine sensitization are coupled to the stimulation of -opioid receptors, these receptors being the most involved in morphine sensitization [17,18]. In morphinesensitized animals -opioid receptor autoradiography revealed a

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signicant increase in the caudate putamen, nucleus accumbens shell, prefrontal and frontal cortex, medial thalamus, hypothalamus, and central gray matter [19]. Furthermore, nitric oxide also mediates some of the behavioral effects of morphine. Morphine releases nitric oxide (NO) [20], and inhibitors of NO synthase (NOS) decrease morphine tolerance [21] and/or inhibit the development and expression of morphine withdrawal syndrome [22]. It has also been reported that NG-nitro-L-arginine methyl ester (L-NAME) attenuates the development of sensitization to the locomotor-stimulating effect of morphine [23,24] and cocaine [24]. Opiates exert differential effects on seizure threshold based on their dose and the animal model used [25]. Thus, in chemical models of seizures, morphine generally induces an anticonvulsant effect at lower doses, but is proconvulsant at higher doses [26,27]. Moreover, endogenous opioids protect against seizures induced by electroconvulsive shock [28,29], and have been implicated in the anticonvulsant properties of acute stress [30]. Both the anti- and proconvulsant effects of morphine in the chemical and electrical models of seizures can be reversed by -opioid receptor antagonists [26,31]. This study investigated if the morphine sensitization affects seizure threshold. We also investigated the inuence of an opioid receptor antagonist or a nitric oxide synthase inhibitor on seizure threshold in morphine-sensitized animals. 2. Methods 2.1. Subjects Male 6- to 8-week-old NMRI mice weighing 2330 g (Pasteur Institute of Iran) were used throughout this study. Animals were housed in groups of four or ve and were allowed free access to food and water except for the short time that animals were removed from their cages for testing. All behavioral experiments were conducted between 10:00 AM and 1:00 PM with normal room light (12-hour regular light/dark cycle) and temperature (22 1 C). All procedures were carried out in accordance with the institutional guidelines for animal care and use. Each mouse was used only once, and each treatment group consisted of at least eight animals. 2.2. Drugs The following drugs were used: morphine sulfate, pentylenetetrazole, naltrexone, and L-NAME (Sigma, UK). All drugs were dissolved in physiological saline. All injections were administered in a volume of 10 mL/kg. Appropriate vehicle controls were run for each experiment. In all experiments, morphine was administered subcutaneously and all other drugs were administered intraperitoneally. To assess clonic seizure threshold, pentylenetetrazole was administered intravenously (0.5%). 2.3. Procedure In the rst step of this study, six groups of mice received six different doses of morphine. In each group of experiments, which included three subgroups of mice, sensitization was produced by daily injection of saline in subgroups 1 and 2 and of 0.1, 0.5, 1, 5, 15, or 30 mg/kg morphine in subgroup 3, for 10 days (sensitization period), followed by a 10-day washout period. Then the challenge test was performed using saline in subgroup 1 and morphine (0.1, 0.5, 1, 5, 15, or 30 mg/kg) in subgroups 2 and 3. As a control, in the seventh group, saline was administered both in the sensitization period and in the challenge test [32]. To assess clonic seizure threshold, pentylenetetrazole was administered intravenously after 60 minutes. In studies on the effect of L-NAME or naltrexone on development of sensitization, L-NAME (20 mg/kg) or naltrexone (10 mg/kg) was co-administered daily with morphine (0.5 mg/kg) during the whole

10-day sensitization period. Ten days after administration of the last sensitization dose, the challenge test was performed using morphine (0.5 mg/kg). Then clonic seizure threshold was assessed intravenously.

2.4. Determination of seizure threshold The primary objective of this study was to examine the modulation of susceptibility to myoclonic seizures induced by PTZ, which is a standard experimental model of clinical myoclonic petit mal seizures with both face and construct validity [33,34] in morphine-sensitized animals. To assess seizure susceptibility, we used the more sensitive method of intravenous administration of PTZ, which allows better detection of modulatory effects on convulsive tendencies [34]. The PTZ threshold was determined by inserting a 30-gauge buttery needle into the tail vein of unrestrained freely moving mice and infusing PTZ (0.5%) at a constant rate of 0.5 mL/minute. Infusion was halted when forelimb clonus followed by full clonus of the body was observed; the dose of PTZ administered (mg/kg mouse weight) was taken as an index of clonic seizure threshold. As such, seizure threshold is dependent on the dose of PTZ administered and time.

2.5. Statistical analysis Data are expressed as the mean SEM clonic seizure threshold for each experimental group. One-way analysis of variance (ANOVA) followed by a TukeyKramer multiple comparison test was used to analyze the data. P b 0.05 was considered the signicance level between groups.

3. Results 3.1. Effect of different doses of morphine on induction of sensitization in seizure threshold 3.1.1. Low doses Fig. 1a illustrates morphine sensitization in the PTZ-induced clonic seizure threshold. There was no signicant difference between groups of mice that received saline in the sensitization period and morphine 0.1, 0.5, or 5 mg/kg in the challenge test. But seizure threshold was increased in the groups that received morphine 0.1 mg/kg in both the sensitization period and the challenge test (P b 0.05). Seizure threshold also increased in the group that received morphine 0.5 mg/kg in both the sensitization period and challenge test (P b 0.001). Subcutaneous administration of morphine (1 mg/kg) in the challenge test increased seizure threshold compared with saline treatment when saline was injected in the sensitization period, whereas administration of morphine (1 mg/kg) in both the sensitization period and the challenge test did not alter seizure threshold compared with saline treatment (P b 0.01).

3.1.2. High doses Administration of saline in the sensitization period and 15 mg/kg morphine in the challenge test or administration of 15 mg/kg morphine in both the sensitization period and challenge test caused no signicant difference in seizure threshold compared with administration of saline in both the sensitization period and challenge test. Administration of saline in the sensitization period and 30 mg/kg morphine in the challenge test or administration of 30 mg/kg morphine in both the sensitization period and challenge test decreased seizure threshold compared with administration of saline in both the sensitization period and challenge test (P b 0.05) (Fig. 1b).

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a
Clonic Seizure Threshold (mg/kg)
100 90 80 70 60 50 40 30 20 10 0 Saline 0.1 0.5 1 5 0.1 0.5 1 5

period decreased seizure threshold compared with administration of morphine 0.5 mg/kg alone in the sensitization period (Fig. 2).
* * ***

4. Discussion This study has demonstrated that subcutaneous administration of morphine (0.1 and 0.5 mg/kg) induces sensitization in PTZ-induced clonic seizures in mice. Intraperitoneal administration of L-NAME, a nonselective inhibitor of nitric oxide synthase, or naltrexone, an opioid receptor antagonist, along with morphine inhibited morphineinduced sensitization in PTZ-induced seizure threshold. The repeated administration of opioid agonists or psychomotor stimulants can result in enhancement of some of the behavioral effects of these drugs, typically referred to as behavioral sensitization [35,36]. Most investigators have studied the motor or reward effects of morphine in morphine-sensitized animals, but to the best of our knowledge there is no study on the inuence of morphine sensitization on seizures. On the other hand, various studies have shown that opioids alter seizure susceptibility in different experimental paradigms [25,26,37,38]. In this study, for the rst time we have shown that mice given morphine (0.1 or 0.5 mg/kg) exhibit a much greater increase in PTZ-induced seizure threshold than those receiving chronic saline treatment. This difference in response to PTZ between the groups indicates the presence of sensitization. The group that received morphine (1 mg/kg) only in the challenge test had an increased seizure threshold compared with the saline group. Morphine and other -opioid receptor agonists have dual anticonvulsant and proconvulsant effects on seizures induced by the inhibitors of -aminobutyric acid (GABA) transmission like pentylentetrazole (PTZ), bicuculline, and picrotoxin [25,26,39]. In these models, opioids generally induced an anticonvulsant effect at lower doses, but were proconvulsant at higher doses [26,27]. Therefore, morphine at a dose of 1 mg/kg could act as an anticonvulsant in this experiment, while this dose did not induce sensitization. We also demonstrated that morphine 30 mg/kg, which is a high dose of morphine, acts as proconvulsant, which is consistent with previous results. High doses of morphine (15 or 30 mg/kg) did not induce sensitization in clonic seizures. We also showed that administration of naltrexone, an opioid receptor antagonist, along with morphine during the sensitization period, which blocked the morphine-induced sensitization by changing seizure threshold, is receptor specic. It has been also reported that both the anti- and proconvulsant effects of morphine in the chemical and electrical models of seizures are reversible by -opioid receptor antagonists [26,31] showing that the effect of morphine on modulation of seizure susceptibility is receptor dependent. On the other hand, Vigano et al. [19] reported that in a model of morphine sensitization that elicited a complex behavioral syndrome in rats, receptor autoradiography revealed a signicant receptor increase in different

Morphine (mg/kg) (challenge)

Morphine (mg/kg) (Sensitization+Challenge)

b
Clonic Seizure Threshold (mg/kg)
60 50 40 30 20 10 0 Saline

15

30

15

30

Morphine (mg/kg) (challenge)

Morphine (mg/kg) (Sensitization+Challenge)

Fig. 1. Effects of morphine sensitization on PTZ-induced clonic seizure threshold. Three groups of animals received daily injections of saline, saline, and morphine for 10 days (sensitization period) and were injected with saline, morphine, and morphine, respectively. Both low (0.1, 0.5, 1, and 5 mg/kg) (a) and high (15 and 30 mg/kg) (b) doses of morphine were used. Data are expressed as the mean SEM clonic seizure threshold for at least eight mice. *P b 0.05, **P b 0.01, and ***P b 0.001 compared with saline/saline group.

3.2. Effect of L-NAME on morphine-induced sensitization in seizure threshold Intraperitoneal administration of L-NAME along with subcutaneous administration of morphine 0.5 mg/kg in the sensitization period decreased seizure threshold compared with administration of morphine 0.5 mg/kg alone in the sensitization period (Fig. 2). 3.3. Effect of naltrexone on morphine-induced sensitization in seizure threshold Intraperitoneal administration of naltrexone along with subcutaneous administration of morphine 0.5 mg/kg in the sensitization

Clonic Seizure Threshold (mg/kg)

100 90 80 70 60 50 40 30 20 10 Saline Mor (0.5) Mor(0.5) Mor(0.5)+L-NAME(20) Mor(0.5)+NTX(10) Mor(0.5) Mor(0.5)

* **

0 Sensitization Saline Challenge Saline

Mor(0.5)

Fig. 2. Effect of L-NAME (20 mg/kg) or naltrexone (10 mg/kg) on development of morphine sensitization in PTZ-induced clonic seizure threshold. L-NAME or naltrexone was coadministered daily with morphine (0.5 mg/kg) over the whole 10-day sensitization period. Data are expressed as the mean SEM clonic seizure threshold for at least eight mice. *P b 0.05 and **P b 0.01 compared with morphine/morphine group.

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parts of the brain including caudate putamen, nucleous accumbens shell, prefrontal and frontal cortex, medial thalamus, hypothalamus, and central gray matter. Therefore, our results from the concomitant administration of naltrexone and morphine are in agreement with the aforementioned reports indicating that morphine-induced sensitization that occurs in PTZ-induced clonic seizures could be morphine receptor dependent. Nitric oxide, a highly reactive messenger molecule that is synthesized in a number of tissues including the brain, is regarded as a neuronal messenger or neurotransmitter in the central nervous system [4042]. NO has been implicated in morphine-induced peripheral analgesia [43,44], as well as tolerance and dependence [21,45]. Morphine and NO have also been linked in the regulation of gastrointestinal function [46], food intake [47], and morphine-induced suppression of splenic lymphocyte proliferation [48]. Morphine and nitric oxide seem to be coupled in several different processes [21,43,46]. Morphine can stimulate nitric oxide release in different tissues including rat median eminence, providing a role for NO in neurotransmitter release [49]. We also showed that both acute and chronic administration of NOS inhibitors signicantly inhibited the modulation of PTZ seizure threshold by morphine, indicating that nitric oxide is involved in both phases of morphine-induced modulation of PTZ-induced seizures. Intraperitoneal administration of L-NAME along with subcutaneous administration of morphine 0.5 mg/kg in the sensitization period decreased seizure threshold compared with administration of morphine 0.5 mg/kg alone in the sensitization period. These ndings are in line with previous studies showing that nitric oxide is implicated in the development of morphine-induced sensitization in rewarding effects [50], formalin test effects [51], state-dependent learning [52], and locomotion [23,53]. In conclusion, morphine in lower doses induced sensitization in PTZ-induced clonic seizures in mice. Interaction with receptors or nitric oxide might be involved in the morphine-induced sensitization in PTZ-induced clonic seizures. References
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