Roles of different subtypes of opioid receptors in mediating the ventrolateral orbital cortex opioid-induced inhibition of mirror-neuropathic pain in the rat

Neuroscience 144 (2007) 1486 –1494

ROLES OF DIFFERENT SUBTYPES OF OPIOID RECEPTORS IN MEDIATING THE VENTROLATERAL ORBITAL CORTEX OPIOID-INDUCED INHIBITION OF MIRROR-NEUROPATHIC PAIN IN THE RAT M. ZHAO,a J. Y. WANG,b H. JIAa AND J. S. TANGa*

and Leichnetz, 1981; Craig et al., 1982), a region that modulates nociception (Sandkühler and Gebhart, 1984; Fields and Basbaum, 1999). Studies in our laboratory have shown that electrolytic lesion of the VLO or microinjection of GABA into the VLO eliminates the Sm-mediated antinociception (Zhang et al., 1995, 1998b, 1999), while electrical or chemical stimulation of the VLO depresses spinal and trigeminal nocifensive reflexes, such as tail flick (TF) reflex and jaw-opening reflex. Similarly, these anti-nociceptive effects can be eliminated by lesion or functional block of PAG (Zhang et al., 1997a,b, 1998a). These data suggest that the VLO is a part of the endogenous antinociception loop consisting of a spinal cord–Sm–VLO– PAG–spinal cord. Furthermore, microinjection of morphine or the highly selective ␮-opioid receptor agonist endomorphin-1 into the VLO depresses the TF reflex, formalininduced nociceptive behavior, and c-fos expression in the spinal dorsal horn neurons, as well as neuropathy-evoked nociception. These effects can be blocked by non-selective opioid receptor antagonist naloxone or selective ␮-receptor antagonist ␤-funaltrexamine (␤-FNA) (Huang et al., 2001, 2002; Xie et al., 2004; Zhao et al., 2006a). These studies suggest that the VLO is involved in opioid receptormediated anti-nociception in acute and chronic inflammatory and neuropathic pain. In addition, it has been proposed that these anti-nociceptive effects may be produced by activation of the VLO–PAG brainstem descending inhibitory system and depression of the nociceptive transmission at the spinal level. However, it is unclear whether opioid agonists and their receptors in the VLO also have a role in the inhibition of the mirror hypersensitivity induced by chronic neuropathic pain. Therefore, the current study was undertaken to examine the effect of opioid agonists microinjected into the VLO on mirror neuropathic pain (MNP) and to determine the influence of different subtypes of opioid receptor antagonists on this effect in the rat L5 and L6 spinal nerve ligation (SNL) model of neuropathic pain.

a

Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an Jiaotong University School of Medicine, Yanta Road West 76, Xi’an, Shaanxi 710061, PR China

b

Department of Immunology and Pathogenic Biology, Xi’an Jiaotong University School of Medicine, Xi’an, Shaanxi 710061, PR China

Abstract—Previous studies have demonstrated that opioid receptors in the prefrontal ventrolateral orbital cortex (VLO) are involved in anti-nociception. The aim of this current study was to examine whether opioid receptors in the VLO have effects on the hypersensitivity induced by contralateral L5 and L6 spinal nerve ligation (SNL), termed as mirror neuropathic pain (MNP) in the male rat. Morphine (1.0, 2.5, 5.0 ␮g) microinjected into the VLO contralateral to the SNL depressed the mechanical paw withdrawal assessed by von Frey filaments and the cold plate (4 °C)-induced paw lifting in a dose-dependent manner on the side without SNL. These effects were antagonized by microinjection of the non-selective opioid receptor antagonist naloxone (1.0 ␮g) into the same VLO site. Microinjection of endomorphin-1 (5.0 ␮g), a highly selective ␮-opioid receptor agonist, and [D-Ala2, 5 D-Leu ]-enkephalin (DADLE, 10 ␮g), a ␦-/␮-receptor agonist, also depressed the MNP. The effects of both drugs were blocked by selective ␮-receptor antagonist ␤-funaltrexamine (␤-FNA, 3.75 ␮g), but the effect of the DADLE was not influenced by the selective ␦-receptor antagonist naltrindole (5.0 ␮g). Microinjection of the ␬-opioid receptor agonist spiradoline mesylate salt (U-62066) (100 ␮g) had no effect on the MNP. These results suggest that the VLO is involved in opioid-induced inhibition of the MNP and the effect is mediated by ␮- (but not ␦- and ␬-) opioid receptors. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: mirror neuropathic pain, hypersensitivity, ventrolateral orbital cortex, opioid, opioid receptor, rat.

The prefrontal ventrolateral orbital cortex (VLO) receives projections from the spinal and medullary dorsal horn lamina I via the thalamic nucleus submedius (Sm) (Craig and Burton, 1981; Craig et al., 1982; Yoshida et al., 1991, 1992; Coffield et al., 1992) and contains neurons that project bilaterally to the periaqueductal gray (PAG) (Hardy

EXPERIMENT PROCEDURES Animals

*Corresponding author. Tel: ⫹86-29-82655172; fax: ⫹86-29-82656364. E-mail address: [email protected] (J.-S. Tang). Abbreviations: DADLE, [D-Ala2, D-Leu5]-enkephalin; ␤-FNA, ␤-funaltrexamine hydrochloride; IL-1␤, interleukin-1␤; MNP, mirror neuropathic pain; NMDA, N-methyl-D-aspartic acid; NPL, number of paw liftings; PAG, periaqueductal gray; PWT, paw withdrawal threshold; p38 MAPK, p38 mitogen-activated protein kinase; Sm, thalamic nucleus submedius; SNL, spinal nerve ligation; TF, tail flick; U-62066, spiradoline mesylate salt; VLO, ventrolateral orbital cortex.

The experiments were performed on male Sprague–Dawley rats (180 –200 g) provided by the Experimental Animal Center of Shaanxi Province, Xi’an, China. The experimental protocol was approved by the Institutional Animal Care Committee of the University. According to the ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983), all efforts were made to minimize the number of animals used and any distress of the animals.

0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.11.009

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SNL The neuropathic pain model was established by ligating the right L5 and L6 spinal nerves as reported previously (Kim and Chung, 1992). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally; SCRC, Shanghai, China) and a dorsal midline incision was made from L3 to S2. The right L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk and the incision was then closed in two layers. The rats were allowed to recover from anesthesia in an observation chamber with a warming light. The rats with sham ligation were treated in the same way as all other groups except that the spinal nerves were not ligated. The animal model of the primary and mirror hypersensitivity was considered to be established successfully if the hind paw withdrawal threshold (PWT) was significantly attenuated on both sides in response to von Frey filament stimulation and the number of paw liftings (NPL) was significantly increased on both sides during a 5 min observation period on a 4 °C cold plate. Only the rats that met these criteria were used.

Catheterization and drug administration Two weeks after the SNL, rats were anesthetized with sodium pentobarbital again and a stainless steel guide cannula (0.8 mm in diameter) was stereotaxically implanted at a position 2.5 mm dorsal to the VLO contralateral to the nerve injury at the following coordinates: 3.0 –3.7 anterior to Bregma, 1.5–2.5 lateral, 4.0 –5.0 from cortical surface (Paxinos and Watson, 1986), and the guide cannula was fixed on the skull (Xie et al., 2004). One week later (i.e. three weeks after the SNL), rats were lightly anesthetized with Alyrane (Baxter Caribe Inc., Guayama, Puerto Rico, USA), and a 1.0 ␮l microsyringe (0.4 mm in diameter) with the tip extending 2.5 mm beyond the end of the guide cannula was inserted into VLO through the guide cannula. Drugs dissolved in normal saline (0.5 ␮l) were then slowly infused into the VLO over 60 s. Drugs used in this study included morphine hydrochloride (1.0, 2.5 and 5.0 ␮g, Shenyang Medicament Co., Shenyang, China), naloxone hydrochloride (1.0 ␮g, RBI/Sigma Co., St. Louis, MO, USA), endomorphin-1 (5.0 ␮g, RBI/Sigma), [D-Ala2, D-Leu5]enkephalin (DADLE; 10 ␮g, RBI/Sigma), Spiradoline mesylate salt (U-62066; 100 ␮g, RBI/Sigma), ␤-FNA (3.75 ␮g, RBI/Sigma), and naltrindole hydrochloride (5.0 ␮g, RBI/Sigma). Of these drugs, except the ␤-FNA which was administered 24 h prior to agonist application (Ward et al., 1982), all other antagonists used were administered 5 min prior to agonist application. Endomorphin-1, DADLE, ␤-FNA and naltrindole were freshly prepared. A time interval of 2 days was allowed before the same animals were tested again. Doses of drugs were chosen according to previous studies (Kanarek et al., 2001; Wright and Ingenito, 2001; Dipirro and Kristal, 2004; Xie et al., 2004) and the same volume of 0.9% saline was injected in control experiments.

full access to the paws from underneath. Behavioral accommodation was allowed until cage exploration and major grooming activities ceased for approximately 20 min. Ten von Frey filaments (Stoelting Company, Wood Dale, IL, USA) with approximately equal logarithmic incremental (0.17) bending force were chosen (von Frey numbers: 3.61, 3.84, 4.08, 4.17, 4.31, 4.56, 4.74, 4.93, 5.07, and 5.18, equivalent to: 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, and 15.0 g, respectively). Starting with filament 4.31, which is one of the middle of the series of filaments, von Frey filaments with different intensities were applied repeatedly in a time interval of 2 s from underneath perpendicularly to the mid-plantar of the hind paw with sufficient force to cause slight bending against the paw for approximately 6 – 8 s. The pattern of positive and negative responses was recorded and converted into a 50% threshold using the formula given by Dixon (1980) and Chaplan et al. (1994).

Cold plate test Cold-induced pain was determined as described by Jasmin et al. (1998) and Sun et al. (2004). Briefly, the rat was placed on a metal plate kept at a cold temperature (4⫾1 °C), and covered with a transparent plastic box (280⫻250⫻210 mm). The number of times the rat lifted its hind paw from the cold plate during 5 min was recorded in a time interval of 10 min throughout a 50 min observation period.

Histology At the end of the experiment, the drug injection site was marked by injection of Pontamine Sky Blue dye (0.5 ␮l, 2% in 0.5 M sodium acetate acid). Under deep anesthesia, the animal was perfused transcardially with 0.9% normal saline followed by 10% formalin. The brain was then removed and fixed in fresh formalin for 3–7 days. One hundred micron sections were cut with a freezing microtome, mounted, and stained with Cresyl Violet. The spread of the drug following intracerebral microinjection with a volume of 0.5 ␮l was about 0.5 mm from injection site, as reported previously (Zhang et al., 1998b).

Data analysis All values were expressed as mean⫾S.E.M.. A linear regression was used to assess the correlation between the effects shown by the area between the time course curve and the baseline and the doses of morphine. The differences in entire observation time or at each time point among different groups were tested statistically by two-way repeated measures analysis of variance (two-way RM ANOVA) followed by post hoc Fisher LSD tests in which it was available (Milligan et al., 2003; Yang et al., 2005). P⬍0.05 was considered to be statistically significant.

RESULTS

Behavioral tests One week after catheterization (i.e. three weeks after the SNL), behavioral testing was performed during the day portion of the circadian cycle (8:00 AM to 6:00 PM) at room temperature (22 °C). All rats were habituated in the experimental arena (20 min for mechanical test and 5 min for cold test) daily during the 3 days before any tests. The animal behaviors were measured blindly by an experimenter before and 5, 15, 25, 35, 45, and 55 min after drug administration.

Tactile test The PWT in response to mechanical stimulation (von Frey filaments) was measured using the up– down method (Dixon, 1980; Chaplan et al., 1994). The rat was placed in a transparent plastic box (280⫻250⫻210 mm) with a metal wire mesh floor that allowed

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General Three weeks after the SNL, the ipsilateral PWT (2.62⫾0.11 g, n⫽23) and the NPL (14.82⫾0.74, n⫽27) and the contralateral PWT (9.00⫾0.36 g, n⫽24) and the NPL (6.93⫾0.35 n⫽22) were significantly different from those observed from sham groups (14.40⫾0.58 g and 1.83⫾0.31 for ipsilateral paw, n⫽6; 13.59⫾0.39 g and 1.75⫾0.22 for contralateral paw, n⫽6) (P⬍0.001), suggesting that when the ipsilateral primary hypersensitivity is induced after SNL, the mirror image hypersensitivity (i.e. MNP) was also induced simultaneously, although the degree of change was significantly smaller than on the ipsilateral side (P⬍0.001), as shown in Fig. 1A and B.

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and the saline-treated groups (P⬍0.05 and P⬍0.01, respectively). The number of the cold plate-evoked paw lifting in the 5.0 ␮g morphine group was significantly smaller than those in the 1.0 ␮g morphine and the salinetreated groups (P⫽0.045 and P⬍0.009, respectively). However, 1.0 ␮g morphine applied to the VLO had no effect on the MNP, either in the tactile test or the cold plate test. Naloxone (1.0 ␮g), a non-selective opioid receptor antagonist, microinjected into the VLO site 5 min prior to morphine (5.0 ␮g) administration antagonized the morphine-induced inhibition of the MNP in both the tactile test and the cold plate test. As shown in Fig. 3A and B, both the time course curves (i.e. saline, naloxone plus morphine and only morphine-treated groups) were significantly different among treatments (F(2, 85)⫽10.41, P⫽0.001; F(2, 75)⫽7.74, P⫽0.005). During the 50 min observation period, the mean PWT in the naloxone plus morphine group (8.47⫾0.71 g,

Fig. 1. Bar graphs showing the changes of the PWT (A) and the NPL (B) ipsilateral and contralateral to the SNL side. *** P⬍0.001, compared with that of the sham test; ### P⬍0.001, compared with that of the ipsilateral paw.

Microinjection of saline into the VLO contralateral to the SNL had no significant (P⬎0.05) effect on the MNP with the PWT (9.01⫾0.69 g, n⫽6) and the NPL (7.10⫾0.64, n⫽6). Effect of naloxone on morphine-induced inhibition of mirror hypersensitivity Morphine microinjected into the VLO contralateral to the SNL side significantly depressed the mechanical and cold MNP induced by SNL in a dose-dependent manner (r⫽0.909, P⫽0.047; r⫽0.948, P⫽0.026). After morphine (1.0, 2.5, 5.0 ␮g) injections, the mean PWTs were 8.99⫾0.75, 11.60⫾1.10, and 12.04⫾0.31 g (n⫽6) and the mean NPLs were 6.28⫾0.79, 4.97⫾0.85, and 4.06⫾0.62 (n⫽6) during the 50-min observation period, respectively. As shown in Fig. 2A and B, both the time course curves (i.e. saline, different doses of morphine-treated groups) were significantly different between treatments (F(3, 100)⫽5.30, P⫽0.007 for PWTs; F(3, 100)⫽3.30, P⫽0.04 for the NPLs). In the tactile test, the depressive effects induced by 5.0 ␮g and 2.5 ␮g morphine were significantly larger than those of the 1.0 ␮g morphine

Fig. 2. Effects of different doses of morphine (Mor) microinjected into the VLO on the mirror image hypersensitivity induced by SNL in the rat. (A) Tactile test: ** P⬍0.01, 5.0 ␮g morphine group compared with 1.0 ␮g morphine group and saline group; # P⬍0.05, 2.5 ␮g morphine group compared with 1.0 ␮g morphine group and saline group. (B) cold plate test: ** P⬍0.01, 5.0 ␮g morphine group compared with saline group; # P⬍0.05, 5.0 ␮g morphine group compared with 1.0 ␮g morphine group during the 50 min observation period. * P⬍0.05, compared with saline group; ⫹ P⬍0.05, compared with 1.0 ␮g morphine group at those time points (two-way RM ANOVA with a post hoc Fisher LSD test).

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endomorphin-1-treated only groups) were significantly different among treatments (F(2, 75)⫽33.08, P⬍0.001; F(2, 75)⫽8.54, P⫽0.003). After microinjection of endomorphin-1, the mean PWT (13.78⫾0.50 g, n⫽6) and the mean NPL (3.83⫾0.48, n⫽6) during the 50 min observation period were significantly different from the saline group (P⬍0.001). After ␤-FNA pretreatment, the endomorphin-1 induced inhibition of the MNP was significantly blocked (P⫽0.001, P⫽0.012) with the mean PWT (8.80⫾0.43 g, n⫽6) and the mean NPL (6.11⫾0.56, n⫽6), which were not significantly different from the saline-treated group (P⬎0.05) (Fig. 4A and B). Effects of ␤-FNA and naltrindole on DADLE-evoked inhibition of mirror hypersensitivity DADLE, a ␦- and partial ␮-opioid receptor agonist (10 ␮g) microinjected into the VLO contralateral to the SNL significantly depressed the MNP induced by SNL. The depressive effect could be antagonized by microinjection of

Fig. 3. Effects of naloxone (Nal) microinjected into VLO on the morphine (Mor, 5.0 ␮g)-induced inhibition of the mirror image hypersensitivity induced by SNL in the rats. (A) Tactile test; (B) cold plate test. ** P⬍0.001, morphine group compared with saline group; ⫹⫹⫹ P⬍0.001 (in A) and ⫹ P⬍0.05 (in B), naloxone plus morphine group compared with only morphine group during the 50 min observation period. * P⬍0.05, compared with saline group; # P⬍0.05 compared with morphine group at those time points (two-way RM ANOVA with a post hoc Fisher LSD test).

n⫽8) was significantly smaller (P⬍0.001) than those of the morphine only group. The mean NPL in the naloxone plus morphine group (6.22⫾0.35, n⫽6) was significantly larger (P⫽0.015) than those of the morphine only group. However, the mean PWT and NPL were not significantly different from the saline-treated group. Naloxone (1.0 ␮g) alone injected into the VLO had no effect on the MNP. The PWT (8.33⫾1.02 g, n⫽8) was not significantly different from the saline-treated group (P⬎0.05) (not shown). Effect of ␤-FNA on endomorphin-1 induced inhibition of mirror hypersensitivity Endomorphin-1 (5.0 ␮g), a highly selective ␮-opioid receptor agonist, microinjected into the VLO contralateral to the SNL depressed the mechanical and cold MNP induced by SNL. These effects can be effectively blocked by ␤-FNA (3.75 ␮g), a selective ␮-opioid receptor antagonist, administered into the same VLO site 24 h prior to endomorphin-1 administration. As shown in Fig. 4A and B, both the time course curves (i.e. saline, ␤-FNA⫹endomorphin-1 and

Fig. 4. Effects of endomorphin-1 (EM-1, 5.0 ␮g) microinjected into the VLO on the mirror image hypersensitivity induced by SNL in the rat and influence of ␤-FNA on this effect. (A) Tactile test; (B) cold plate test. *** P⬍0.001, EM-1 group compared with saline group; ### P⬍0.001 (in A) and ## P⬍0.01 (in B) EM-1 plus ␤-FNA group compared with only EM-1 group during the 50 min observation period. * P⬍0.05, compared with saline group; # P⬍0.05, compared with EM-1 group at those time points (two-way RM ANOVA with a post hoc Fisher LSD test.)

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␤-FNA (3.75 ␮g) 24 h prior to DADLE administration, but this effect was not influenced by pretreatment with the selective ␦-opioid receptor antagonist naltrindole (5.0 ␮g). As shown in Fig. 5A and B, the time course curves were significantly different among treatments in both tactile test and cold plate test (F(3, 100)⫽16.99, P⬍0.001; F(3, 100)⫽5.31, P⫽0.007). After DADLE injection, the mean PWT (12.37⫾0.66 g, n⫽6) and the mean NPL (3.67⫾0.71, n⫽6) during the 50 min observation period were significantly different from the saline group (P⬍0.001, P⫽0.007, respectively). After pre-treatment with ␤-FNA, the mean PWT (7.86⫾0.61 g) was smaller (P⬍0.001) and the mean NPL (6.81⫾0.89) was larger (P⬍0.01) than those of the DADLE only group, but not significantly different from the saline-treated group (P⫽0.152 and P⫽1.000, respec-

Fig. 6. Effects of U-62066 microinjected into the VLO on the mirror image hypersensitivity induced by SNL in the rat. (A) Tactile test; (B) cold plate test. There is no statistical difference between the U-62066 group and the saline control group (two-way RM ANOVA with a post hoc Fisher LSD test).

tively). However, after pretreatment with naltrindole, the mean PWT (12.07⫾0.32 g, n⫽6) and the mean NPL (3.86⫾0.90, n⫽6) were not significantly different from the DADLE only group (P⫽1.000). Effects of U-62066 on mirror hypersensitivity U-62066 (100 ␮g), a selective non-peptide ␬-opioid receptor agonist, microinjected into the VLO contralateral to the SNL had no effect on the MNP induced by SNL. After U-62066 injection, the mean PWT (8.75⫾0.51 g, n⫽5) and the mean NPL (6.87⫾0.67, n⫽5) were not significantly different from the saline-treated group (F(1, 45)⫽0.001, P⫽0.976 for tactile test; F(1, 45)⫽0.04, P⫽0.845 for cold plate test), as shown in Fig. 6A and B. Fig. 5. Effects of 10 ␮g DADLE microinjected into the VLO on the mirror image hypersensitivity induced by SNL in the rat and influences of ␤-FNA and naltrindole (Nat) on this effect. (A) Tactile test; (B) cold plate test. *** P⬍0.001 (in A) and ** P⬍0.01 (in B), DADLE group compared with saline group; ⫹⫹⫹ P⬍0.001 (in A) and ⫹⫹ P⬍0.01 (in B), DADLE plus ␤-FNA group compared with only DADLE group during the 50 min observation period. * P⬍0.05, DADLE group compared with saline group; # P⬍0.05, DADLE plus ␤-FNA group compared with DADLE only group at those time points. There is no statistical difference between the DADLE plus Nat group and the only DADLE group (two-way RM ANOVA with a post hoc Fisher LSD test).

DISCUSSION Mirror image hypersensitivity induced by L5/L6 SNL Results in this present study have indicated that three weeks after the SNL, the mechanical PWT contralateral to the SNL is significantly smaller and the cold-evoked paw lifting times larger than those obtained from the sham tests, suggesting that the primary and mirror hypersensi-

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tivities can be produced simultaneously in the bilateral hind paw in the SNL rat, although the magnitude of the MNP behavior is smaller than that seen in the primary hind paw. This result is consistent with that reported in the partial sciatic nerve injury model by Sinnott et al. (1999). MNP has also been reported in several other experimental animal models of neuropathic pain and pathological pain conditions, such as chronic sciatic nerve constriction injury (Attal et al., 1990), partial sciatic nerve section (Seltzer et al., 1990), trauma of the peripheral nerves (Zimmermann, 2001; Chacur et al., 2001) as well as carrageenan and bee venom evoked inflammatory pain. (Coderre and Melzack, 1985; Kayer and Guilbaud, 1987; Chen et al., 2000). It suggests that the MNP may be a universal phenomenon in many chronic nerve injury and inflammatory pain models, as observed in patients with chronic pain. Previous studies have indicated that the primary, secondary and referred mirror-image pain may share some common neural mechanisms at both peripheral and spinal levels (Urban et al., 1999; Chen et al., 2000). MNP is developed by temporal spinal summation of ongoing inputs of primary C-fiber afferents from the injured site or adjacent to injured site (Chen et al., 2001). However, once developed, it is independent to the ongoing primary afferent input. It is maintained by central plasticity changes, i.e. central sensitization (Chen et al., 2001). Some studies have indicated that peripheral nerve lesion can affect contralateral non-lesioned neurons in the spinal dorsal horn, which cover a wide range of outcome measures ranging from in situ hybridization and immunohistochemistry to anatomical re-arrangement (Koltzenburg et al., 1999). Also, there is evidence showing that brainstem descending facilitatory system from the rostral medial medulla (RMM) contributes to the secondary as well as referred mirrorimage pain, but not the primary one (Urban et al., 1999; Chen et al., 2003). However, a central change mediated by spinal commissural interneurons in the processing has been highly favored (Koltzenburg et al., 1999) which is also dependent upon activation of spinal N-methyl-D-aspartic acid (NMDA) and non-NMDA receptor (Koltzenburg et al., 1999; Chen et al., 2000). Recently, some reports showed that immune factors can contribute to the MNP. Milligan et al. (2003) were the first to identify the spinal mediators of the mirror image low-threshold mechanical allodynia. They found that both ipsilateral and mirror-image sciatic inflammatory neuropathy (SIN)-induced allodynia can be reversed by the intrathecal delivery of fluorocitrate, a glial metabolic inhibitor, or CNI-1493, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK) pathways. Furthermore, Yang et al. (2005) have found that an intracisternal injection of SB203580, a p38 MAPK inhibitor, produces a significant inhibition of interleukin-1␤ (IL-1␤)induced mechanical allodynia or mirror-image mechanical allodynia produced by 10 pg of IL-1␤. These results suggest that central MAPK participates in the primary and mirror-image allodynia. However, these data could not completely explain the underlying neural mechanisms of the MNP. Thus, further experiments were needed for clarifying this mechanism.

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Role of opioid in the VLO-mediated inhibition of mirror hypersensitivity Our current study has clearly shown that morphine administrated into the contralateral VLO depresses the mechanical and cold hypersensitivities contralateral to the SNL, and both effects can be effectively antagonized by nonselective opioid receptor antagonist naloxone administrated into the same VLO site. It suggests that the VLO is involved in the opioid-receptor-mediated anti-nociceptive effect on the mirror image hypersensitivity. The results of this study are in accordance to those reported in our laboratory that microinjection of morphine into the VLO depresses the TF reflex, formalin-evoked nociceptive behavior, nociceptive response of neurons and c-fos expression in the spinal dorsal horn, as well as the ipsilateral primary allodynia in the SNL rat (Huang et al., 2001, 2002; Xie et al., 2004; Zhao et al., 2006a,b). Moreover, Al Amin et al. (2004) have also demonstrated that continuous perfusion with morphine of the VLO produces significant and naloxone-reversible depression of ipsilateral tactile and cold allodynia in the spinal nerve injury model. Together with those previous studies, our present results suggest that the VLO is involved in the opioid-receptor-mediated antinociceptive effects not only on the primary acute, tonic or inflammatory, and neuropathic pain, but also on the mirror image allodynia/hyperalgesia in neuropathic pain. To our best knowledge, our data provide the first evidence for opioid (morphine) inhibition of the mirror image pain at the cerebral level. Furthermore, our current results have showed that opioid receptor antagonist naloxone alone applied to the VLO does not alter the mechanical mirror image hypersensitivity, which suggests that the MNP induced by the SNL is not a result of deactivation of opioid receptors in the VLO and the effect of naloxone on the morphine-induced inhibition of the MNP is not due to facilitation of the nociceptive response in the rat SNL model. This result is consistent with that of naloxone alone applied to the VLO (Zhao et al., 2006a) on the primary allodynia in SNL rats. It suggests that the opioid receptors in the VLO lack a tonic inhibitory action on either the mirror or primary neuropathic pain. It is also noted that in the present study, the drug injection is limited in the VLO contralateral to the nerve injured side, but we believe that the ipsilateral VLO may also have a similar effect on the nociceptive responses, since a comparable study (Zhang et al., 1996) has indicated that stimulation of the ipsilateral Sm, a region that projects to VLO, can inhibit the nociceptive response of the spinal dorsal horn neurons similar to that of stimulation of the contralateral Sm, which may be due to the VLO projecting bilaterally into the lateral or ventrolateral part of the PAG (Hardy and Leichnetz, 1981; Craig et al., 1982). Roles of different opioid receptor subtypes in VLO opioid-evoked inhibition of mirror hypersensitivity The present study has extended the abovementioned findings by showing that the highly selective ␮-opioid receptor agonist endomorphin-1 microinjected into the VLO con-

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tralateral to the SNL depresses the MNP induced by SNL, and this inhibition can be effectively blocked by the selective ␮-opioid receptor antagonist ␤-FNA. Furthermore, application of ␤-FNA, but not naltrindole, a selective ␦-opioid receptor antagonist, into the VLO selectively blocks the ␦and ␮-opioid receptor agonist DADLE-induced inhibition of the MNP, although DADLE has a higher affinity for ␦-receptor than ␮-receptor (Smith et al., 1988). It suggests that ␮- but not ␦-opioid receptors in the VLO are involved in the inhibition of the MNP in the nerve injury model. This notion is also supported by previous anatomic findings showing that ␮-opioid receptors are distributed in the VLO of the prefrontal cortex (Mclean et al., 1986; Mansour et al., 1987; Delfs et al., 1994; Burkey et al., 1996) and the endomorphin-1-like immunoreactivity is widely and densely distributed in the intracerebral pain modulation system (Martin et al., 1999). Moreover, systemic application of ␮-opioid receptor agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin) (Desmeules et al., 1993) or intrathecal application of endomorphin (Han et al., 2005) depresses the mechanical allodynia in the paw opposite to the peripheral sciatic mononeuropathy or complete Freund’s adjuvant (CFA) injection side in rats. The findings in the present study further suggest that the VLO is involved in the opioid-induced inhibitory effect on the MNP induced by the SNL. In addition, our present study indicated that the ␦- and ␬- opioid receptors in the VLO may not play any role in the VLO opioid-induced inhibition of the MNP in the SNL rat. It is consistent with our previous studies that ␦- and ␬-opioid receptors are not involved in the VLO-mediated-antinociception on the persistent pain behavior induced by formalin (Xie et al., 2004) and the ipsilateral allodynic behavior caused by the SNL (Zhao et al., 2006a). However, Desmeules et al. (1993) have reported that systemic administration of the ␦-opioid agonist BUBU [tyr-D-Ser(O-t-butyl)Gly-Phe-Leu-Thr] and the ␬-opioid agonist U-69593 [(⫹)-(5␣,7␣,8␤)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro [4,5]dec-8-yl]-benzeneacetamide] depresses the mechanical pain-like behaviors in the side contralateral to the lesioned nerve. The difference may result from activation of ␬- and ␦-opioid receptors in the regions other than the VLO due to different route of drug administration. Possible mechanisms of VLO opioid-evoked anti-mirror hypersensitivity There is evidence indicating that descending inhibition may be involved in modulating the secondary pain in neuropathic pain. Bain et al. (1998) have demonstrated that in SNL rats, spinal transection completely abolishes primary neuropathic painlike behaviors in the ipsilateral paw, but causes normal tail to begin flicking upon innocuous probing (secondary allodynia). Furthermore, Monhemius et al. (2001) have provided evidence that the persistent nociceptive inputs can activate the PAG brainstem descending inhibitory system. They demonstrate that in rats with partial sciatic nerve ligation but not normal rats, the response to formalin injection in the contralateral paw is significantly depressed, and microinjection of GABA into the PAG significantly increased the response of the contralateral paw

to formalin. It suggests that a tonic descending inhibition of the MNP has been developed in the PAG after the partial sciatic nerve ligation. Therefore, it is reasonable to propose that the VLO opioid-induced antinociception on the MNP as well as the primary one may be produced by activation of the VLO–PAG brainstem descending inhibitory system and inhibition of the nociceptive transmission at the spinal cord level. Since the direct action of opioid on neurons is to hyperpolarize the cell membrane due to a increased K⫹ conductance and to reduce transmitter release secondary to inhibition of a voltage-dependent calcium conductance, it has been suggested that the excitatory effects of opioid on neurons may result from inhibition of an inhibitory GABAergic interneuron (Heinricher et al., 1992, 1994; Vaughan et al., 1997). The notion has been supported by evidence showing that the GABAergic interneurons and the ␮-opioid receptors are distributed widely in the VLO (Mclean et al., 1986; Esclapez et al., 1987; Mansour et al., 1987; Delfs et al., 1994; Burkey et al., 1996). More importantly, we have found that most of the GABAergic neurons and their terminals express ␮-opioid receptors (Huo et al., 2005). These anatomic studies have pointed out that a local neuronal network consisting of inhibitory GABAergic and opioidergic neurons and their terminals as well as the projection neurons may exist in the VLO. Indeed, microinjection of the GABAA receptor antagonist into the VLO dose-dependently inhibits the TF reflex in the rat, and increases the antinociceptive effects of morphine microinjected into the VLO, while application of GABAA receptor agonist into the VLO attenuates this morphine-induced inhibition (Qu et al., 2006). These results not only indicate the existence of such a local neuronal network as stated above, but also suggest that GABAergic modulation is involved in mediating the VLO opioid-induced antinociceptive effect. It is possible that local application of opioid agents or stimulation-evoked endogenous opioid peptide released from opioidergic neurons directly inhibits the inhibitory action of the GABAergic interneurons on the output neurons projecting to the PAG. Such a disinhibitory effect may lead to activation of the VLO–PAG brainstem descending inhibitory system and depression of the nociceptive transmission at the spinal cord level.

CONCLUSION In conclusion, the findings of this study suggest that the VLO is involved in the opioid-induced anti-mirror-imagehypersensitivity and this effect is mediated by ␮- (but not ␦and ␬-) opioid receptors in the SNL rats. GABAergic disinhibition may be also involved in the VLO opioid-induced inhibition of the MNP as well as the primary one. Acknowledgments—The authors wish to thank Drs. D. Y. Yao and N. Chung for their expert help in reviewing the manuscript. The project was supported by the National Natural Science Foundation of China (No. 30270453: 30570592).

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REFERENCES Al Amin HA, Atweh SF, Baki SA, Jabbur SJ, Saade NE (2004) Continuous perfusion with morphine of the orbitofrontal cortex reduces allodynia and hyperalgesia in a rat model for mononeuropathy. Neurosci Lett 364:27–31. Attal N, Jazat F, Kayser V, Guilbaud G (1990) Further evidence for pain-related behaviors in a model of unilateral peripheral mononeuropathy. Pain 41:235–251. Bain D, Ossipov MH, Zhong C, Malan TP, Porreca F (1998) Tactile allodynia, but not thermal hyperalgesia, of the hindlimb is blocked by spinal transaction in rats with nerve injury. Neurosci Lett 241:79 – 82. Burkey AR, Carstens E, Wenniger JJ, Tang JW, Jasmin L (1996) An opioidergic cortical antinociception triggering site in the agranular insular cortex of the rat that contributes to morphine antinociception. J Neurosci 16:6612– 6623. Chacur M, Milligan ED, Gazda LS, Armstrong C, Wang H, Tracey KJ, Maier SF, Watkins LR (2001) A new model of sciatic inflammatory neuritis (SIN): induction of unilateral and bilateral mechanical allodynia following acute unilateral peri-sciatic immune activation in rats. Pain 94:231–244. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL (1994) Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53:55– 63. Chen HS, Chen J, San YY (2000) Contralateral heat hyperalgesia induced by unilaterally intraplantar bee venom injection is produced by central changes: a behavioral study in the conscious rats. Neurosci Lett 284:45– 48. Chen HS, Chen J, Guo WG, Zheng MH (2001) Establishment of bee venom-induced contralateral heat hyperalgesia in the rat is dependent upon central temporal summation of afferent input from the site of injury. Neurosci Lett 298:57– 60. Chen HS, Li MM, Shi J, Chen J (2003) Supraspinal contribution to development of both tonic nociception and referred mirror hyperalgesia: a comparative study between formalin test and bee venom test in the rat. Anesthesiology 98:1231–1236. Coderre TJ, Melzack R (1985) Increased pain sensitivity following heat injury involves a central mechanism. Behav Brain Res 15:259 –262. Coffield JA, Bowen KK, Miletic V (1992) Retrograde tracing of projections between the nucleus submedius, the ventrolateral orbital cortex and the midbrain in the rat. J Comp Neurol 132:488 – 499. Craig AD, Wiegand SJ, Price JL (1982) The thalamocortical projection of the nucleus submedius in the cat. J Comp Neurol 206:28 – 48. Craig AD, Burton H (1981) Spinal and medullary lamina I projection to nucleus submedius in medial thalamus: a possible pain center. J Neurophysiol 45:443– 465. Delfs JM, Kong H, Mestek A, Chen Y, Yu L, Reisine T, Chesselet MF (1994) Expression of mu opioid receptor mRNA in rat brain: an in situ hybridization study at the single cell level. J Comp Neurol 345:46 – 68. Desmeules JA, Kayser V, Guilbaud G (1993) Selective opioid receptor agonist modulate mechanical allodynia in an animal model of neuropathic pain. Pain 53:277–285. Dipirro JM, Kristal MB (2004) Placenta ingestion by rats enhances ␦and ␬-opioid antinociception, but suppresses ␮-opioid antinociception. Brain Res 1014:22–33. Dixon WJ (1980) Efficient analysis of experimental observation. Annu Rev Pharmacol Toxicol 20:441– 462. Esclapez M, Campistron G, Trottier S (1987) Immunocytochemical localization and morphology of GABA-containing neurons in the prefrontal and frontoparietal cortex of the rat. Neurosci Lett 15:131–136. Fields HL, Basbaum AI (1999) Central nervous system mechanisms of pain modulation. In: Textbook of pain, 4th edition (Wall PD, Melzack R, eds), pp 309 –329. New York: Churchill Livingstone.

1493

Han XZ, Li HL, Wang ZM, Li YQ (2005) Involvement of spinal endomorphin 2 in mirror-image pain induced by peripheral inflammation. Chin J Neuroanat 21:299 –304. Hardy SGP, Leichnetz GR (1981) Frontal cortex projections to the periaqueductal gray in the rat: a retrograde and orthograde horseradish peroxidase study. Neurosci Lett 23:13–17. Heinricher MM, Morgan MM, Fields HL (1992) Direct and indirect action of morphine on medullary neurons that modulate nociception. Neuroscience 48:533–543. Heinricher MM, Morgan MM, Tortorici V, Fields HL (1994) Disinhibition of off-cells and antinociception produced by an opioid action within the rostral ventromedial medulla. Neuroscience 63:279 –288. Huang X, Tang JS, Yuan B, Jia H (2001) Morphine applied to the ventrolateral orbital cortex produces a naloxone-reversible antinociception in the rat. Neurosci Lett 299:189 –192. Huang X, Tang JS, Yuan B, Jia H (2002) Inhibitory effect of morphine microinjection into the ventrolateral orbital cortex on c-fos expression of neurons in spinal dorsal horn induced by formalin-test in rats. J Xi’an Med Univ 23:104 –107. Huo FQ, Wang J, Tang JS, Jia H (2005) GABAergic neurons express ␮-opioid receptors in the ventrolateral orbital cortex of the rat. Neurosci Lett 382:265–268. Jasmin L, Kohan L, Franssen M, Janni G, Goff JR (1998) The cold plate as a test of nociceptive behaviors: description and application to the study of chronic neuropathic and inflammatory pain models. Pain 75:367–382. Kanarek RB, Mandillo S, Wiatr C (2001) Chronic sucrose intake augments antinociception induced by injections of mu but not kappa opioid receptor agonists into the periaqueductal gray matter in male and female rats. Brain Res 920:97–105. Kayer V, Guilbaud G (1987) Local and remote modifications of nociceptive sensitivity during carrageenin-induced inflammation in the rats. Pain 28:99 –107. Kim SH, Chung JM (1992) An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 50:355–363. Koltzenburg M, Wall PD, McMahon SB (1999) Does the right side know what the left is doing? Trends Neurosci 22122–22127. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1987) Autoradiographic differentiation of ␮-, ␦-, ␬-opioid receptors in the rat forebrain and midbrain. J Neurosci 7:2445–2464. Martin SS, Gerall AA, Kastin AJ, Zadina JE (1999) Differential distribution of endomorphin-1 and endomorphin-2-like immunoreactivities in the CNS of the rodent. J Comp Neurol 405:450 – 471. Mclean S, Rothman RB, Herkenham M (1986) Autoradiographic localization of ␮- and ␦-opioid receptors in the forebrain of the rat. Brain Res 378:49 – 60. Milligan ED, Twining C, Chacur M, Biedenkapp J, O’Connor K, Poole S, Tracey K, Martin D, Maier SF, Watkins LR (2003) Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J Neurosci 23:1026 –1040. Monhemius R, Green DL, Roberts MHT, Azami J (2001) Periaqueductal gray mediated inhibition of responses to noxious stimulation is dynamically activated in a rat model of neuropathic pain. Neurosci Lett 298:70 –74. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd ed. Sydney: Academic Press. Qu CL, Tang JS, Jia H (2006) GABAergic modulation involvement in the ventrolateral orbital cortex morphine-induced antinociception in the rat. Brain Res 1073–C 1074C:281–289. Sandkühler J, Gebhart GF (1984) Characterization of inhibition of a spinal nociceptive reflex by stimulation medially and laterally in the midbrain and medulla in the pentobarbital-anesthetized rat. Brain Res 305:67–76. Seltzer Z, Dubner R, Shir Y (1990) A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 43:205–218.

1494

M. Zhao et al. / Neuroscience 144 (2007) 1486 –1494

Sinnott CJ, Garfield JM, Strichartz GR (1999) Differential efficacy of intravenous lidocaine in alleviating ipsilateral versus contralateral neuropathic pain in the rats. Pain 80:521–531. Smith DJ, Perrotti JM, Crisp T, Cabral ME, Long JT, Scalzitti JM (1988) The ␮opiate receptor is responsible for descending pain inhibition originating in the periaqueductal gray region of the rat brain. Eur J Pharmacol 156:47–54. Sun RQ, Wang HC, Wan Y, Jing Z, Luo F, Han JS, Wang Y (2004) Suppression of neuropathic pain by peripheral electrical stimulation in rats: ␮-opioid receptor and NMDA receptor implicated. Exp Neurol 187:23–29. Urban MO, Zahn PK, Gebhart GF (1999) Descending facilitatory influences from the rostral medial medulla mediate secondary, but not primary hyperalgesia in the rat. Neuroscience 90:349 –352. Vaughan CW, Ingram SL, Connor MA, Christie MJ (1997) How opioid inhibits GABA-mediated neurotransmission. Nature 390:611– 614. Ward SJ, Portoghese PS, Takemori AE (1982) Pharmacological characterization in vivo of the novel opiate, beta-funaltrexamine. J Pharmacol Exp Ther 220:494 – 498. Wright RC, Ingenito AJ (2001) Prevention of isolation-induced hypertension by intrahippocampal administration of a nonpeptide kappaopioid receptor agonist. Hippocampus 11:445– 451. Xie YF, Wang J, Huo FQ, Jia H, Tang JS (2004) ␮ But not ␦ and ␬ opioid receptor involvement in the ventrolateral orbital cortex opioid-evoked antinociception in formalin test rats. Neuroscience 126:717–726. Yang CS, Jung CY, Ju JS, Lee MK, Ahn DK (2005) Intracisternal administration of mitogen-activated protein kinase inhibitors reduced IL-1␤-induced mirror-image mechanical allodynia in the orofacial area of rats. Neurosci Lett 387:32–37. Yoshida A, Dostrovsky JO, Sessle BJ, Chiang CY (1991) Trigeminal projections to the nucleus submedius of the thalamus in the rat. J Comp Neurol 307:609 – 625. Yoshida A, Dostrovsky JO, Chiang CY (1992) The afferent and efferent connections of the nucleus submedius in the rat. J Comp Neurol 324:115–132.

Zhang YQ, Tang JS, Yuan B, Jia H (1995) Inhibitory effects of electrical stimulation of thalamic nucleus submedius area on the rat tail flick reflex. Brain Res 696:205–212. Zhang YQ, Tang JS, Yuan B, Jia H (1996) Inhibitory effects of electrical stimulation of thalamic nucleus submedius on the nociceptive responses of spinal dorsal horn neurons in the rat. Brain Res 73:16–24. Zhang YQ, Tang JS, Yuan B, Jia H (1997a) Inhibitory effects of electrically evoked activation of ventrolateral orbital cortex on the tail-flick reflex are mediated by periaqueductal gray in rats. Pain 72:127–135. Zhang S, Tang JS, Yuan B, Jia H (1997b) Involvement of the frontal ventrolateral orbital cortex in descending inhibition of nociception mediated by the periaqueductal gray. Neurosci Lett 224:142–146. Zhang S, Tang JS, Yuan B, Jia H (1998a) Inhibitory effects of electrical stimulation of ventrolateral orbital cortex on the rat jaw-opening reflex. Brain Res 823:359 –366. Zhang S, Tang JS, Yuan B, Jia H (1998b) Inhibitory effects of glutamate-induced activation of thalamic nucleus submedius are mediated by ventrolateral orbital cortex and periaqueductal gray in rats. Eur J Pain 2:153–163. Zhang S, Tang JS, Yuan B, Jia H (1999) Electrically-evoked inhibitory effects of the nucleus submedius on the jaw-opening reflex are mediated by ventrolateral orbital cortex and periaqueductal gray matter in the rat. Neuroscience 93:867– 875. Zhao M, Wang JY, Jia H, Tang JS (2006a) ␮- But not ␦- and ␬-opioid receptors in the ventrolateral orbital cortex mediate opioid-induced antiallodynia in a rat neuropathic pain model. Brain Res 1076:68 –77. Zhao M, Li Q, Tang JS (2006b) The effects of microinjection of morphine into thalamic nucleus submedius on formalin-evoked nociceptive responses of neurons in the rat spinal dorsal horn. Neurosci Lett 401:103–107. Zimmermann M (1983) Ethical guideline for investigations of experimental pain in conscious animals. Pain 16:109 –110. Zimmermann M (2001) Pathobiology of neuropathic pain. Eur J Pharmacol 429:23–37.

(Accepted 8 November 2006) (Available online 19 December 2006)