Activation of P2X7 receptors in the midbrain periaqueductal gray of rats facilitates morphine tolerance

Pharmacology, Biochemistry and Behavior 135 (2015) 145–153

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Activation of P2X7 receptors in the midbrain periaqueductal gray of rats facilitates morphine tolerance Zhi Xiao a,⁎, You-Yan Li b, Meng-Jie Sun b a b

Research Center for Medicine & Biology, Zunyi Medical University, Zunyi, Guizhou 563003, PR China Graduate School, Zunyi Medical University, Zunyi, Guizhou 563003, PR China

a r t i c l e

i n f o

Article history: Received 4 February 2015 Received in revised form 27 May 2015 Accepted 4 June 2015 Available online 6 June 2015 Keywords: Morphine Tolerance P2X7 receptor Midbrain periaqueductal gray

a b s t r a c t Opiates such as morphine exhibit analgesic effect in various pain models, but repeated and chronic morphine administration may develop resistance to antinociception. The purinergic signaling system is involved in the mechanisms of pain modulation and morphine tolerance. This study aimed to determine whether the P2X7 receptor in the ventrolateral midbrain periaqueductal gray (vlPAG) is involved in morphine tolerance. Development of tolerance to the antinociceptive effect of morphine was induced in normal adult male Sprague–Dawley (SD) rats through subcutaneous injection of morphine (10 mg/kg). The analgesic effect of morphine (5 mg/kg, i.p.) was assessed by measuring mechanical withdrawal thresholds (MWTs) in rats with an electronic von Frey anesthesiometer. The expression levels and distribution of the P2X7 receptor in the vlPAG was evaluated through Western blot analysis and immunohistochemistry. The acute effects of intra-vlPAG injection of the selective P2X7 receptor agonist Bz-ATP, the selective P2X7 receptor antagonist A-740003, or antisense oligodeoxynucleotide (AS ODN) targeting the P2X7 receptor on morphine-treated rats were also observed. Results demonstrated that repeated morphine administration decreased the mechanical pain thresholds. By contrast, the expression of the P2X7 receptor protein was up-regulated in the vlPAG in morphine tolerant rats. The percent changes in MWT were markedly but only transiently attenuated by intra-vlPAG injection of Bz-ATP (9 nmol/0.3 μL) but elevated by A-740003 at doses of 10 and 100 nmol/0.3 μL. AS ODN (15 nmol/0.3 μL) against the P2X7 receptor reduced the development of chronic morphine tolerance in rats. These results suggest that the development of antinociceptive tolerance to morphine is partially mediated by activating the vlPAG P2X7 receptors. The present data also suggest that the P2X7 receptors may be a therapeutic target for improving the analgesic effect of morphine in treatments of pain when morphine tolerance occurs. © 2015 Published by Elsevier Inc.

1. Introduction Morphine is an effective treatment for acute and chronic pain syndromes, particularly for chronic diseases accompanied with intractable pain (Berrios et al., 2008; Cherubino et al., 2012). Although systemic administration of morphine effectively relieves pain, prolonged morphine exposure may lessen analgesic effects and thus limit efficacy as a therapeutic method. Morphine tolerance mechanisms are complex and involve several regulatory factors, including functional changes in receptors, such as opioid and glutamate receptors (Inturrisi, 2005; Sanchez-Blazquez et al., 2013; Williams et al., 2013), and content alterations in neurotransmitters (Ossipov et al., 2005; Toda et al., 2009; Ueda and Ueda, 2009). Nevertheless, the exact mechanisms underlying morphine tolerance remain to be elucidated. The midbrain periaqueductal gray (PAG) is a strategic site in the endogenous nociceptive modulatory system (Millan, 2002; Tavares and

⁎ Corresponding author. E-mail address: [email protected] (Z. Xiao).

http://dx.doi.org/10.1016/j.pbb.2015.06.002 0091-3057/© 2015 Published by Elsevier Inc.

Lima, 2007). During pain modulation, PAG integrates somatic and autonomic responses to nociceptive and other stress stimuli (Behbehani, 1995; Fields, 2000). PAG, particularly the ventrolateral periaqueductal gray (vlPAG) region (Macey et al., 2015; Mehalick et al., 2013), is also an essential neural circuit for opioid-mediated analgesia (Basbaum et al., 1978; Fyfe et al., 2010; Tortorici et al., 2003). Adenosine 5′-triphosphate (ATP) is released by neuronal and nonneuronal cells (Koles et al., 2007); in addition to being an intracellular energy source, ATP is an important neurotransmitter or neuromodulator that activates cation-permeable ion channels (P2X receptors) and G-protein-coupled receptors (P2Y receptors) on the cell surface (Burnstock, 2006). The P2X7 receptor subtype, an ATP-gated nonselective cation channel (Sperlagh et al., 2006), is widely distributed in vivo, and its expression is altered in many pathophysiological processes, such as inflammation, pain, and cancer (Alves et al., 2013; Di Virgilio, 2012; Franceschini and Adinolfi, 2014). Zhou et al. (2010) found that microglial cells in the rat spinal cord expressed the P2X7 receptor, and the protein level of this receptor was upregulated after chronic exposure to morphine. Suppression of P2X7 receptor activation also significantly attenuated the loss of morphine analgesic potency.

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Although activation of the spinal P2X7 receptor contributes to the development of morphine tolerance, the role of this receptor in morphine tolerance at the supraspinal levels has not been studied. Given these findings, we hypothesized that P2X7 receptor in the vlPAG may play an essential role in morphine tolerance in rats. Therefore inhibiting the activation of the P2X7 receptor may reverse morphine tolerance and potentiate morphine-induced antinociception in rats. This idea was tested in this study. 2. Materials and methods 2.1. Animals and ethics Male Sprague–Dawley rats weighing 220 ± 10 g were purchased from the Center of Laboratory Animals, Third Military Medical University (Chongqing City, China). In a temperature-controlled room (25 ± 1 °C), the rats were housed in groups of four or five per cage under natural light/dark cycle. Food pellets and water were given to the rats ad libitum throughout the experiments, except during behavioral testing periods. All studies were approved by the Institutional Animal Care and Use Committee of the Zunyi Medical University and performed in strict compliance with the Ethical Issues of the International Association for the Study of Pain. Efforts were exerted to reduce the number of animals used and to minimize their suffering. 2.2. Induction of morphine tolerance The rat model of morphine tolerance was established as previously described (Chen et al., 2012). Morphine was given subcutaneously (s.c.) twice daily (with a 12-h dosing interval) from day 1 to day 9 at 10 mg/kg body weight to establish systemic analgesic tolerance. As previous studies demonstrated that 10 mg/kg is sufficient to induce systemic morphine tolerance in male rats, we used this dose in the experiment (Chen et al., 2012). To evaluate the development of morphine tolerance, we assessed morphine antinociception to mechanical stimuli at 30 min after an acute test dose (5 mg/kg) of morphine was intraperitoneally (i.p.) given. Morphine analgesic effects before and after a defined period of tolerance induction were compared. Baseline nociceptive thresholds were measured 15 min before subcutaneous injection of morphine, and the development of tolerance was monitored 30 min after an acute intraperitoneal injection of morphine. 2.3. Electronic pressure meter test of mechanical withdrawal threshold (MWT) After acclimatization to the testing apparatus (30 min a day for three consecutive days), the rats were tested for mechanical allodynia. To determine MWT, we placed each rat in an individual transparent Plexiglass cage (18 cm × 12 cm × 12 cm) with a wire mesh floor in a quiet room. The rats were allowed to explore and groom until settling down. The test involved evoking a hindpaw flexion reflex (paw withdrawal) with a hand-held force transducer (IITC 2390 series electronic von Frey anesthesiometer, Life Science Instruments, US) equipped with a 0.5 mm2 contact area polypropylene tip. The investigator was trained to perpendicularly apply the tip to the central area of the hind paw with gradually increasing pressure. Endpoint was characterized by removal of the paw, in which the animal actively lifted the whole paw on the tip of the anesthesiometer. Positive responses included prolonged hind paw withdrawal, licking or biting of the hind paw, or shaking the paw with high amplitude movements in response to the stimulus. Each hind paw was measured five times in grams, and the average values of five measurements were regarded as the paw MWT (Cunha et al., 2004). The development of morphine-induced tolerance was detected by measuring percent changes in MWT after a challenge injection of morphine (5 mg/kg, i.p.) and calculated as follows: MWT (%) = (tested threshold − basal threshold) / basal threshold × 100.

2.4. Surgical and microinjection procedures Anesthesia was induced through i.p. injection of 4% chloral hydrate (10 mL/kg body weight). The rats were mounted on a stereotaxic frame (Narishige SR-5R, Tokyo, Japan). The skull was exposed, and the bregma was located. A stainless steel guide cannula (0.8 mm o.d.) was inserted unilaterally into the vlPAG and fixed to the skull by using dental zinc cement and jeweler's screws. The stereotaxic coordinates for the vlPAG were 7.90 mm posterior to the bregma, 0.80 mm lateral to the midline, and 6.00 mm ventral to the skull surface. A dummy cannula was inserted into the guide cannula at the time of surgery to minimize occlusion. Skull screws and dental acrylic were used to hold the cannulae securely in place. After removal from the stereotaxic apparatus, the rats were (i.p.) administered with 1 mL of 0.9% sterile saline to prevent dehydration and then placed in a thermally controlled cage to avoid hypothermia until complete anesthetic recovery. Prior to any experiments, the animals were allowed to recover from the implantation surgery for 5 d and were monitored for signs of motor deficiency. Rats with any neurological deficits caused by the surgical procedure were excluded from the experiments. On the day of intra-vlPAG injection, the rats were transferred from the main holding area to the laboratory and were left undisturbed for 1 h prior to drug administration. Each rat was lightly restrained, and a 32-gauge injection cannula (1.0 mm longer than the guide cannula) was inserted into the guide cannula. The injection cannula was connected to a 5-μL Hamilton microsyringe. A total volume of 0.3 μL was injected over 3 min, and the injector was left in place for an additional 2 min before slow removal to ensure complete drug diffusion. Successful infusion was confirmed by monitoring the movement of a small air bubble in the microsyringe. After the experiments, the animals were anesthetized through i.p. injection of 4% chloral hydrate (20 mL/kg body weight) and then intracardially perfused with physiological saline (0.9% NaCl) and 4% paraformaldehyde solution. The needle position of the cannula was visually confirmed with 0.1 μL of 2% Evans blue infusion through the microinjection cannula. Administration sites were verified through histological examination and plotted on coronal maps adapted from the atlas of Paxinos and Watson (Paxinos and Watson, 2007). Only rats with whole microinjection sites within the vlPAG were included in analysis. Animals with cannulae located outside the vlPAG (e.g., dorsal lateral PAG, lateral PAG, dorsal raphe nucleus) were considered “cannula misses” and discarded (Fig. 1). 2.5. Immunohistochemistry The distribution of the P2X7 receptor in the vlPAG was detected through immunohistochemistry. The animals were sacrificed by an overdose of 4% chloral hydrate anesthesia (20 mL/kg body weight, i.p.), and then perfused with 150 mL of physiological saline (NS) in the aorta through the left ventricle, followed by 200 mL of 4% paraformaldehyde in 0.2 M phosphate buffer (PB). Rat brains were removed and post-fixed in 4% paraformaldehyde in PB for 4–6 h, and then transferred to 30% sucrose solution for 48–60 h before being cryoprotected. The PAGs were serially cut at a thickness of 25 μm with a cryostat (Leica CM1950, Germany) along the axis. One of every 5–6 sections through the vlPAG was collected for immunohistochemical analysis. After incubation with 3% H2O2 in 0.01 M phosphate-buffered saline (PBS) and 3% bovine serum albumin (BSA) in 0.1 M PBS, the sections were incubated with the primary antibody rabbit anti-rat P2X7 polyclonal antiserum (1:400; Abcam Corporation, USA) for the P2X7 receptors. After 1 h of incubation at 37 °C, the sections were stained overnight at 4 °C. Subsequently, 5% goat anti-rabbit serum (Beijing Zhongshan Biotech Co., China) was used as the secondary antibody. The tissue was immunoreacted for the P2X7 receptor in 0.05% diaminobenzidine tetrahydrochloride (DAB), 0.03% H2O2, and 0.2% nickel ammonium sulfate (DAB–Ni) in 0.01 M PBS for 2–3 min to yield a black reaction product. Finally, the sections were mounted on glass sides,

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Mouse anti-rat β-actin primary antibody (1:2000; Novus Biologicals, USA) was included as a control for protein loading. The membranes were then incubated in goat anti-rabbit horseradish peroxidase (HRP)- or goat anti-mouse HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology, CA) for 2 h at room temperature before the blots were visualized in enhanced chemiluminescence solution (Amersham Pharmacia Biotech, UK) and exposed to X-ray films. The developed X-ray films were scanned for data analysis. Protein levels were normalized to β-actin as the loading control. Relative optical density (ROD) of the protein bands was measured after subtracting the film background. Data are expressed as mean ratio ± S.E.M. of the P2X7/βactin protein. 2.7. Experimental design and drugs

Fig. 1. (A) Schematic diagram of the microinjection sites for saline, Bz-ATP, A-740003 and oligodeoxynucleotides (ODNs). Dots represent the corresponding sites identified histologically through Evans blue dye microinjection. (B) Photograph of an injection site in the vlPAG. Microinjection sites were mostly distributed within the vlPAG region. Data from rats with an injection site outside the vlPAG region were discarded.

dehydrated through ascending series of ethanol solutions and xylene, air-dried, and coverslipped. To confirm the specificity of immune labeling, we exposed the control slides to diluted normal goat serum, instead of the primary antibody. For quantification, the images of positive stained vlPAG sections were analyzed using a Leica Q500IW image analysis system. The whole mount preparations were used for quantitative analysis as described previously (Van Nassauw et al., 2001). The immunoreactive positive neuron bodies in the vlPAG were counted per visual field (0.3 mm2) on sections with 25 μm thickness. Only cells with a distinct cell body and clear cellular boundary were included in the counts. Cell measurements were obtained using the mean value of five separate high-power fields per section, and 10 sections per brain were assayed. Cell density was expressed as cells per square millimeter under 40× objective (total magnification 400×). 2.6. Western blot Animals were anesthetized by an overdose of chloral hydrate (20 mL/kg body weight, i.p.), and the vlPAG tissue was rapidly removed. The collected tissue samples were homogenized in a lysis buffer containing a mixture of protease inhibitors (Roche, Mannheim, Germany) and phenylmethylsulfonyl fluoride (PMSF) (Sigma, St Louis, Missouri, USA). Protein samples (20 μg/lane) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5%–12% gels, Bio-Rad, Canada) and transferred onto polyvinylidene fluoride membranes (Sigma Aldrich, USA). The membranes were blocked with 5% non-fat milk and then incubated overnight at 4 °C with a primary antibody (rabbit anti-rat P2X7 receptor, 1:600; Abcam Corporation, USA).

The experiments consisted of four series. In series 1, changes in MWT values were determined after repeated administration of morphine. The rats were randomly and equally divided into three groups according to a random number table: normal group, saline group, and morphine group (n = 12). Morphine (10 mg/kg, s.c.) was administered to rats in the morphine group twice a day for 9 d. In the saline group, the rats were injected with 1 mL/kg physiological saline solution (0.9% NS), instead of morphine, with the same schedule. The normal group served as control subjects. All experimental rats were subjected to MWT test daily. In series 2, alterations in P2X7 receptor expression in the vlPAG were observed. Six rats from each group in the first series were used for immunohistochemical analysis, and the remaining six rats for Western blot analysis. In series 3, we observed the effects of intra-vlPAG microinjection of 2′(3′)-O-(4-Benzoylbenzoyl)-ATP triethylammonium salt (Bz-ATP) or (N-(1-(Honore et al.)-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl) acetamide) (A-740003) on the MWT values of rats subjected to repetitive morphine treatment. Briefly, 32 rats received morphine treatment (10 mg/kg, s.c.) twice a day for 4 d and were then randomly divided into four groups: morphine + Bz-ATP group, morphine + 1 nmol A-740003 group, morphine + 10 nmol A-740003 group, and morphine + 100 nmol A-740003 group (n = 8 per group). The morphine + Bz-ATP group received intra-vlPAG injection of 0.3 μL of Bz-ATP (9 nmol). The other three groups received intra-vlPAG injection of 0.3 μL A-740003 (1, 10, and 100 nmol). The doses of the experimental drugs were selected based on our preliminary experiment results. The agonist and antagonist pretreatments were administered 30 min before a test dose injection of morphine (5 mg/kg, i.p.). MWT values were measured 30 min after injection of the test dose of morphine. In series 4, changes in MWT in response to ODNs were determined. A total of 24 normal rats were divided into three groups: saline + morphine group, mismatch ODN + morphine group (MM ODN + morphine), and antisense ODN + morphine group (AS ODN + morphine) (n = 8 per group). The MM ODN + morphine and AS ODN + morphine groups were given ODN (15 nmol/0.3 μL) through vlPAG microinjection once daily for 5 d and morphine (10 mg/kg, s.c.) twice daily for nine consecutive days. In the saline + morphine group, the rats were injected with 0.3 μL of sterile physiological saline (0.9% NS), instead of ODN, with the same schedule. On day 9 after chronic morphine treatment, the rats received a single injection of a test dose of morphine (5 mg/kg, i.p.). MWT values were then measured. At the end of the experiment, rats from all groups were used for Western blot assay. Morphine hydrochloride was purchased from Shengyang First Pharmaceutical Factory (Shengyang City, China). Bz-ATP and A-740003 were purchased from Sigma Chemical Co. and diluted in normal saline. Bz-ATP and different doses of A-740003 were administered through intra-vlPAG injection in a volume of 0.3 μL. Phosphorothioate-modified oligonucleotides of the rat P2X7 receptor were synthesized and purified by Sangon Biological Engineering Technology Co. (Shanghai City,

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China). According to a previous report (Kong et al., 2005), the sequences were designed as follows: P2X7 receptor antisense ODN: 5′-TTG ATG GTG CCG TAA TTC ACG CTC T-3′ targeted to the nucleotide sequence 186 through 210 that directly follows the initiation codon of the rat P2X7 receptor; and mismatch ODN: 5′-AAT TAC ACA GTA AGC GAA CTT AGC C-3′. A database search using the BLAST program indicated that the antisense sequence was specific for the rodent P2X7 receptor. Nevertheless, the search did not identify the corresponding rodent sequence for the mismatch sequence. Antisense and mismatch ODNs for the P2X7 gene were dissolved in double distilled water to a concentration of 50 nmol/μL. ODNs were aliquoted and stored at −20 °C, and oligonucleotide treatments were performed as previously described (Xiao et al., 2010).

3.2. Observation of changes in P2X7 receptor expression in the vlPAG after morphine tolerance through immunohistochemistry Nine days after chronic morphine treatment, the immunohistochemistry results manifested a relatively weak P2X7 receptor-specific immunoreactivity in the vlPAG in rats in the normal and saline group; moreover, statistically insignificant differences were detected between the two groups (P N 0.05). In the morphine group, immunohistochemical data showed that similar to behavioral changes, P2X7 receptor expression was markedly up-regulated and densely expressed within the vlPAG on day 9 post-chronic morphine treatment compared with that in the normal group (P b 0.001). This finding suggests the involvement of vlPAG P2X7 receptor activation in the development of morphine tolerance (Fig. 3A and B).

2.8. Statistical analysis Experimental data were processed using GraphPad Prism (version 6.01; GraphPad Software, Inc., CA) and SPSS 16.0 (SPSS Inc., USA). All data were presented as mean ± standard deviation. Two-way ANOVA was used to analyze the time course of morphine tolerance and the effect of drugs on the development of morphine tolerance. Post hoc comparisons were calculated using Fisher's (LSD) post hoc tests. P b 0.05 was considered statistically significant.

3. Results

3.3. Observation of the protein levels of the P2X7 receptor in the vlPAG through Western blot Immunoblots from vlPAG homogenates revealed the presence of an immunopositive band of the P2X7 receptor in the normal, saline, and morphine groups. The results were quantified based on the ROD of the immunoblot bands compared with β-actin calculated from the densitometric quantification of the bands. No significant differences were detected between the normal and saline groups (P N 0.05). The protein level of the P2X7 receptor was significantly higher in the morphine group than those in the normal and saline groups at 9 d after chronic morphine treatment (all P b 0.001; Fig. 4A and B).

3.1. Development of tolerance to analgesia produced by repeated morphine injection in rats Before subcutaneous injection of morphine (day 0), the percentage change in MWT was not significantly different among the three experimental groups (P N 0.05). In the morphine group, the analgesic effect induced by a test dose (5 mg/kg, i.p.) of morphine gradually decreased after the rats received multiple subcutaneous injections of 10 mg/kg morphine twice daily and was nearly abrogated on day 9. This phenomenon was considered morphine tolerance. By contrast, the percentage changes in MWT in the saline group did not significantly change compared with that in the normal group at all observation time points (all P N 0.05). Moreover, basal MWT did not significantly change during this time period (Fig. 2).

Fig. 2. Percentage changes in MWT of the experimental rats subjected to repeated morphine injections. Rats were administered with morphine (10 mg/kg, s.c.) twice daily for 9 d. The antinociceptive effect of morphine was measured through the paw withdrawal test after treatment with a test dose of morphine treatment (5 mg/kg, i.p.). The analgesic efficacy of the test dose of morphine significantly decreased after repeated morphine administration (**P b 0.01, ***P b 0.001 compared with the normal group).

Fig. 3. Alterations in P2X7 receptor expression in the vlPAG after chronic morphine tolerance. Photomicrographs illustrate P2X7 receptor immunostaining in the vlPAG in the normal (Fig. 3A-a), saline (Fig. 3A-b), and morphine groups (Fig. 3A-c). Scale bars = 50 μm. Graph shows the number of P2X7-positive cells in the vlPAG (***P b 0.001 compared with the normal or saline group; n = 6) (Fig. 3B).

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Fig. 4. Up-regulation of P2X7 receptor protein level in the vlPAG induced by chronic morphine treatments. Western blot analysis detected a protein band of approximately 75 kDa, which coincides with the known molecular weight of the P2X7 receptor (Fig. 4A). β-Actin was used as the loading control. The protein levels of the P2X7 receptor in different groups were expressed as ROD (***P b 0.001 compared with the normal or saline group) (Fig. 4B).

3.4. Effects of the P2X7 receptor agonist and antagonist pretreatments on chronic morphine tolerance The effects of the P2X7 receptor agonist and antagonist on morphine tolerance were examined on rats treated with morphine for 4 d. Based on the above-mentioned result (Results 3.1), repetitive morphine treatments (10 mg/kg, s.c., twice daily) for 4 days produced a significant morphine tolerance and the percent change in MWT at this time point served as the baseline. First, the effect of P2X7 receptor activation in the vlPAG on the percent change in MWT was tested. Intra-vlPAG injection of Bz-ATP (9 nmol/0.3 μL) was administered 30 min before injection of a test dose of morphine (5 mg/kg, i.p.). The result showed that the percentage change in MWT in rats in the morphine + Bz-ATP group decreased from 35.2% ± 7% (before morphine i.p. injection) to 13.8% ± 4% at 30 min post-morphine i.p. injection. The percent change reached the lowest point at 60 min post-morphine i.p. injection and underwent a relative “plateau” period until the end of the observation time. Intra-vlPAG injection of Bz-ATP evidently but only transiently promoted morphine analgesic tolerance. Second, the effects of various doses of A-740003 on the percent change in MWT were tested. The results showed that intra-PAG injection of 1 nmol A-740003 had no effect on the percent change in MWT value in morphine-tolerant rats as evidenced by the value similar to the baseline level at all observation time points. Moreover, 10 or 100 nmol A-740003 microinjection considerably and dose-dependently retained morphine antinociceptive effects to mechanical stimuli, especially in the morphine + 100 nmol A740003 group. These results indicate that activation or inhibition of the P2X7 receptor in the vlPAG was sufficient to promote or attenuate the analgesic effect of morphine, at least temporarily, when morphine tolerance occurs (Fig. 5).

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Fig. 5. Effects of Bz-ATP or A-740003 on the development of morphine tolerance after chronic repetitive morphine treatments. Rats were pre-treated with Bz-ATP (9 nmol) or A-740003 (1, 10, and 100 nmol). The percent change in MWT at 4 d post-chronic repetitive morphine treatment served as the baseline (dotted line). The percent changes in MWT were significantly attenuated after intra-vlPAG injection of 9 nmol Bz-ATP in the Bz-ATP + morphine group but elevated by 10 and 100 nmol A-740003 in the morphine + 10 nmol A-740003 and morphine + 100 nmol A-740003 groups. Microinjection of 1 nmol A-740003 did not significantly affect the antinociceptive potency of morphine (***P b 0.001, *P b 0.05 compared with the baseline level; ###P b 0.001, #P b 0.05 compared with the morphine + 1 nmol A-740003 group; ▲▲▲P b 0.001, compared with morphine + 10 nmol A-740003 group; n = 8).

3.5. AS ODN prevented morphine tolerance In this experimental procedure, a reversal effect of intra-vlPAG injection of AS ODN targeting the P2X7 receptor on chronic morphine tolerance was observed. P2X7 receptor-targeting ODN was used to confirm the specific action on the P2X7 receptor. Western blot analysis indicated that delivery of AS ODN (15 nmol/0.3 μL) for five consecutive days significantly downregulated P2X7 receptor expression in the vlPAG compared with delivery of saline + morphine (all P b 0.001), which indicates knockdown efficiency. The protein expression levels of the vlPAG P2X7 receptor were not significantly different between the saline + morphine and MM ODN + morphine groups (P N 0.05). A significant reversal effect of morphine-induced tolerance was observed in the AS ODN + morphine group compared with that in the saline + morphine group (P b 0.001). No significant difference was detected between the saline + morphine and MM ODN + morphine groups in terms of the percent change in MWT (P N 0.05).

4. Discussion The present set of experiments tested the hypothesis that activation of the P2X7 receptors in the vlPAG contributes to chronic morphine tolerance in rats. The results show that repetitive morphine application could induce morphine tolerance in rats and dramatically elevate the expression levels of the P2X7 receptor in the vlPAG after 9 d of subcutaneous injection of morphine. Furthermore, the role of activation of the vlPAG P2X7 receptor in morphine tolerance was confirmed using a pharmacological approach. Microinjection of Bz-ATP, a selective agonist of the P2X7 receptor, into the vlPAG significantly but only transiently attenuated morphine-induced antinociception. By contrast, vlPAG microinjection of the P2X7 receptor selective antagonist A-740003, the analgesic effect of morphine was temporarily maintained. It was also found that AS ODN against the P2X7 receptor reduced the development

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Fig. 6. P2X7 receptor antisense, but not mismatch ODN, at 15 nmol/0.3 μL intra-vlPAG injection once daily for 5 d reduced morphine tolerance in rats. Western blot showed the down-regulation of the P2X7 receptor protein level by intra-vlPAG delivery of AS ODN targeting the P2X7 gene for five consecutive days (***P b 0.001, compared with the saline + morphine group). The P2X7 receptor protein level in the MM ODN + morphine group was not significantly different compared with that in the saline + morphine group (Fig. 6A and B). The percent changes in MWT of the AS ODN + morphine group was higher than those in the saline + morphine and MM ODN + morphine groups (**P b 0.01, ***P b 0.001, compared with the saline + morphine group). The percent changes in MWT in the MM ODN + morphine group were insignificant compared with those in the saline + morphine group (Fig. 6C).

of chronic morphine tolerance in rats. These data demonstrate the close relationship between the purinergic signaling system and morphine tolerance in the vlPAG.

Morphine is a highly potent opioid analgesic and a psychoactive drug that is widely used to treat diverse pain states. However, the analgesic effect gradually subsides following repetitive administrations. The occurrence of diminishing levels of analgesic effect limits the clinical efficacy of morphine as a therapeutic agent. Many receptors and neurotransmitters have been proposed to account for morphine tolerance mechanisms; the following factors are believed to be involved in morphine-induced analgesia tolerance: decoupling, internalization and/or down-regulation of mu-opioid receptor (Sim-Selley et al., 2007; Smith et al., 2002); up-regulation of N-methyl-D-aspartate (NMDA) receptor function (Adam et al., 2006, 2008; Allen and Dykstra, 2000); down-regulation of glutamate transporters (Bogulavsky et al., 2009; Popik et al., 2000); increased nitric oxide production (Watkins et al., 2005); and numerous neuropeptides, such as nociceptin/orphanin (Linz et al., 2014) and cholecystokinin octapeptide (Munro et al., 1998). Over recent years, several researchers reported that consistent with neuronal changes, glial cells in the central nervous system (CNS), such as microglia and astrocytes, were activated after morphine tolerance was established (Harada et al., 2013; Watkins et al., 2005, 2007). As PAG receives differential projections from the brain stem and other higher brain structures, the sources of afferent projections to PAG are extensive, thereby allowing this midbrain region to be influenced by motor, sensory, and limbic structures (Beitz, 1982; Millan, 2002). Consistently, studies have shown that significant changes or enhanced activities in the vlPAG mediated the development of morphine tolerance. After repeated administrations of morphine into the vlPAG, morphine tolerance may rapidly develop (Morgan et al., 2006). In rats, chronic subcutaneous injections of morphine may cause tolerance to subsequent test doses of morphine, but this effect can be eliminated by intra-vlPAG injections of the opioid receptor antagonist naltrexone (Lane et al., 2005). Additionally, a recent study found that mouse midbrain (mainly in the PAG) astrocytes play an important role in the development of analgesic tolerance to morphine by releasing various inflammatory cytokines and neurotrophic factors (Harada et al., 2013). These findings indicated that the mechanisms in the PAG responsible for morphine tolerance are complicated. On the basis of the signal transduction mechanisms and characteristic molecular structures, the P2 purinoceptor can be divided into the P2X receptors (P2X1–7) and P2Y receptors (P2Y1, 2, 4, 6, 11, 12, 13, 14) (Chen et al., 1995; Fredholm, 1995; Illes and Ribeiro, 2004). With techniques such as mRNA analysis (Yu et al., 2008) and whole-cell patchclamp (Sanchez-Nogueiro et al., 2014), Western blot and immunohistochemistry results confirmed that the P2X7 receptors are localized in the rodent CNS neurons (Diaz-Hernandez et al., 2009). Previous works have also suggested the expression of P2X7 receptor on CNS glia cells included astrocytes and microglial cells (Hashioka et al., 2014; Trang et al., 2012; Ying et al., 2014). Compared with other P2X receptors, P2X7 receptor have a lower affinity for ATP (Surprenant and North, 2009), indicating that their activation mostly occurs in pathological conditions associated with enhanced extracellular ATP levels. In peripheral tissues, P2X7 receptors mediate inflammation, cancer, cell proliferation, and apoptosis (Burnstock, 2007). In the nervous system, they are involved in the modulation of neurotransmitter release, as well as microglial and astroglial activation (Sperlagh et al., 2006). The activation of P2X7 receptor on neuronal or non-neuronal cells is related to many brain disorders such as trauma (Kimbler et al., 2012; O'Hare Doig and Fitzgerald, 2015), Alzheimer's disease (Diaz-Hernandez et al., 2012; Sanz et al., 2014), Parkinson's disease (Carmo et al., 2014; Liu et al., 2013), and multiple sclerosis (Grygorowicz et al., 2011). In morphine tolerance, the intrathecal microdialysis of the P2X receptor antagonist 2′,3′-O-(2,4,6-trinitrophenyl) (TNP)-ATP can attenuate morphine tolerance via down-regulation of the expression of the N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B in the synaptosomal membrane and via inhibition of excitatory amino acids released in morphine-tolerant rats (Tai et al., 2010). After chronic

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exposure to morphine, the protein expression of the P2X7 receptor in spinal microglia was up-regulated and morphine tolerance was developed. Intrathecal administration of Brilliant Blue G (BBG), a potent P2X7 receptor inhibitor, or RNA interference targeting the spinal P2X7 receptor significantly attenuated the loss of morphine analgesic potency, up-regulated P2X7 receptor expression, and activated microglia (Zhou et al., 2010). Consistent with previous reports, we also found that the expression of the vlPAG P2X7 receptor significantly increased in morphine tolerant rats but remained unchanged in saline-injected rats; however, the exact cell type of the expression site was not determined and must be further investigated. In CNS, ATP is localized in synaptic or astrocyte vesicles and coreleased with noradrenaline, acetylcholine, or other substances under nerve stimulations (Burnstock, 2004). Under such circumstances, high concentrations of ATP may be released into the pericellular space. The limited extracellular space in the CNS causes the efflux of these compounds, resulting in sufficiently high concentrations to activate lowaffinity receptors, such as P2X7. Therefore, we believed that the afferent fiber endings of the PAG released more available ATP for the vlPAG when rats were exposed to repetitive morphine treatments, but an in vivo measure of extracellular ATP concentration is clearly demanded further investigations. Proinflammatory cytokines play a vital role in morphine tolerance (Hutchinson et al., 2008; Shen et al., 2012). Ghavimi et al. (2015) found that the activities of proinflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6) and nuclear factorkappa B increased in the cerebral cortex of morphine-tolerant rats. On the one hand, the increased proinflammatory cytokines can interact with the opioid receptors and promote morphine tolerance. Chen (Chen et al., 2007) found that activation of the CX3CL1/fractalkine receptor diminished the effect of mu, delta, and kappa opioid agonists on their receptors in the PAG of rats. On the other hand, proinflammatory cytokines can up-regulate P2X7 receptor expression and increase sensitivity to extracellular ATP in morphine tolerant rats (Humphreys and Dubyak, 1998; Narcisse et al., 2005). The activated P2X7 receptor can initiate secretion of several proinflammatory substances, such as IL-1β, IL-18, TNF-α (Bartlett et al., 2014; Ferrari et al., 1997). This positive-feedback between P2X7 receptor activation and proinflammatory cytokines could accelerate morphine analgesic tolerance. As mentioned above, the activation of the P2X7 receptor is involved in a myriad of physiological and pathophysiological processes. In the current study, the agonist and antagonist of P2X7 receptor were used to understand its precise role in the development of analgesic tolerance to morphine. Following vlPAG microinjection of the P2X7 receptor selective agonist Bz-ATP (9 nmol), analgesic effect induced by challenge morphine (5 mg/kg, i.p.) was significantly but temporarily attenuated. This finding suggests that activation of the P2X7 receptor in the vlPAG is sufficient for morphine tolerance. A-740003 is structurally unrelated to the P2X7 receptor antagonists and exhibits therapeutic effect on neuropathy-induced mechanical allodynia (Honore et al., 2006; McGaraughty et al., 2007). The current study illustrated that intravlPAG injection of A-740003 (10 and 100 nml) partly and transiently reversed morphine tolerance. The AS ODN technology is a widely used method for evaluating the in vivo contributions of many proteins involved in pain behavior (Stone and Vulchanova, 2003). Previous studies found that AS ODN could be absorbed by PAG cells, and the expression of endogenous molecules in the PAG could be successfully down-regulated by microinjection of AS ODN (Maeda et al., 2005; Oka et al., 2001; Xiao et al., 2010). In the current study, AS ODN targeting the P2X7 receptor was used to obtain in vivo evidence regarding the role of the receptor in morphine tolerance development. Microinjection of the P2X7 receptor AS ODN, not MM ODN, resulted in down-regulation of P2X7 gene expression in the vlPAG and successfully attenuated the development of morphine tolerance in rats. In summary, the present results clearly illustrated that activation of the P2X7 receptor in the vlPAG play an important role in the

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development of chronic tolerance to morphine antinociception in rats. Intra-vlPAG injection of the P2X7 receptor selective antagonist A740003 (only temporarily) or P2X7 receptor AS ODN can significantly maintain the analgesic effect of morphine. These findings may clarify the mechanisms underlying the development of analgesic tolerance to morphine. From the perspective of reducing and/or abolishing the side effects of morphine, the use of the combination of morphine and the P2X7 receptor antagonist may be an effective therapeutic approach for pain management. Abbreviations A-740003 (N-(1-{[(cyanoimino) (5-quinolinylamino) methyl] amino}2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide) AS ODN antisense oligodeoxynucleotide ATP adenosine triphosphate BSA bovine serum albumin Bz-ATP 2′(3′)-O-(4-benzoylbenzoyl)-ATP CNS central nervous system DAB diaminobenzidine MM ODN mismatch oligodeoxynucleotide MWT mechanical withdrawal threshold NS physiological saline PAG midbrain periaqueductal gray PB phosphate buffer PBS phosphate-buffered saline vlPAG ventrolateral midbrain periaqueductal gray PAG midbrain periaqueductal gray PB phosphate buffer PBS phosphate-buffered saline vlPAG ventrolateral midbrain periaqueductal gray Conflict of interest statement The authors declare no conflict of interest. Acknowledgments The study was supported by the National Natural Science Foundation of China (grant number 31160207). References Adam, F., Bonnet, F., Le Bars, D., 2006. Tolerance to morphine analgesia: evidence for stimulus intensity as a key factor and complete reversal by a glycine site-specific NMDA antagonist. Neuropharmacology 51, 191–202. Adam, F., Dufour, E., Le Bars, D., 2008. The glycine site-specific NMDA antagonist (+)HA966 enhances the effect of morphine and reverses morphine tolerance via a spinal mechanism. Neuropharmacology 54, 588–596. Allen, R.M., Dykstra, L.A., 2000. Role of morphine maintenance dose in the development of tolerance and its attenuation by an NMDA receptor antagonist. Psychopharmacology (Berl.) 148, 59–65. Alves, L.A., Bezerra, R.J., Faria, R.X., Ferreira, L.G., da Silva Frutuoso, V., 2013. Physiological roles and potential therapeutic applications of the P2X7 receptor in inflammation and pain. Molecules 18, 10953–10972. Bartlett, R., Stokes, L., Sluyter, R., 2014. The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease. Pharmacol. Rev. 66, 638–675. Basbaum, A.I., Clanton, C.H., Fields, H.L., 1978. Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems. J. Comp. Neurol. 178, 209–224. Behbehani, M.M., 1995. Functional characteristics of the midbrain periaqueductal gray. Prog. Neurobiol. 46, 575–605. Beitz, A.J., 1982. The organization of afferent projections to the midbrain periaqueductal gray of the rat. Neuroscience 7, 133–159. Berrios, I., Castro, C., Kuffler, D.P., 2008. Morphine: axon regeneration, neuroprotection, neurotoxicity, tolerance, and neuropathic pain. P. R. Health Sci. J. 27, 119–128. Bogulavsky, J.J., Gregus, A.M., Kim, P.T., Costa, A.C., Rajadhyaksha, A.M., Inturrisi, C.E., 2009. Deletion of the glutamate receptor 5 subunit of kainate receptors affects the development of morphine tolerance. J. Pharmacol. Exp. Ther. 328, 579–587. Burnstock, G., 2004. Cotransmission. Curr. Opin. Pharmacol. 4, 47–52. Burnstock, G., 2006. Historical review: ATP as a neurotransmitter. Trends Pharmacol. Sci. 27, 166–176. Burnstock, G., 2007. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev. 87, 659–797.

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