European Neuropsychopharmacology (2014) 24, 1557–1566

www.elsevier.com/locate/euroneuro

Uridine decreases morphine-induced behavioral sensitization by decreasing dorsal striatal dopamine release possibly via agonistic effects at GABAA receptors Ping Liua, Chunfu Wua, Wu Songa, Lisha Yua, Xiaofeng Yanga, Rongwu Xiangb, Fang Wanga, Jingyu Yanga,n a

Department of Pharmacology, Shenyang Pharmaceutical University, Box 31, 103 Wenhua Road, 110016 Shenyang, PR China b Mathematics Teaching & Research Section, Shenyang Pharmaceutical University, 110016 Shenyang, PR China Received 20 December 2013; received in revised form 14 June 2014; accepted 20 June 2014

KEYWORDS

Abstract

Uridine; β-alanine; Morphine; Dopamine; Dorsal striatum; Microdialysis

Uridine, a potential endogenous neuromodulator, has been demonstrated to interact with the dopaminergic system and to regulate dopamine-related behaviors. The present study investigated the effects of uridine on morphine-induced hyperactivity and behavioral sensitization and on modulating dopaminergic neurotransmission in mice, which may help to understand how uridine and its metabolites act as modulators of the GABAA receptors. The results showed that either systemic (30 or 100 mg/kg) or central (30, 100 or 300 nM) uridine administration significantly attenuated the hyperactivity induced by acute morphine treatment in mice. Intracerebroventricular administration of uracil and β-alanine also inhibited morphine-induced hyperactivity. Uridine, a known modulator of the GABA receptors, increased the extracellular levels of GABA in the brain. In addition, the GABAA receptors antagonist bicuculline significantly attenuated the effects of uridine on morphine-induced hyperactivity, suggesting that the GABAA receptors potentially mediate the effects of uridine and its metabolites on morphine-related activity. It was also observed that morphine-induced locomotor sensitization was abolished after chronic uridine treatment. In vivo microdialysis demonstrated that uridine reversed morphine-induced dopamine release in the dorsal striatum of morphine-sensitized mice. In conclusion, these data suggest that the therapeutic effects of uridine and its metabolites on morphine-induced hyperactivity and

n

Corresponding author. Tel./fax: +86 24 23986339. E-mail address: [email protected] (J. Yang).

http://dx.doi.org/10.1016/j.euroneuro.2014.06.010 0924-977X/& 2014 Elsevier B.V. and ECNP. All rights reserved.

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1.

Introduction

Repeated intermittent administration of morphine causes an augmentation of its psychomotor-stimulatory effects, termed behavioral sensitization (Hao et al., 2007a; Kalivas et al., 1998), which provides a practical animal model for screening drugs that have the potential to modulate drug craving (Shuto et al., 2006). The neurobiological mechanisms underlying behavioral sensitization are thought to be associated with alterations in GABA receptor activity and dopamine (DA) transmission in some specific brain regions, including those mediating incentive motivation and drug reward (Ojanen et al., 2003). It has been reported that GABA receptor agonists abolish morphine-induced behavioral sensitization via inhibiting DA neurotransmission (Leite-Morris et al., 2002). Uridine, besides its classic pyrimidine metabolism function, is a potent neuromodulator in the central nervous system (CNS) and there is evidence that it has neuroregulatory effects, such as sleep-promoting and antiepileptic actions, improving memory function and affecting neuronal plasticity, etc. (Dobolyi et al., 2011; Yamamoto et al., 2011). It has been shown that uridine is able to alter central dopaminergic activity and dopaminergic-mediated behaviors, as well as to interact with GABA binding sites (Guarneri et al., 1983, 1985; Kimura et al., 2001). For example, in rodents, chronic uridine treatment modulates amphetamine-induced hyperactivity and enhances the release of DA in the striatum (Myers et al., 1993), reduces rotation after repeated injections of methamphetamine (Myers et al., 1995), and modulates the rate of recovery of striatal DA receptors after their irreversible blockade (Farabegoli et al., 1988). Collectively, the reduction in the number of DA binding sites, the reduction in DA release, and modulation of haloperidol-induced catalepsy are all indicative of a dopaminergic antagonist property of uridine (Myers et al., 1993). The striatum is one of the nuclei of the CNS in which endogenous opioids and their receptors (μ, δ and κ) are abundantly expressed (Mansour et al., 1995). Our previous study has shown that acute morphine treatment induced endogenous uridine release in mouse dorsal striatum (Song et al., 2013). There is also evidence that the extracellular levels of uracil, the main metabolite of uridine, are modulated by morphine in mice (Kulkarni et al., 1997; Wang et al., 2009). Thus, it is assumed that uridine in the CNS may be involved in morphine-related neurological and behavioral symptoms. Furthermore, uridine or its metabolites may be endogenous ligands for GABAergic receptors (Connolly and Duley, 1999), and the generation of uridine metabolites is involved in the physiological and pathological effects of uridine (Peters et al., 1987a, 1987b). Uridine is degraded to uracil by uridine phosphorylase (Upase), while uracil is further broken down to β-alanine by

β-ureidopropionase (DPD) (Ipata et al., 2010; Yamamoto et al., 2011). β-alanine did fulfill the characteristics of a neurotransmitter, and it was structurally similar to GABA, modulated GABA uptake and exerted some pharmacological actions on GABA receptor-mediated processes (Tiedje et al., 2010). Accordingly, in the present study we investigated whether uridine was able to circumvent acute morphineinduced hyperactivity and established behavioral sensitization. In addition, we employed in vivo microdialysis and HPLC to observe the effects of uridine and its metabolites on the changes in extracellular DA levels in the dorsal striatum of freely moving mice.

2. 2.1.

Experimental procedures Animals

Male KM mice, 6 to 8 weeks old, weighing 20–25 g (Baudry et al., 2010), were supplied by the Experimental Animal Centre of Shenyang Pharmaceutical University. The mice were maintained under standard housing conditions in a 12 L:12 D light/dark cycle. Experiments were performed during the light phase. The mice were given free access to food and water throughout the experiments. All animals were treated in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China.

2.2.

Drugs

Drugs were obtained from the following sources: morphine hydrochloride from Shenyang First Pharmaceutical Factory (China); DA, uridine, uracil and bicuculline from Sigma (St. Louis, MO, USA); suramin from J&K Chemical Company (China). Artificial cerebrospinal fluid (ACSF) contained 147 mM NaCl, 4 mM KCl, and 2.3 mM CaCl2. For intraperitoneal (i.p.) injection, drugs were dissolved and adjusted to the final concentrations with 0.9% saline and injected in a volume of 1.0 ml/kg, in which the dose of morphine was quantified by hydrochloride; for intracerebroventricular (i.c.v.) administration, drugs were dissolved in ACSF. All the other chemicals used were of the highest available purity.

2.3.

I.c.v. injection

Mice were anesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a Kopf stereotaxic apparatus (David Kopf, Tijunga, CA, USA). A 26-gauge stainless steel guide cannula was inserted into the lateral cerebral ventricle (A/P 0.2 mm, M/L +1.0 mm, D/V 2.5 mm, Fig. 1B) unilaterally according to the mouse brain atlas (Paxinos and Franklin 2001) and was fixed to the skull with dental cement. When the dental cement had hardened, a dummy cannula, cut to the same dimension as the guide cannula, was inserted to seal the top of the guide cannula to prevent clogging and minimize possible risk of infection. The mice were allowed to recover for 5 days, before injection was made by inserting a 33-gauge stainless steel injector tube into the guide cannula. The injector tube was

Uridine decreases morphine-induced behavioral sensitization by decreasing dorsal striatal dopamine release possibly 1559

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Fig. 1 The location of the i.c.v. injection (panels A and C) and microdialysis probe (panels B and D) in the dorsal striatum and the induction of morphine behavioral sensitization of mice. Effects of acute morphine on locomotor activity in mice. Locomotor activity was recorded for 120 min immediately after acute administration of saline or morphine (10 and 20 mg/kg, i.p.), n =7–8, statistical analysis was performed by a one-way ANOVA with post-hoc Tukey's HSD test. nnPo0.01, nnnPo0.001 vs. saline group (panel E). Effects of a challenge injection of morphine (10 mg/kg, i.p.) on day 13 on locomotor activity of mice treated with morphine or saline from day 1 to day 7. Morphine was given once daily for 7 days followed by 5 days of withdrawal. On day 1, day 7 and day 13, locomotor activity was recorded for 120 min immediately after morphine injection. Results were expressed as mean locomotor counts (mean7S.E.M.) in 120 min (n=8). Locomotor activity was analyzed using a Student's t-test to compare the difference between two experimental groups on day 1, day 7 and day 13. nnPo0.01 vs. saline group (panel F).

attached to a PE-10 tubing fitted to a 10-μl Hamilton syringe (Hamilton, NV, USA). 1 μl of solution was infused into the lateral cerebral ventricle over 5 min by a microinfusion pump (Razel Scientific Instruments, St. Albans, VT, USA) (Qi et al., 2008). All the dose of drug given by i.c.v. injection was quantified by molar, the dosing volume was 1 μl.

2.4.

Behavioral sensitization schedule

2.4.1. Locomotor activity procedure Locomotor activity was determined using an area sensor composed of eight cages of equal size under low illumination. The protocol for locomotion sensitization induced by morphine was the same as we

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previously reported (Hao et al., 2007b), including 7 days of morphine treatment once daily, followed by 5 days of withdrawal. Morphine challenge (10 mg/kg, i.p.) testing was performed on day 13. Locomotor activity was monitored on day 1, day 7 and day 13. Mice were injected with morphine or saline in their home cages. Immediately after the morphine injection, the mice were placed into the locomotion chambers to record locomotor activity for 2 h. In the saline control group of mice, the treatment was identical except that morphine was replaced by saline during the drug injections. Finally, morphine (10 mg/kg) challenge injections were given on day 13 in both the morphine- and saline-treated groups.

2.4.2. Effects of uridine, uracil or β-alanine on acute morphine-induced increases in locomotor activity To investigate the effects of uridine on acute morphine-induced locomotion, different doses of uridine were centrally (10, 30, 100 and 300 nM, i.c.v.) or systemically (10, 30, 100 and 1000 mg/kg, i. p.) injected into mice 30 min before morphine (10 mg/kg) or saline administration. The effects of the uridine metabolites, uracil (10, 30, 100 and 300 nM, i.c.v.) and β-alanine (10, 30, 100 and 300 nM, i. c.v.), were also investigated. Bicuculline and suramin were administered 15 min before saline or uridine injection in mice with morphine-induced hyperactivity. Locomotor activity was measured immediately after morphine or saline administration.

2.4.3. Effects of uridine on morphine-induced behavioral sensitization Following the above procedure, mice in the uridine +morphine group were injected with uridine for 12 days (during the 7 days of morphine treatment and 5 days of withdrawal). On day 13, the test session began with a 15-min habituation period, followed by the administration of morphine (10 mg/kg). The locomotor activity of the mice was measured for 120 min.

2.5.

In vivo microdialysis

2.5.1. Implantation of the dialysis probe and brain dialysis The procedure used to prepare and implant the dialysis probe was carried out in the laboratory, as described previously (Wu et al., 1988). On the test day, the mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and implanted with Hospal AN 69 dialysis fibers (310 mm o.d., 200 mm i.d., Dasco, Bologna, Italy) transversally through the dorsal striatum (coordinates: A +0.6 mm from the bregma, V 3.5 mm from the occipital bone, Fig. 1B) (Giuffrida et al., 1999; Ito et al., 2002). The dialysis probes were covered with Super-Epoxy glue over their whole length except for the zones (2.0 mm wide) which were to be positioned in the dorsal striatum. This kind of probe has a total dialysis length of 4.0 mm and can collect neurochemicals (such as DA and uridine) from both sides of the dorsal striatum. The sample collection tube was removable, successive dialysis samples could be accurately extracted from the same mice. The animals were individually housed in a plastic cages and left to recover after the surgery. The dialysis samples were collected for 180 min. Brain microdialysis was performed about 12 h after probe implantation. The perfusion fluid was pumped through the dialysis probe at a flow rate of 2 ml/ min. After about 2 h, successive dialysis samples were collected and analyzed every 20 min. Test solutions were continuously pumped through the dialysis probe when the release of neurochemicals became stable in the last three samples with a deviation from average less than 10%. At the end of the experiments, the position of the dialysis fiber was verified via frozen coronal sections (20 mm in thickness) using a freezing microtome (AS-620, Shandon, USA) and mice with the correct probe placements were included in the final analysis.

2.5.2. Analytical procedure DA levels were measured using a HPLC system (LC-10AD pump, Shimadzu Co., Kyoto, Japan) equipped with a reversed-phase ODScolumn (Diamonsil™, 5 μm, C18, 150  4.6 mm, Dikma Co., Beijing, China) and an electrochemical detector (L-ECD-10A; Shimadzu Co.). The column temperature was maintained at 37 1C. The detector was set at +0.60 V (Sun et al., 2011). The mobile phase (85 mM citrazinic acid, 100 mM sodium acetate anhydrous, 0.2 mM Na2EDTA, and 1.2 mM sodium 1-octanesulfonate adjusted to pH 3.7) was delivered at 1.0 ml/min by the HPLC. The levels of uridine and uracil were measured by HPLC-UV (250  4.6 mm, platisil ODS, 5 μm particle size column, DIKMA Technologies, China) at a flow rate of 0.8 ml/min. Formate buffer (0.02 M) containing 2% acetonitrile, pH 4.45, was used for elution. The flow rate was 0.8 ml/min. Column temperature was 0–5 1C. UV detection was performed at 260 nm. β-alanine and GABA were measured using HPLC-FD (150  4.6 mm, C18, 5 μm particle size column, Agilent Technologies, USA) coupled to a fluorescence detector (excitation wavelength: 340 nm, emission wavelength: 450 nm, RF-10AxL, Shimadzu, Japan) (Qi et al., 2012), following precolumn derivatization with OPA (Hao et al., 2005). The OPA solution was prepared as follows: 9 mg OPA was dissolved in 167 ml methanol, then 1.5 ml 0.4 M sodium borate and 7 ml 2-mercaptoethanol were added and mixed well. The solution was stored at 4 1C for one week and protected from light. Gradient elution was used to separate the mixture of amino acids. Mobile phase A consisted of 0.1 M sodium acetate buffer, pH 6.7 while mobile phase B consisted of 97.5% methanol and 2.5% tetrahydrofuran. The flow rate of the pump was set at 1.0 ml/min. Both mobile phases were passed through a 0.22 mm filter and the column temperature was maintained at 37 1C.

2.6.

Statistical analysis

Statistical analysis was carried out using SPSS 13.0 software for Windows (SPSS Inc., Chicago, IL, USA). All values were expressed as mean7S.E.M. In the behavioral experiments, Student's t-test was used to compare the difference between two experimental groups, while one-way ANOVA was used to compare the differences among three or more groups, with post-hoc Tukey's HSD test for individual group comparisons. The interactions between every two factors were analyzed by two-way ANOVA. The level of significance was set at Po0.05. For microdialysis experiments, the levels of DA, uridine, uracil, β-alanine and GABA were expressed as the percentage change compared with the respective basal value, which was calculated as the mean of three consecutive samples before drug administration with a deviation from average less than 10%. The data of DA release were analyzed by two-way ANOVA with ‘group’ as the between factor and ‘time’ as the within factor with post-hoc Tukey's HSD test; the mean DA% change was analyzed by Student's t-test to compare the difference between morphine group and saline group. For each time point, a one-way ANOVA was used with post-hoc Tukey's HSD test. Other data were analyzed by one-way ANOVA with post-hoc Tukey's HSD test. The level of significance was set at Po0.05.

3.

Results

3.1. Effects of uridine on hyperactivity induced by acute morphine As shown in Fig. 1E, acute injection of morphine (10 or 20 mg/kg, i.p.) significantly increased locomotor activity compared with saline injection [F (2, 19) = 8.68, Po0.001].

Uridine decreases morphine-induced behavioral sensitization by decreasing dorsal striatal dopamine release possibly 1561 As shown in Fig. 2A, a single systemic administration of uridine (30 or 100 mg/kg, i.p.) inhibited the hyperactivity induced by morphine (10 mg/kg, i.p.) [F (4, 30) = 5.13, Po0.05 for 30 mg/kg uridine, Po0.01 for 100 mg/kg uridine]. When used alone, uridine did not affect the locomotor activity [F (4, 29) =0.57, P40.05]. The basal concentrations of nucleoside in 40 ml dialysate collected every 20 min after the implantation of probes were as follows in the drug-naïve group: uridine: 270.2774.10 nM; uracil: 1.4870.16 mM; β-alanine: 13.2173.82 mM and GABA: 34.7573.27 mM. As shown in Fig. 2B, uridine caused a marked increase of 380.66% in uridine levels at 40 min (Po0.001), a 473.84% elevation of uracil at 80 min (Po0.001), a slight but significant 27% elevation of β-alanine (Po0.05) and a 96% rise in GABA at 80 min (Po0.01). Fig. 2C and D shows the effects of bicuculline or suramin on the responses induced by uridine on morphine-induced hyperactivity. A two-way ANOVA indicated a significant interaction between uridine and bicuculline [uridine: F (1, 32) = 38.02, Po0.01; bicuculline: F (2, 32) =2.25, P40.05; interaction uridine  bicuculline: F(2, 32) = 4.42, Po0.05], post-hoc analysis confirmed that bicuculline was able to dose-dependently block the inhibitory effects of uridine on morphine-induced sensitization (Fig. 2C). However, suramin, a P2Y receptor antagonist, when given 15 min prior to uridine, failed to attenuate the effects of uridine on morphine-induced increases in locomotion. The data of DA release caused by morphine challenge in the dorsal striatum were analyzed by in vivo microdialysis to investigate the mechanisms by which repeated administration of uridine reversed the established behavioral sensitization to morphine. The basal concentration of DA in the dialysate in the dorsal striatum was 1.0770.24 nM in the drug-naïve group. As shown in Fig. 2E, acute morphine treatment (10 mg/ kg, i.p.) increased DA release in the dorsal striatum maximally to 187.04713.70% of the basal level at 140 min. Acute administration of uridine (100 mg/kg) 30 min before morphine treatment had a tendency to attenuate morphine-induced DA release without statistical significance (0–180 min)[F (1, 18 )= 4.24, P = 0.062]; significantly decreased the mean DA% change (100–180 min) from 178.57711.00 to 141.1979.44. When each time point was analyzed by one-way ANOVA with post-hoc Tukey's HSD test, uridine was found to significantly reduce DA release after uridine injection at 100, 140, 160 and 180 min.

3.2. Effects of uridine metabolites on hyperactivity induced by acute morphine To observe the effects of intracerebroventricular administration of uridine on hyperactivity induced by acute morphine treatment, locomotor activity was tested. As shown in Fig. 3A, a single central administration of uridine (10, 30, 100 or 300 nM, i.c.v.) inhibited the hyperactivity induced by morphine (10 mg/kg, i.p.), [F (4, 29) = 3.26, Po0.05 for 30 nM uridine, Po0.01 for 100 and 300 nM uridine]. Intracerebroventricular administration of uridine caused a marked increase of 246% in the levels of β-alanine at

60 min (Po0.01) and 205% elevation of GABA at 60 min (Po0.01) in the dorsal striatum (in Fig. 3B). As shown in Fig. 3C and D, both uracil and β-alanine significantly attenuated morphine-induced hyperactivity [for uracil: F (4, 35) = 5.85, Po0.05 at 100 nM, Po0.01 at 300 nM; for β-alanine: F (4, 35) = 7.81, Po0.05 at 100 nM, Po0.01 at 300 nM].

3.3. Effects of uridine on morphine-induced behavioral sensitization As shown in Fig. 1F, chronic administration of morphine once daily for 7 consecutive days induced an enhancement of the locomotor activity between the first and last day of morphine treatment. After a 5-day drug-free period, mice were challenged with morphine (10 mg/kg, i.p., day 13) and locomotor activity was recorded for 120 min. It was observed that morphine challenge significantly increased locomotor activity compared to that in the drug-naïve group (Po0.01), confirming that behavioral sensitization to morphine was established. To observe the effects of uridine on the development of morphine-induced behavioral sensitization, uridine was injected for 12 days (morphine was administered concurrently on the first 7 days, followed by 5 days of withdrawal). As shown in Fig. 4A, concomitant injection of uridine (10, 30, 100 or 300 mg/kg, i.p.) significantly inhibited the expression of morphine-induced behavioral sensitization when uridine was administered for 12 days (during the 7 days of morphine treatment and 5 days of withdrawal) [F (4, 32) = 7.43, Po0.01]. As shown in Fig. 4B, uridine alone, when administered repeatedly for 7 days or 12 days, did not affect locomotor activity [7 days: F (4, 32) = 0.68, P40.05; 12 days: F (4, 32) = 0.17, P40.05]. The basal concentrations of nucleosides in the morphinesensitized group (day 13, β-alanine: 12.8773.91 mM and GABA: 31.8476.12 mM) were not changed, and like the single administration of uridine, as shown in Fig. 4C, the last administration of uridine caused a marked increase of 404.38% in the levels of uridine at 40 min (Po0.001), 481.23% elevation of uracil at 80 min (Po0.001), 156.79% elevation of β-alanine (Po0.05) and 235.84% of GABA (Po0.01). The basal concentration of DA in the morphine-sensitized group (day 13, 1.4170.45 nM) was not significantly different from that in the control group, as shown in Fig. 4D. The data of DA release in the dorsal striatum after morphine challenge were analyzed on day 13 and were found to be significantly attenuated by uridine administration in morphine-sensitized animals [F (1, 17) = 9.03, Po0.01 for 12 days uridine administration]; the mean DA% change (100– 180 min) was significantly decreased from 276.30712.96 to 185.19714.72, and then each time point was analyzed by one-way ANOVA with post-hoc Tukey's HSD test; uridine also significantly reduced DA release after uridine injection at 100 min.

4.

Discussion

The major findings of the present study are: (a) acute and chronic uridine administration inhibited morphine-induced

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Fig. 2 Effects of uridine on acute morphine-induced hyperactivity. Uridine was administered systemically (10, 30, 100 and 300 mg/ kg, i.p., panel A), 30 min before morphine (10 mg/kg) or saline injection. The elevation of extracellular uridine, uracil, β-alanine and GABA concentrations in the dorsal striatum during uridine administration systemically (100 mg/kg, i.p., panel B) was investigated by microdialysis, the data were analyzed by one-way ANOVA with post-hoc Tukey's HSD test. Bicuculline (0.1 and 0.2 μM, i.c.v., panel C) and suramin (0.5 and 1 μM, i.c.v., panel D) were administered 15 min before saline or uridine injection in mice with morphine-induced hyperactivity. The effects of uridine on DA release in the dorsal striatum induced by acute morphine treatment were investigated by pretreatment with uridine (100 mg/kg, i.p.) 30 min prior to morphine injection, the data of DA release were analyzed by two-way ANOVA with ‘group’ as the between factor and ‘time’ as the within factor with post-hoc Tukey's HSD test; the mean DA% change was analyzed by Student's t-test to compare the difference between morphine group and saline group. For each time point, a one-way ANOVA was used with post-hoc Tukey's HSD test (panel E). The data were expressed as means7S.E.M. (n =7–10 per group). Locomotor activity was recorded continuously for 120 min, one-way ANOVA was used to compare the differences among three or more groups, with post-hoc Tukey's HSD test for individual group comparisons. The interactions between every two factors were analyzed by two-way ANOVA. nPo0.05, nnPo0.01 vs. saline group; + Po0.05, + + Po0.01, + + + Po0.001 vs. basal level (100%, 0 min); ♯Po0.05, ♯♯Po0.01 vs. morphine group; $Po0.05, $$Po0.01 vs. uridine (100 mg/kg) group. To evaluate the interaction between groups, these data were analyzed by two-way ANOVA followed by Tukey's HSD test.

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Fig. 3 Effects of uridine metabolites on morphine-induced hyperactivity. Uridine was administered centrally (10, 30, 100 and 300 nM, i.c.v., panel A) 30 min before morphine (10 mg/kg) or saline injection. The elevation of extracellular β-alanine and GABA concentrations in the dorsal striatum during uridine administration centrally (100 nM, i.c.v., panel B), the data were analyzed by one-way ANOVA with post-hoc Tukey's HSD test. The effects of two uridine metabolites, uracil (10, 30, 100 and 300 nM, i.c.v., panel C) and β-alanine (10, 30, 100 and 300 nM, i.c.v., panel D) were also investigated. The data were expressed as means7S.E.M. (n =6–8 per group). Locomotor activity was recorded continuously for 120 min, one-way ANOVA with post-hoc Tukey's HSD test for individual group comparisons. nnPo0.01 vs. saline group; + Po0.05, + + Po0.01, + + + Po0.001 vs. basal level (100%, 0 min); ♯ Po0.05, ♯♯Po0.01 vs. morphine group.

hyperactivity and the expression of behavioral sensitization to morphine, respectively; (b) uridine increased the extracellular levels of GABA in the brain, while the GABAA receptor antagonist bicuculline attenuated the effects of uridine, suggesting that the GABAA receptor may be the target of uridine; (c) in acute morphine-induced hyperactivity, both uracil and β-alanine mimicked the effects of centrally-administered uridine to inhibit morphine-induced hyperactivity, suggesting that uridine metabolites can play similar roles with uridine; (d) the enhancement of morphine-induced DA release in the dorsal striatum was reduced by pretreatment of morphine-sensitized mice with uridine. These findings demonstrated that the reversal of behavioral sensitization to morphine by uridine and its metabolites can be attributed in part to the attenuation of enhanced DA release associated with the GABAA receptor after morphine challenge, which implied that uridine might be a potential candidate for treatment of opioid abuse. In the present study, concomitant injection of uridine with morphine inhibited morphine-induced locomotor activity increase, indicating the antagonistic effects of uridine on morphine-induced behavioral sensitization. To determine whether uridine injection increases GABA levels in the extracellular space, we measured the GABA levels after uridine administration. Uridine (100 mg/kg) greatly increased

GABA levels in the dorsal striatum, by 95.83% (acute) or 135% (chronic) compared to controls. Predictably, a significant interaction was observed between uridine and the GABAA receptor antagonist bicuculline, indicating that the reversal effects of uridine on acute morphine-induced hyperactivity and established behavioral sensitization may be associated with the GABAA receptor. Indeed, several observations have shown the interaction of uridine with GABA binding sites in rat cerebellar membranes, frontal cortex, hippocampus and thalamus (Guarneri et al., 1983, 1985). Another hypothesis is that the action of uridine is mediated via pyrimidine receptors, because uridine is known to increase the levels of UTP in the central nervous system, inducing a variety of cellular effects through activation of the uracil-nucleotide responsive subtypes of G-protein-coupled P2Y receptors (P2Y2, P2Y4, P2Y6 and P2Y14). However, suramin, a P2Y receptor antagonist, failed to attenuate the effects of uridine, suggesting that P2Y receptors were not involved in these effects. A commonly accepted theory is that enhanced DA transmission in the CNS is consistently associated with the expression of behavioral sensitization (Wolf, 1998). Consequently, manipulations of the dopaminergic system could be an effective means to counteract the neural processes involved in opiate addiction (Cordonnier et al., 2007).

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Fig. 4 Effects of chronic uridine treatment on morphine-induced behavioral sensitization. After 5-day withdrawal from repeated morphine administration, all mice were given a morphine challenge injection (10 mg/kg, i.p.). Uridine (10, 30, 100 and 300 mg/kg, i.p.) was given for 12 days (7-day morphine +5-day withdrawal) (panel A). The effects of chronic uridine administration on locomotor activity in drug-naïve mice were also investigated on day 7 and day 13 (panel B). The elevation of extracellular uridine, uracil, βalanine and GABA concentrations in the dorsal striatum during the last uridine treatment (100 mg/kg, i.p., panel C) was investigated by microdialysis, the data were analyzed by one-way ANOVA with post-hoc Tukey's HSD test. The effects of daily administration of uridine given 30 min before morphine injection for 12 days (7days with morphine and 5 days during withdrawal) on DA release induced by morphine challenge (100 mg/kg, i.p., panel D) were investigated. On the test day (day 13), a challenge dose of morphine was given, the data of DA release were analyzed by two-way ANOVA with ‘group’ as the between factor and ‘time’ as the within factor with post-hoc Tukey's HSD test; the mean DA% change was analyzed by Student's t-test to compare the difference between morphine group and saline group. For each time point, a one-way ANOVA was used with post-hoc Tukey's HSD test. All the data were expressed as means7S.E.M. (n =6–8 per group). nnPo0.01 vs. saline group; + Po0.05, + + Po0.01, + + + Po0.001 vs. basal level (100%, 0 min); ♯Po0.05, ♯♯Po0.01 vs. morphine group.

Emerging evidence suggests that uridine, a neuroactive molecule, is involved in dopaminergic activity and related behaviors distinct from its role in pyrimidine metabolism (Connolly and Duley, 1999). Studies have demonstrated that chronic uridine administration reduced DA release evoked by amphetamine and haloperidol (Agnati et al., 1989; Farabegoli et al., 1988). Evidence also exists that chronic administration of uridine markedly decreases the number of striatal DA binding sites (Farabegoli et al., 1988). In addition, uridine-treated rats with unilateral striatal lesions show a decreased rotation induced by the DA precursor L-dopa or repeated injections of methamphetamine (Myers et al., 1995). Thus, it is reasonable to assume that uridine might have an effect on dopaminergic neurotransmission to affect the development of morphine behavioral sensitization. In microdialysis experiments, uridine alone failed to alter extracellular DA, but significantly reduced morphine-

induced DA release in the dorsal striatum of morphinesensitized mice, the mean DA% change (100–180 min) was more significantly decreased, demonstrating a neuromodulatory property of this substance in the brain. These findings, at least in part, are consistent with previous studies showing that uridine interacts with the dopaminergic system to mediate locomotor activity (Agnati et al., 1989; Farabegoli et al., 1988; Myers et al., 1993, 1995). To obtain insight into the relevant metabolic processes, we measured the extracellular levels of uridine and its metabolites uracil and β-alanine in the dorsal striatum after i.p. injection of uridine. Uridine levels were raised 4-fold, uracil levels were raised 5-fold and β-alanine levels were raised by 26.79%, the increases in extracellular β-alanine in the dorsal striatum after the last uridine injection of chronic uridine administration were also observed, indicating the extremely rapid turnover of uridine in the brain. In order to explore the

Uridine decreases morphine-induced behavioral sensitization by decreasing dorsal striatal dopamine release possibly 1565 action of uridine, i.c.v. injection of uridine was used to prevent its metabolism in the liver. However, i.c.v. injections of uridine could also increase the amounts of its metabolites. In the present study, i.c.v. injection of both uracil and β-alanine inhibited morphine-induced increases in locomotion. Therefore, it is speculated that uridine may directly impact on GABAA receptors in the CNS, and it is likely that further breakdown products of uridine can play similar roles on morphine-induced hyperactivity. It is known that β-alanine and GABA have a very similar chemical structure. The uptake or transport mechanisms for β-alanine might thus share similarities with those for GABA (Borycz et al., 2012). Both systemic and i.c.v. injections increased the extracellular levels of β-alanine in the dorsal striatum. β-alanine produced by uridine may be related to GABA uptaking. The effects of morphine on endogenous uridine release in the dorsal striatum have been investigated with the aim of understanding whether uridine does indeed influence morphineinduced neurobehaviors (Song et al., 2013). It has been shown that systemic administration of morphine produced an approximately 2-fold increase in dorsal striatal uridine release. The present data showed that a high dose of uridine (100 mg/kg) caused a 4-fold increase in uridine levels in the brain. The present data together with our previous findings clearly demonstrated that endogenous uridine release correlates with morphine administration, and might be sufficient to modulate morphine-induced reward-related behaviors. Since uridine, as a dietary component, is not toxic and can access the brain from the plasma through transporters (Kovacs et al., 2010), it is an appealing candidate for the treatment of drug abuse. Although the current state of knowledge about non-adenosine nucleosides makes it difficult to conclude how uridine influences the dopaminergic system, our findings strengthened several different lines of research concerning the regulatory roles of uridine in the CNS. However, to gain detailed information about the pharmacological roles of uridine, the direct target proteins and other proteins downstream of uridine should be further elucidated. In conclusion, the present study revealed for the first time that uridine and its metabolites are able to circumvent morphine-induced hyperactivity and behavioral sensitization, presumably by modulating the dopaminergic system possibly via agonistic effects at GABAA receptors in the dorsal striatum. This work clearly implicates these substances as potential anti-opioid candidates.

Role of the funding source This project was supported by National Natural Science Foundation of China (No. 81373383). The National Natural Science Foundation of China had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Contributors Jing-Yu Yang designed and supervised the study. Chun-Fu Wu has taken part in the design of the experiment. Ping Liu, Wu Song, Li-Sha Yu, Xiao-Feng Yang and Fang Wang carried out the experiments. Rong-Wu Xiang undertook the statistical analyses. Ping Liu

wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.

Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgments The authors gratefully acknowledge financial support from National Natural Science Foundation of China (No. 81373383).

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