Agmatine inhibits morphine-induced locomotion sensitization and morphine-induced changes in striatal dopamine and metabolites in rats

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Abstract

The effects of agmatine on morphine-induced locomotion sensitization and morphine-induced changes in extracellular striatal dopamine (DA) and DA metabolites were studied. The locomotor response to morphine challenge (3 mg/kg, s.c.) was enhanced in rats 3 days after repeated morphine administration, indicating development of locomotion sensitization. In vivo microdialysis demonstrated a significant increase in striatal basal levels of the DA metabolites DOPAC and HVA, but not in DA itself, and an increase in DA response to morphine challenge in rats 3 days after withdrawal. Agmatine (1, 10, 80 mg/kg) inhibited morphine-induced locomotion sensitization and the changes in DA noted above. Idazoxan attenuated the effects of agmatine on locomotion, suggesting that the effects are mediated by imidazoline receptors. In addition, repeated morphine also increased the expression of tyrosine hydroxylase mRNA in the VTA after 4 days of morphine pretreatment, while decreasing the expression of dynorphin mRNA at 3 days after withdrawal. Agmatine inhibited morphine-induced changes in dynorphin, but not in tyrosine hydroxylase mRNA expression. These data suggest that agmatine, likely by activating imidazoline receptors, inhibits morphine-induced locomotion sensitization and morphine-induced changes in extracellular DA and in dynorphin expression. Thus, agmatine deserves further study as an anti-opioid medication.

Introduction

Opioid dependence is a chronic brain disease characterized by high rates of relapse after abstinence (Leshner, 1997). Robinson and Berridge, 1993, Robinson and Berridge, 2003 proposed that repeated drug reward (“liking”) and craving (“wanting”) after abstinence might lead to sensitization in brain reward systems, e.g. the mesolimbic dopamine (DA) system, which is different from the system underlying opiate withdrawal (Wise, 1996, Nestler, 2004). The mesolimbic DA system originates from DA neurons in the ventral tegmental area (VTA) of the midbrain and projects predominantly to the forebrain nucleus accumbens (NAc) and prefrontal cortex (PFC) (Wise, 1996). Opioid reinforcement has been thought to be mediated by opioid-induced inhibition of GABAergic interneurons in the VTA, which leads to disinhibition (or activation) of VTA DA neurons and an increase in DA release in the NAc (Johnson and North, 1992, Xi and Stein, 2002, De Vries and Shippenberg, 2002). Further, repeated administration of morphine may enhance the disinhibition of DA neurons and increase their sensitivity to stimulation, and therefore augment opioid addiction and relapse (Volpicelli et al., 1999, Nestler, 2004). Opioid withdrawal or withdrawal-induced negative reinforcement has been thought to be a major factor in opioid dependence and relapse (Koob et al., 1989, Koob and Le Moal, 1997). However, this hypothesis has been challenged by findings that naloxone-precipitated withdrawal failed to evoke reinstatement of opioid-seeking behavior in rats that previously self-administered morphine, whereas a challenge injection of heroin did evoke reinstatement. Such findings suggest that the positive reinforcing effect of opioids, rather than withdrawal, may be the major reason for opioid craving and relapse (Shaham et al., 1996, Shalev et al., 2002).

Current studies indicate that many non-opioid chemicals such as NMDA receptor antagonists, nitric oxide synthase (NOS) inhibitors or N-type calcium channel blockers can modulate or antagonize the pharmacological actions of opioids (Su et al., 2003, Heidbreder and Hagan, 2005). Agmatine is an endogenous, non-opioid neurotransmitter or neuromodulator (Reis and Regunathan, 1999), which can block NMDA receptors (Gibson et al., 2002) and calcium channels (Weng et al., 2003), and also inhibit NOS activity (Li et al., 1999c). It was reported that systemic administration of agmatine significantly enhances morphine analgesia (Li et al., 1999a, Yesilyurt and Uzbay, 2001, Ruiz-Durantez et al., 2003), inhibits the development of tolerance to morphine analgesia (Kolesnikov et al., 1996, Li et al., 1998, Li et al., 1999b, Fairbanks and Wilcox, 1997), attenuates ethanol and morphine withdrawal syndromes (Uzbay et al., 2000, Aricioglu-Kartal and Uzbay, 1997, Li et al., 1998, Li et al., 1999b), and inhibits opioid (fentanyl) self-administration (Morgan et al., 2002). In addition, agmatine also inhibits the acquisition, expression or reinstatement of morphine-induced conditioned place preference (Wei et al., 2005). These effects were blocked selectively by blockade of imidazoline receptors, but not by blockade of other receptors or proteins (Reis and Regunathan, 2000, Su et al., 2000, Su et al., 2003, Wu et al., 2005), suggesting imidazoline receptor-mediated effects by agmatine.

Locomotion sensitization is another commonly used animal model to study neural mechanisms underlying drug craving and relapse (Robinson and Berridge, 1993, Stewart and Badiani, 1993). The repeated, intermittent administration of opioids or psychostimulants produces persistent increases in locomotion and incentive motivation (Vanderschuren and Kalivas, 2000), manifested by increased locomotor activity and other behavioral responses upon re-exposure to opioids or psychostimulants (Kornetsky, 2004). There is increasing evidence that the mesolimbic DA system plays an essential role in the mediation of locomotion sensitization to morphine (Vanderschuren and Kalivas, 2000). In the present study, we first employed in vivo microdialysis with HPLC techniques to observe repeated morphine-induced changes in basal levels of DA and its metabolites (DOPAC, HVA) and in the DA response upon re-exposure to morphine. Then, we observed the effects of agmatine on morphine-induced locomotion and locomotion sensitization as well as on changes in extracellular striatal DA. In addition, evidence also shows that repeated administration of morphine increases the expression of transcription factor cAMP-response-element-binding protein (CREB) and ΔFosB, a truncated splice variant of the FosB gene, which may subsequently alter the expression of tyrosine hydroxylase (TH) and dynorphin (Dyn) mRNA (Kreek, 1997, Berke and Hyman, 2000, Nestler et al., 2001). Dyn is co-existed with GABA in GABA-containing projection neurons in the NAc and dorsal striatum and it acts as a negative feedback mechanism: it is released from the terminals of neurons from the striatum and activates κ opioid receptors located on nerve terminals and cell bodies of DA-containing neurons in the VTA and inhibits their activity (Hyman, 1996, Kreek, 1997, Shippenberg and Rea, 1997). ΔFosB, by inhibiting Dyn expression, may down-regulate this feedback loop and enhance the rewarding properties of drugs of abuse. On the other hand, CREB may enhance Dyn expression and thereby attenuate the rewarding properties of drugs of abuse (Nestler et al., 2001). TH, the rate limiting enzyme in DA biosynthesis, is another possible target gene of ΔFosB and CREB. Thus, Dyn and TH might be important factors that regulate brain DA levels. So we further observed the effects of agmatine on such repeated morphine-induced changes in VTA TH and striatal Dyn mRNA levels.

Section snippets

Animals

Male Wistar rats (Beijing Animal Center, China) initially weighing 200–240 g were used in the present study. The rats were grouped 6 per cage and acclimated to the housing conditions and handled daily for 3–4 days before the experiments began, to minimize handling stress during testing. Animals were maintained on a 12 h light/dark cycle (lights on between 7:00 A.M. and 7:00 P.M.) and given ad libitum access to food and water, in strict compliance with the guidelines set for the use of

Effect of agmatine on morphine-induced locomotor activity

As shown in Fig. 1A, acute morphine (10 mg/kg morphine, no agmatine, on day 1) significantly increased locomotor activity, when compared with saline in drug naïve rats (t = 4.45, P < 0.01, n = 8). Co-administration of agmatine (1, 10, 80 mg/kg) with morphine mildly inhibited morphine-induced increase in locomotor activity in rats [F(3,27) = 2.72, P = 0.06, n = 7–8]. Individual group comparisons using the Student's t-test showed that agmatine, at 80 mg/kg, significantly inhibited morphine-induced increases

Discussion

The present study starts by confirming previous reports (Powell and Holtzman, 2001) that 4 days of repeated morphine produces an increase in rat locomotion in response to morphine challenge 3 days after morphine withdrawal, suggesting the development of locomotion sensitization. Agmatine pretreatment significantly inhibited morphine-induced locomotion sensitization, which was blocked by pretreatment with idazoxan, a selective imidazoline receptor antagonist. Agmatine itself did not alter

Role of the funding source

This project was supported by the National Basic Research Program of China (No. 2003CB515400) and National Natural Science Foundation of China (No. 30472017). The roles of the two funding sources are both basic research work.

Contributors

Xiao-Li Wei has contributed to most of the works including behavioral study and HPLC detection. Rui-Bin Su has taken part in the design of the whole experiment and participated in the work of HPLC detection. Ning Wu has participated in some of the work of dopamine determination. Xin-Qiang Lu has attended the behavioral study. Jian-Quan Zheng has taken part in the design of the whole experiment and helped to modify the manuscript carefully. Jin Li has taken part in the design of the whole

Conflict of interest

There is not any conflict of interest about this work.

Acknowledgements

This project was supported by the National Basic Research Program of China (No. 2003CB515400) and National Natural Science Foundation of China (No. 30472017). We thank Dr. Eliot L. Gardner at the National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health in the USA for his constructive comments and suggestions during the preparation of the manuscript.

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