Pathophysiology 16 (2009) 37

Treadmill exercise reduces self-administration of morphine in male rats

Mahmoud Hosseini a, , Hojjat Allah Alaei b , Asieh Naderi b , Mohammad Reza Shari b , Reza Zahed c
a

Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran b Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran c Tehran University of Medical Sciences, Tehran, Iran Received 5 October 2008; accepted 23 November 2008

Abstract Exercise can activate the same pathways as morphine. The aim of the present study was to clarify the effect of short-term and mid-term exercises on the self-administration of morphine in rats. Male Wistar rats were initially trained to receive small pellets of food by pressing the active lever in self-administration apparatus. Rats were divided into 4 groups: Saline, Morphine, Exercise 1 (11 days) and Exercise 2 (30 days). Their jugular vein was cannulated. The animals were placed in self-administration apparatus and allowed to self-administer morphine (0.5 mg per infusion all test groups) or saline (Saline group) during consecutive days, for 2 h/sessions. In the group 1 the rats were running before each session of self-administration and of group Exercise 2, 30 days before surgery as well as before each session. The pressing numbers of active and passive levers in each group and among different groups were compared. The number of active lever pressing of Morphine group was signicantly higher than Saline group (p < 0.001). In Exercise 1 and Exercise 2 groups, the number of active lever pressing was signicantly lower than Morphine group (p < 0.001). As exercise can activate many neurotransmitter systems involved in the addiction process and increase the release of endorphins, it is likely that could decrease the morphine self-administration in this experimental setup. 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Exercise; Morphine; Rat; Self-administration

1. Introduction In the general population the prevalence of addiction varies 316% [1]. It has been suggested that opiate abuse causes long-lasting neural changes in the brain that underpin the behavioral abnormalities associated with cognitive decits, tolerance, and dependence [2]. Addiction is a chronic disorder that requires long-term treatment. Medications have been developed that add to the benets of psychosocial interventions for the prevention of relapse. These medications generally reduce drug craving and reduce the likelihood of relapse to compulsive drug use. Nevertheless treatments most of these methods have given unsatised results until now [1]. According to the results of previous studies, several neurotransmitter systems, including dopamine, glutamate, acetylcholine, GABA, histamine and nitric oxide

Corresponding author. Fax: +98 511 8828564. E-mail address: hosseinim@mums.ac.ir (M. Hosseini).

have been implicated in rewarding properties of morphine [3,4]. Regular physical exercise promotes neural health and function. Both animal and human studies have shown that exercise facilitates recovery from injury [5] and improves cognitive function [68]. Physical exercise also inuences the central dopaminergic, noradrenergic, and serotonergic systems [911]. In particular, exercise leads to the release of certain neurotransmitters in the brain that alleviate both physical and mental pain. Additionally, it is one of the few ways to generate new neurons. Much of the research done in this area has focused on running, but all types of aerobic exercise provide benets. Exercise exerts its effects on the brain through several mechanisms, including neurogenesis, mood enhancement, and endorphin release [12]. Recent ndings have shown that endorphin levels, primarily beta-endorphin, are elevated with exercise [13] in relation to the intensity of the exercise. Others have not, however, found evidence for this relation [14]. More specically, it

0928-4680/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2008.11.001

M. Hosseini et al. / Pathophysiology 16 (2009) 37

has been proposed that elevation in -endorphin produces an opiate-like euphoria leading to positive mood states including the so-called runners hige [12]. Findings from several studies have also led some investigators to propose that exercise produces cross-tolerance to opioid agonists [13]. This hypothesis is supported by studies demonstrating that chronic and voluntary wheel running decreases the antinociceptive response to mu-opioid drugs [14,15]. In contrast, chronic and voluntary exercise slightly increases aversive responses to the kappa opioid spiradoline in the CPP task, while simultaneously decreasing the antinociceptive response to spiradoline [16]. It has been suggested, if exercise can produce catecholamines (when production is reduced by drug use) and act as a catalyst in promoting neuronal growth, then exercise could be used as a valuable aid to help people to recover from addiction [17]. On the other hand some of evidences show that exercise can change the level of some neurotransmitters involved in rewarding properties of morphine. Recently it has been shown that long-term compulsive exercise reduces the rewarding efcacy of 3,4-methylene dioxymethamphetamine (MDMA) and also could abolish acute MDMA-stimulated dopamine release in nucleus accumbens [18]. Therefore the aim of the present study was try to clarify the effect treadmill running on morphine self-administration in rats.

cannula was connected to an infusion pump via a swivel, allowing the animal to move relatively freely. Pressing of the active lever, marked by a red light, resulted in a 10 s infusion of 0.1 ml uid through an infusion pump. The uid was saline in the Saline group and Morphine (5 mg/ml) in the other groups. Depression of the active lever during this time (10 s) did not affect the infusion of the drug. Pressing of the passive lever had no programmed consequences. In this study, the number of lever pressing was regarded as a measure of the reinforcing action of the drug [19,20]. 2.3. Experimental design Male rats (n = 32) were randomly selected, and were divided into 4 groups: (I) Saline group, which received 0.1 ml saline in the self-administration sessions. (II) Morphine group, which received 0.1 ml morphine (5 mg/ml) during the self-administration sessions. (III) Exercise 1 group (an 11-day exercise), rats were running for 90 min daily on the treadmill and upon a 30-min break, they were placed in the self-administration apparatus and received morphine as same as the animals of Morphine group. (IV) Exercise 2 group (a 30-day exercise), the animals were running for 30 days before cannulation and then before each morphine self-administration session. 2.4. Training phase

2. Materials and methods 2.1. Animals and their housing Before surgery, male Wistar rats (250300 g) were grouphoused and received food and water ad libitum. They were maintained under a daynight cycle with lights on between 07:00 and 19:00. Ten days before starting the experiments, the daynight cycle was reversed, and the animals were tested in the dark phase. Before the tests, food and water were available ad libitum in the home cages. During the training phase and the rst 6 days of the experimental period, they had food restriction in their cages. Experimental plan was approved by the Isfahan University Committee on Animal Research. 2.2. Self-administration apparatus Briey, to aid in acquisition of drug self-administration, rats were initially trained to press a lever using food as reinforcement before being surgically implanted with a chronic intravenous (iv) jugular catheter. Training and testing were done in standard operant conditioning cages (21 cm 21 cm 28 cm) placed in a sound-attenuated room, ventilated with fans, based on the method used previously by others [19,20] with minor modications. The apparatus was equipped with active and passive levers, 2 cm above the oor, with a red light located 4 cm above the active lever. The iv

Ten days before the experiments, the animals were transferred to a special room and the daynight cycle was reversed (light on at 7 p.m.), and the experiments were carried out during the dark phase. Before the surgery, the training program was started after 24 h food restriction. The animals were placed in the self-administration apparatus where a lever lled with food pellets was available. Each lever pressing resulted in the delivery of a 100-mg pellet [20]. Each rat allowed self-training until 40 pellets being received. Following acquisition of lever pressing behavior, rats were returned to ad libitum food and allowed to gain their weight for 3 days, before the surgery was done [20,21]. 2.5. Wheel running Before running on the treadmill a few days before the beginning of the experiments the rats were introduced with the apparatus to learn how to run on it. Doing that identied the sick or lazy rats which then excluded them from the next steps. The running period for exercise 1 group was 90 min for 11 days with the level slope of 15 and the rate of 15 m/min [22,23] and then after a 30-min break the rats were inserted in a self-administration apparatus. In Exercise 2 group, the rats ran 90 min each day for 30 days, then iv cannulation was carried out and then they were allowed to recover from surgery and nally, they were placed in the self-administration apparatus. The animals of this group ran 90 min before each session. The rats in Saline and Morphine

M. Hosseini et al. / Pathophysiology 16 (2009) 37

groups ran 15 min 3 days a week to experience the similar situations. 2.6. Surgical procedures Animals were anaesthetized with intraperitoneal injection of ketamine (150 mg/kg) and rampon (0.1 mg/kg), and a cannula was inserted into the jugular vein. The cannula was guided subcutaneously up to the skull and connected to polyethylene tubing where it was xed to a metal tube and secured to the skull with small screws, xed with dental acrylic cement. After surgery, rats were given 300,000 units of procaine penicillin G (i.p.) to prevent infection. The animals were allowed 57 days to recover from surgery [24]. 2.7. Self-administration phase Seven days after recovery and following 24 h of food restriction, rats were placed in the operant chambers where a lever lled with food pellets was available. Each active lever pressing resulted in the delivery of a 100-mg pellet. The jugular cannula of rats was connected to an infusion pump, and the animals were placed in the self-administration apparatus for 2 h each day on an FR-1 schedule [20]. The trained animals were allowed to press active and passive levers freely. By pressing the active lever, the rats received 0.1 ml of morphine or saline and small pellets in the rst 69 days, and saline or morphine without pellets in the nal 5 days of the experiment. Pressing the passive lever did not deliver uid or food. In the rst 69 of self-administration period, the availability of food was restricted in order to reduce body weight by 15% which has been shown to facilitate the initiation of intravenous self-administration [21]. Changes of less than 15% in the number of injection in the last 3 days were regarded as baseline. On the next 5 days, the animals had free access to their ad libitum food. Catheters were ushed daily with 0.1 ml saline containing heparin sulfate (50 U/ml) during the recovery period as well as before and after the self-administration sessions. All operant sessions were conducted during the animals dark cycle. Catheter potency was tested by the injection of 0.1 ml of sodium pentobarbital solution (10 mg/ml) into the catheter and observation of animal behavior. Animals with patent catheters exhibit prominent signs of anesthesia (loss of muscle tone) a few seconds after the administration [20]. 2.8. Data analysis Data have been presented as means SEM. The number of active and passive lever pressings of nal 5 days was compared in each group and also the number of active lever pressing between different groups was compared using repeated-measures one-way analysis of variance (ANOVA) and Tukey post hoc comparisons. The criterion for statistical signicance was p < 0.05.

3. Results In Saline group there was no signicant difference between the number of active and passive lever pressing (Fig. 1A). In Morphine group, the number of active lever pressing was signicantly higher than passive one in all days followed (Fig. 1B) (p < 0.001). This indicates that the animals pressed the active lever for morphine. In Exercise 1 (11-day

Fig. 1. The effects of morphine without and with exercise on the active (to receive morphine) and passive lever pressing tested in a morphine self-administration apparatus. Rats were cannulated into jugular veins and received saline (Saline group) or morphine (Morphine, Exercise 1 and Exercise 2 groups) (8 rats in each group) in a morphine self-administration apparatus. The animals of Exercise 1 group were running 90 min before each session and of Exercise 2 group 30 days before surgery as well as before each session. The numbers of active and passive lever pressings were compared in each group using the repeated measure ANOVA. Data are shown as means SEM. (A = Saline group), (B = Morphine group), (C = Exercise 1 group) and (D = Exercise 2 group). The number of active lever pressing in Morphine and Exercise groups was higher than passive ones but in Saline group there was no difference between active and passive lever pressing.

M. Hosseini et al. / Pathophysiology 16 (2009) 37

Fig. 2. Results of active lever pressing between 4 groups (Saline, Morphine, Exercise 1 and Exercise 2) (8 animals each). See also the legend in Fig. 1. The number of active lever pressing of Morphine group was higher than Saline group and the number of active lever pressing in Exercise 1 and Exercise 2 groups was lower than morphine group (p < 0.001).

Fig. 3. Results of passive lever pressing between all groups of animals (Saline, Morphine, Exercise 1 and Exercise 2). See also the legend in Fig. 1. No signicant difference in the number of passive lever pressing was observed between groups.

exercise) and Exercise 2 (30-day exercise) groups the number of active lever pressing was signicantly higher than passive one in all days, but the number of pressings was about half of that in nonexercising Morphine group (Fig. 1C and D) (p < 0.001). The comparison of active lever pressings between Morphine and Saline groups showed that the number of active lever pressing in Morphine group was higher than in the Saline group (Fig. 2, P < 0.001). In the both Exercise groups receiving morphine the pressing of the active lever was also signicantly lower than in the group receiving morphine without exercise, but there was no signicant difference between Exercise 1 and Exercise 2 groups (Fig. 2). As Fig. 3 shows, there were no signicant differences in the number of passive lever pressing between all groups.

4. Discussion The results of present study showed that treadmill running could reduce active lever pressing and thus also in the intake morphine. These results agree with the other studies which showed that treadmill running exercise decreased the 3,4methylenedioxymethamphetamine (MDMA)-induced CPP

in an exercise duration-dependent manner with no evident MDMA-induced CPP in 12-week exercised mice and a moderate magnitude of drug-induced CPP performance in 8-week exercised mice [18]. In another study, experimental rhythmic exercise could activate the central opioid systems [25]. These researchers have suggested that exercise could be used to treat patients with addiction disorder [25]. Physical activity is assumed to maintain and enhance physical and mental health. Long-term regular exercise in human subjects is reported to increase self-esteem and anxiety relive [26]. Previous studies have shown that exercise stimulates the release of endogenous opioid peptides such as endorphins within approximately 30 min from the start of activity. These endorphins tend to minimize the discomfort of exercise and are even associated with a feeling of euphoria. In the present study we showed that physical exercise can also reduce carving behavior of rats for morphine. Some researchers have suggested that the rewarding effect of wheel running is mediated by endogenous opioids i.e. the wheel running must activate at least some of the same systems that are activated by morphine and other opiates [13]. It has been suggested that during chronic, long-term exercise the sensitivity to the effects of morphine and other mu opioids decreases [16]. The result of this study may be at least in part, due to activation of endogenous opioid system by treadmill running. This idea have been conrmed by the other studies, which have shown that CSF beta-endorphin levels were increased by voluntary chronic running in rats, and it remained high for the rst 2 days after the interruption of running [16,26,27]. All of these evidences show that endogenous opioid levels increase after exercise. In other studies the addiction has been attributed to endorphin deciency [3]. In this study, we propose that lower craving to morphine was partly due to the activation of the endogenous opioid system. In addition to endogenous opioid system, physical exercise inuences the central dopaminergic, noradrenergic and serotonergic systems [28]. There is evidence in favor of changes in synthesis and metabolism of monoamines during exercise [28]. Experiments with rats have been demonstrated that exercise affects the regional levels of glutamate, glutamine and GABA [29]. The result of present study may be due to each of these neurotransmitters. However, the dopaminergic mesolimbic system that consist of ventral tegmental area (VTA), nucleus accumbens and medial prefrontal cortex is considered to be crucial in the rewarding actions of opiates and involved in drug dependence [30]. Chen et al. [18] have shown that 12 weeks of exercise intervention abolishes acute MDMA-stimulated dopamine release in nucleus accumbens and also rewarding efcacy of this drug. The result of present study showed that exercise could reduce rewarding properties of morphine. We suggest that the dopaminergic mesolimbic system may have some role in the result of this present study. In 1988, Palmer showed that exercise was a useful component in their alcohol treatment program [31]. Grove and

M. Hosseini et al. / Pathophysiology 16 (2009) 37

Wilkinson [32] indicated a positive effect on craving as well as lower levels of confusion resulting from a 1-week exercise treatment used in a smoking cessation program. Wendt [17] showed that exercise signicantly changed behavior and in a program designed for the participation at least 5 days per week for 40 min per session might yield signicant results for both prevention and recovery. In previous study we showed that treadmill running reversed short-term memory-decit by morphine in rats [23]. In conclusion, the present study showed that short-term as well as moderate-term exercise decreased craving to morphine in rats. We are currently evaluating the endorphin and other neurotransmitter secretions with the aid of micro dialysis method of the brains.

References
[1] S.R. Savage, Long-term opioid therapy: assessment of consequences and risks, J. Pain Symptom Manage. 11 (1996) 274286. [2] J.T. Williams, M.J. Christie, O. Manzoni, Cellular and synaptic adaptations mediating opioid dependence, Physiol. Rev. 81 (2001) 299343. [3] H. Alaei, A.A. Pourshanazari, A. Rafati, Electrical stimulation of nucleus raphe dorsalis changes morphine self-administration and withdrawal symptoms in rats, Pathophysiology 9 (1) (2002) 1. [4] M.R. Zarindast, S. Ahmadi, A. Haeri-Rohani, A. Rezayof, M.R. Jafari, M. Jafari-Sabet, GABA (A) receptor in the basolateral amygdale are involved in mediating morphine reward, Brain Res. 1006 (2004) 4958. [5] B.B. Johansson, A. Ohlsson, Environment, social interaction, and physical activity as determinants of functional outcome after cerebral infarction in the rat, Exp. Neurol. 139 (1997) 322327. [6] D. Sutoo, K. Akiyama, Regulation of brain function by exercise, Neurobiol. Dis. 13 (2003) 114. [7] A.F. Kramer, S. Hahn, N.J. Cohen, M.T. Banich, E. McAuley, C.R. Harrison, J. Chason, E. Vakil, L. Bardell, R.A. Boileau, A. Colcombe, Fitness and neurocognitive function, Nature 400 (1999) 418419. [8] N. Ahmadiasl, H. Alaei, O. Hnninen, Effect of exercise on learning, memory and levels of epinephrine in rats hippocampus, J. Sports Sci. Med. 2 (2003) 106109. [9] L.F. Berkman, High, usual and impaired functioning in communitydwelling older men and women: ndings from the Mac Arthur Foundation Research Network on Successful, Aging J. Clin. Epidemiol. 46 (1993) 11291140. [10] A.F. Schinder, M. Poo, The neurotrophin hypothesis for synaptic plasticity, Trends Neurosci. 23 (2000) 639645. [11] B. Lu, A. Chow, Neurotrophins and hippocampal synaptic transmission and plasticity, J. Neurosci. Res. 58 (1999) 7687. [12] M.K. Mc Govern, The effects of exercise on the brain, Biology 202 (2005) 1. [13] B.T. Lett, V.L. Grant, M.T. Koh, G. Flynn, Prior experience with wheel running produces cross-tolerance to the rewarding effect of morphine, Pharmacol. Biochem. Behav. 72 (2001) 101105. [14] R.B. Kanarek, A.V. Gerestein, R.P. Wildman, W.F. Mathes, K.E. Danci, Choronic running-wheel activity decreases sensitivity to morphineinduced analgesia in male and female rats, Pharmacol. Biochem. Behav. 61 (1998) 1927.

[15] M.A. Smith, M.A. Lyle, Chronic exercise decreases sensitivity to mu opioids in female rats: correlation with exercise output, Pharmacol. Biochem. Behav. 85 (2006) 1222. [16] M.A. Smith, D.L. Yancey, Sensitivity to the effect of opioids in rats with free access to exercise wheels: mu-opioid tolerance and physical dependence, Psychopharmacology 168 (2003) 426434. [17] S. Wendt Michael, Changing brain chemistry with intense exercise for drug addiction prevention and recovery, Research paper presentation for treating addictions in special populations: Research Confronts Reality 78 (2002) 17161751. [18] H.I. Chen, Y.M. Kuo, C.h. Liao, C.J. Jen, A.M. Huang, C.G. Cherng, S.W. Su, L. Yu, Long-term compulsive exercise reduces the rewarding efcacy of 3,4-ethylenedioxymethamphetamine, Behav. Brain. Res. 187 (2008) 185189. [19] H. Alaei, M. Esmaeili, A. Nasimi, A. Pourshanazari, Ascorbic acid decreases morphine self-administration and withdrawal symptoms in rats, Pathophysiology 12 (2005) 103107. [20] H. Sahraei, G. Poorheidari, M. Foadaddini, H. Sahraei, G. Poorheidari, M. Foadaddini, A. Khoshbaten, A. Asgari, A. Noroozzadeh, H. Ghoshooni, S.H. Firoozabadi, M.R. Zarrindast, Effects of nitric oxide on morphine self-administration in rat, Pharmacol. Biochem. Behav. 77 (2004) 111116. [21] H. Sahraei, F. Motamedi, A. Khoshbaten, M.R. Zarrindast, Adenosines A (2) receptors inhibit morphine self-administration in rats, Eur. J. Pharmacol. 383 (1999) 107113. [22] H. Alaei, R. Moloudi, A.R. Sarkaki, H. Azizi-Malekabadi, O. Hanninen, Daily running promotes spatial learning and memory in rats, Pathophysiology 14 (2) (2007) 105108. [23] H. Alaei, L. Borjeian, M. Azizi, S. Orian, A. Pourshanazari, O. Hanninen, Treadmill running reverses retention decit induced by morphine, Eur. J. Pharmacol. 536 (2006) 138141. [24] S.Y. Kim, N. Chudapongse, S.M. Lee, M.C. Levin, J.T. Oh, H.J. Park, I.K. Ho, Proteomic analysis of phosphotyrosyl proteins in morphine-dependent rat brains, Brain Res. Mol. Brain. Res. 133 (2005) 5870. [25] P. Thoren, J.S. Floras, P. Hoffmann, D.R. Seals, Endorphins and exercise: physiological mechanisms and clinical implications, Med. Sci. Sports Exercise 22 (1990) 417428. [26] B.S. Brown, T. Payne, C. Kim, G. Moore, P. Krebs, W. Martin, Chronic response of rat brain norepinephrineand serotonin levels to endurance training, J. Appl. Physiol. 46 (1979) 1923. [27] W.F. Mathes, R.B. Kanarek, Chronic running wheel activity attenuates the antinociceptive actions of morphine and morphine-6-glucoronide administration into the periaqueductal gray in rats, Pharmacol. Biochem. Behav. 83 (2006) 578584. [28] R. Meeusen, K. De Meirleir, Exercise and brain neurotransmission, Sports Med. 20 (1995) 160188. [29] C.Y. Guezennec, A. Abdelmalki, B. Serrurier, D. Merino, X. Bigard, M. Berthelot, C. Pierard, M. Peres, Effects of prolonged exercise on brain ammonia and amino acids, Int. J. Sports Med. 19 (1998) 323327. [30] H. Alaei, M. Hosseini, Captopril attenuate conditioned induced place preference by morphine and morphine withdrawal signs in rat, Pathophysiology 14 (2007) 5560. [31] J. Palmer, N. Vacc, J. Epstein, Adult inpatient alcoholics: physical exercise as a treatment intervention, J. Stud. Alcohol 49 (5) (1988) 418421. [32] J.R. Grove, A. Wilkinson, Effects of exercise on selected correlates of smoking withdrawal, Int. J. Sport Psychol. 24 (1993) 217236.