- Email: [email protected]
The role of dopamine receptors in ventrolateral orbital cortex-evoked antinociception in a rat formalin test model
European Journal of Pharmacology 657 (2011) 97–103
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
The role of dopamine receptors in ventrolateral orbital cortex-evoked antinociception in a rat formalin test model Yong-Hui Dang b, Bo Xing b, Yan Zhao b, Xin-Jie Zhao b, Fu-Quan Huo a, Jing-Shi Tang a,⁎, Chao-Ling Qu a,⁎, Teng Chen b a
Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Xi'an Jiaotong University School of Medicine, Yanta Road West 76#, Xi'an, Shaanxi 710061, China Department of Forensic Medicine, Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Xi'an Jiaotong University School of Medicine, Yanta Road West 76#, Xi'an, Shaanxi 710061, China
b
a r t i c l e
i n f o
Article history: Received 2 August 2010 Received in revised form 29 December 2010 Accepted 27 January 2011 Available online 17 February 2011 Keywords: Ventrolateral orbital cortex Dopamine receptor Antinociception Persistent inflammatory pain Formalin test (Rat)
a b s t r a c t The present study examined the roles of dopamine and D1- and D2-like dopamine receptors in ventrolateral orbital cortex (VLO)-evoked antinociception in rats with persistent inflammatory pain. Following formalin injection into the rat unilateral hindpaw pad, the effects of dopamine receptor agonist and antagonist microinjections into the VLO on nociceptive behavior were observed. Results demonstrated that VLO microinjection of the non-selective dopamine receptor agonist apomorphine (R(−)-apomorphine hydrochloride, 1.0, 2.5 and 5.0 μg) depressed later-phase nociceptive behavior induced by formalin injection; this effect was attenuated by the D2-like dopamine receptor antagonist S(−)-raclopride(+)-tartrate salt (raclopride, 3.0 μg), but not by the D1-like dopamine receptor antagonist R(+)-SCH-23390 hydrochloride (SCH-23390, 1.0 μg). Apomorphine-induced antinociception was mimicked by microinjection of the D2-like dopamine receptor agonist (−)-quinpirole hydrochloride (2.0 and 5.0 μg) into the same VLO site, and this effect was antagonized by raclopride (3.0 μg). In addition, microinjection of the D1-like dopamine receptor agonist R(+)-SKF-38393 hydrochloride (5.0 μg) had no effect on formalin-induced nociceptive behavior during the later phase. However, the D1-like dopamine receptor antagonist SCH-23390 (2.5, 5.0 and 10 μg) depressed nociceptive behavior in a dose-dependent manner. These results suggested that dopamine mediated VLO-induced antinociception via different mechanisms in the persistent inflammatory pain model; D2-like receptors mediated dopamine-induced antinociception, while D1-like dopamine receptors exhibited tonic facilitatory action on nociceptive behavior, thereby blocking D1-like dopamine receptors could induce antinociception. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Previous studies have shown that the ventrolateral orbital cortex (VLO) is part of an endogenous analgesic system, consisting of the spinal cord–thalamic nucleus submedius (Sm)–ventrolateral orbital cortex (VLO)–periaqueductal gray (PAG)–spinal cord loop (Tang et al., 2009). Electrical stimulation of the VLO or microinjection of excitatory amino acid glutamate into the VLO depresses tail flick and jaw opening reflexes, and these effects are eliminated by electrolytic lesions of the PAG or microinjection of inhibitory neurotransmitter γ-aminobutyric acid (GABA) into the PAG (Zhang et al., 1997a,b, 1998a). However,
⁎ Corresponding authors at: Tel.: + 86 29 82655172; fax: + 86 29 82656364. E-mail addresses: [email protected] (J.-S. Tang), [email protected] (C.-L. Qu). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.064
lesions or chemical inhibition of the VLO eliminates the antinociceptive effect induced by Sm activation (Zhang et al., 1995, 1998b, 1999). Anatomic studies have demonstrated that the limbic forebrain area, which includes the VLO, receives input from dopaminergic terminals originating from the ventral tegmental area (VTA) (Benes et al., 1993; Ohara et al., 2003). Behavioral studies have shown that the nucleus accumbens, dorsolateral striatum, rostral agranular insular cortex, and anterior cingulate cortex are involved in dopamine-mediated antinociception (Magnusson and Fisher, 2000; Taylor et al., 2003; Koyanagi et al., 2008; Coffeen et al., 2008; Burkey et al., 1999; López-Avila et al., 2004). A recent study from our laboratory demonstrated that D2-like (D2/D3) dopamine receptors are involved in VLO-evoked descending antinociception in the rat tail flick test (Sheng et al., 2009). However, it remains unknown whether dopamine and its receptors are involved in modulation of persistent inflammatory pain. Formalin injection to the rat unilateral hindpaw pad, which evokes biphasic nociceptive behavior, has been widely used to study persistent inflammatory nociceptive processes and the
98
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
efficacy of analgesic drugs (Dubuisson and Dennis, 1977; Coderre et al., 1993; Abbott et al., 1995; Watson et al., 1997). The present study examined the effects of nonselective dopamine receptor agonist R(−)apomorphine hydrochloride (apomorphine) microinjection into the VLO on formalin-induced nociceptive behavior. In addition, the influence of D2-like and D1-like dopamine receptor antagonists S(−)-raclopride (+)-tartrate salt (raclopride) and R(+)-SCH-23390 hydrochloride (SCH-23390) microinjections on apomorphine-induced effects was analyzed. Furthermore, the effects of the D2-like dopamine receptor agonist (−)-quinpirole hydrochloride (quinpirole), D1 like dopamine receptor agonist R(+)-SKF-38393 hydrochloride (SKF-38393), and antagonist SCH-23390 on formalin-induced nociceptive behavior were examined in the rat. 2. Materials and Methods 2.1. Animals Experiments were performed on male Sprague–Dawley rats (220– 250 g), which were provided by the Experimental Animal Center of Shaanxi Province, China. Experimental protocols were approved by the Institutional Animal Care Committee of Xi'an Jiaotong University and were in accordance with ethical guidelines from the International Association for the Study of Pain (Zimmermann, 1983). All efforts were made to minimize the number of animals used, as well as distress to the animals. 2.2. Intracerebral Guide Cannula Placement The rats were intraperitoneally anesthetized with sodium pentobarbital (50 mg kg−1), and the head was immobilized in a stereotaxic frame. A small craniotomy was performed just above the VLO. A stainless steel guide cannula (0.8 mm in diameter) was stereotaxically inserted, with the tip 2.0 mm dorsal to the VLO, at the following coordinates: 3.2 mm anterior to bregma, 2.5 mm lateral, and 4.5 mm below cortical surface (Paxinos and Watson, 1986). The cannula was then attached to the skull with three microscrews and dental cement. Once the animals recovered from anesthesia, sodium penicillin was administered (0.2 million units/day for 4 days, intraperitoneally) to prevent wounds and intracerebral infections. The animals were carefully nursed and fed in clean cages. 2.3. Formalin Test The formalin test was performed as previously described (Dubuisson and Dennis, 1977; Wheeler-Aceto and Cowan, 1991; Coderre et al., 1993; Abbott et al., 1995; Watson et al., 1997). Each rat was placed in a plastic chamber with a mirror placed under the glass plane at a 45° angle to observe spontaneous activity of the affected paw. The rat received a 50-μl subcutaneous injection of diluted (5%) formalin to the unilateral hindpaw pad, contralateral to the intracerebral cannula, and was immediately returned to the chamber. The number of flinches or shakes of the affected paw from the glass plane, as well as the duration (s) of licking/lifting of the affected paw within a 5-min period, were recorded for 60 min by two experimenters blind to grouping. Formalin was injected 1–2 times in each rat over a 1-week interval to assure disappearance of local inflammation and experimental repeating (second injection site was different from the first injection site) (Li et al., 2007). 2.4. Intracerebral Drug Microinjection The rats were lightly anesthetized with enflurane (Baxter Caribe, Guayama, Puerto Rico, USA), a very short-acting anesthetic, and a 1.0-μl microsyringe, with the tip extending 2 mm beyond the end of the guide cannula, was inserted into the VLO through the guide cannula. The drugs
were dissolved in saline (0.5 μl) and slowly infused through the microsyringe at a constant speed over a 60-s period to observe the effect on formalin-induced nociceptive behavior (agitation). Drugs used in the present study, including the nonselective dopamine receptor agonist apomorphine [R(−)-apomorphine hydrochloride], D2-like (D2/D3) dopamine receptor agonist quinpirole [(−)quinpirole hydrochloride], D1-like (D1/D5) dopamine receptor agonist SKF-38393 [R(+)-SKF-38393 hydrochloride], antagonist SCH-23390 [R (+)-SCH-23390 hydrochloride], and D2-like (D2/D3) dopamine receptor antagonist raclopride [S(−)-raclopride(+)-tartrate salt] were purchased from RBI/Sigma, St. Louis, MO, USA. The drugs were freshly prepared in saline. Agonist was injected into the VLO contralateral to the affected hindpaw, and antagonist was administered 5 min prior to the agonist injection. Drug doses were chosen according to previous studies (Altier and Stewart, 1998; Magnusson and Fisher, 2000; Taylor et al., 2003; Meyer et al., 2009), where they were reported to be effective. Equal volumes of saline were injected into the VLO as vehicle controls. 2.5. Histology At the end of the experiment, the drug injection sites were marked by injection of Pontamine Sky Blue dye (0.5 μl, 2% in 0.5 M sodium acetate solution). Under deep anesthesia, the rats were transcardially perfused with 0.9% normal saline, followed by 10% formalin. The brains were then removed and fixed in 10% formalin solution for 7 days. The brains were cut into 30-μm thick sections using a freezing microtome, and then the slices were stained with cresyl violet. The injecting sites were histologically identified within the VLO, with an example shown in Fig. 1. The drug did not spread N0.5 mm from the injection sites, as previously reported (Qu et al., 2006). 2.6. Data Analysis All values were expressed as mean± S.E.M. Due to a correlation between flinching/shaking and licking/lifting responses during the formalin test, i.e., when duration of licking/lifting paw increased, the amount of flinching/shaking decreased and vice versa, previous reports were references (Wheeler-Aceto and Cowan, 1991; Coderre et al., 1993; Watson et al., 1997). In addition, a new method for determining combined scores according to saline control experiments was utilized: the maximal number of flinching/shaking during a 5-min period (throughout a 60-min observation period) was scored as 5; maximal duration (s) of licking/lifting during an additional 5-min period was also scored as 5. The sum (10 scores) of both values represented maximal nociceptive behavior (agitation). Therefore, the agitation score at each time point was accounted for throughout the 60-min observation period. One-way analysis of variance (ANOVA) was used to analyze differences between various groups during the early phase (0–5-min) of formalin-induced nociceptive behavior. Two-way ANOVA, followed by a post-hoc multiple comparison (Fisher LSD test), was used to analyze differences during the late-phase (20–60-min) observation time and at each time point among different groups. P values b 0.05 were considered to be statistically significant. 3. Results 3.1. Effect of Apomorphine Microinjection into the VLO on Formalin-Induced Agitation Responses Microinjection of apomorphine, a nonselective dopamine receptor agonist (1.0, 2.0, and 5.0 μg, in 0.5 μl, respectively) into the VLO had no effect on formalin-induced agitation responses during the early phase (Fig. 2A and C, P N 0.05). However, during the later phase, formalininduced nociceptive behavior was significantly depressed. As shown in Fig. 2A, time course curves (saline, and different doses of apomorphinetreated groups) were different between treatments (F(3,243) = 18.378,
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
Fig. 1. Photomicrograph showing a location example of an injection site in the ventrolateral orbital cortex (VLO). Arrow points to injection site within VLO. Acb, accumbens nucleus; CI, claustrum; CPu, caudate putamen; fmi, forceps minor corpus callosum; Fr, frontal cortex; VLO, ventrolateral orbital cortex. Scale bar = 1000 μm.
P b 0.001) and across times (F(8,243) = 14.911, P b 0.001). However, there was no interaction between treatment and time (F(24,243) = 1.248, P = 0.202). Further analyses indicated that formalin-induced later phase (20–60 min) nociceptive behavior (agitation score) was significantly reduced with increasing apomorphine doses (Fig. 2C). Detailed comparisons at individual time points and between various groups are shown in Fig. 2A. Microinjection of raclopride, a D2-like dopamine receptor antagonist (3.0 μg), into the VLO at 5-min prior to apomorphine (5.0 μg) injection, significantly reduced apomorphine-induced inhibition of nociceptive
99
behavior during the later phase (F(2,207) = 30.631, P b 0.001), and the agitation score in the raclopride plus apomorphine group was significantly greater (P b 0.01) than the apomorphine group (Fig. 2B and D). Raclopride (3.0 μg) injection alone into the VLO did not influence later phase nociceptive behavior, but rather enhanced early phase nociceptive behavior (Fig. 3A and C). Pretreatment with D1-like receptor antagonist SCH-23390 (1.0 μg) did not influence the inhibitory effect of apomorphine during the later phase. However, during the early phase, it provoked an inhibitory effect on apomorphine; the agitation score in the SCH-23390 plus apomorphine group was significantly less than in the apomorphine group (Fig. 2B and D). It is important to note, however, that 1.0 μg of SCH-23390 had little to no effect on formalin-induced nociceptive behavior when injected alone (Fig. 3B and D). 3.2. Effect of Quinpirole Microinjection into the VLO on Formalin-Induced Agitation Responses The inhibitory effect of apomorphine on formalin-induced nociceptive behavior was mimicked by a microinjection of quinpirole, a D2-like dopamine receptor agonist, into the same VLO site. Quinpirole (2.0 and 5.0 μg) significantly depressed the agitation response during the later phase, but not during the early phase. As shown in Fig. 4A, time course curves (saline and different doses of quinpirole-treated groups) were different between treatments (F(2,144) = 39.416, P b 0.001) and across times (F(8,144) = 13.042, P b 0.001), but displayed no interaction between treatment and time (F(16,144) = 0.936, P = 0.530). Further analyses indicated that different doses of quinpirole had no effect on formalin-
Fig. 2. Effects of microinjection of apomorphine (Apo) into the VLO on formalin-evoked nociceptive behavior (agitation) and the influence of D2- and D1-like dopamine receptor antagonists on apomorphine effects. A and C: effects of different doses (1, 2, and 5 μg) of apomorphine on agitation at those time points (A) and during phase 1 and 2 observation periods, respectively (C). *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05, compared with 1.0 μg apomorphine; +P b 0.05, compared with 2.0 μg apomorphine. B and D: effects of D2- and D1-like dopamine receptor antagonists, raclopride (Rac, 3 μg) and SCH-23390 (SCH, 1.0 μg), on apomorphine (5 μg)-induced inhibition of agitation at those time points (C) and during phase 1 and 2 observation periods, respectively (D). *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05 and ##P b 0.01, compared with 5.0 μg apomorphine.
100
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
Fig. 3. Effects of microinjection of D2- and D1-like dopamine receptor antagonists, raclopride (Rac, 3.0 μg) and SCH-23390 (SCH, 1.0 μg), alone into the VLO on formalin-evoked nociceptive behavior (agitation), respectively. A and B: time course curves showing no effects in all but 20-min time point; C and D: bar graphs showing that raclopride enhances agitation responses in phase 1, but not in phase 2 observation period, while SCH-23390 reduces agitation response in phase 2, but not in phase 1 observation period. *P b 0.05 and **P b 0.01, compared with saline.
induced nociceptive behavior during the early phase, and the agitation score was not significantly different from the saline group. However, during the later phase, agitation scores significantly decreased following increasing quinpirole doses (P b 0.001, Fig. 4B). Detailed comparisons at individual time points and between various groups are shown in Fig. 4A. Microinjection of raclopride, a D2-like dopamine receptor antagonist (3.0 μg), into the VLO at 5 min prior to quinpirole (5.0 μg) injection, significantly reduced quinpirole-induced inhibition of nociceptive behavior during the later phase, and agitation scores in the raclopride plus quinpirole group were significantly larger (P b 0.001) than in the quinpirole group (Fig. 4B and D). 3.3. Effect of Microinjection of SKF-38393 and SCH-23390 into the VLO on Formalin-Induced Agitation Responses Microinjection of SKF-38393 (5.0 μg), a D1-like dopamine receptor agonist, into the VLO had no effect on formalin-induced nociceptive behavior during the later phase. Agitation scores were not significantly different from the saline group during the 20–60 min observation period, as well as at all but the 20-min time point (P N 0.05, Fig. 5A and C). However, during the early phase, SKF-38393 significantly increased nociceptive behavior, and agitation scores were significantly larger than in the saline group (P b 0.001, Fig. 5A and C). Microinjection of SCH-23390, a D1-like dopamine receptor antagonist (2.5, 5.0, and 10 μg), into the VLO significantly decreased the agitation response during the early and later phases. As shown in Fig. 5B, time course curves (saline and different doses of SCH-23390treated groups) were different between treatments (F(3,234) = 49.256, P b 0.001), across times (F(8,243) = 8.282, P b 0.001), and between interactions (F(24,243) = 1.694, P = 0.026). Further analyses indicated that 5.0 and 10 μg SCH-23390 resulted in significantly decreased
agitation scores during the early phase compared with the saline group (P b 0.001). However, agitation scores decreased with increasing SCH-23390 doses during the later phase (P b 0.05 and P b 0.001, Fig. 5D). Detailed comparisons at individual time points and between various groups are shown in Fig. 5B. 4. Discussion Results from the present study demonstrated that microinjection of the nonselective dopamine receptor agonist apomorphine into the VLO significantly depressed formalin-induced nociceptive behavior during the later phase, but not during the early phase. This effect was attenuated by the D2-like dopamine receptor antagonist raclopride, but not by the D1-like dopamine receptor antagonist SCH-23390. Furthermore, the apomorphine effect was mimicked by microinjection of the D2-like dopamine receptor agonist quinpirole into the same VLO site, and this effect was antagonized by raclopride. In addition, raclopride microinjected alone into the VLO did not influence formalin-evoked nociceptive behavior during the later phase, which suggested that the antagonizing effect of raclopride on quinpirole and apomorphineinduced inhibition was not due to a result of a direct raclopride-induced increase of nociceptive behavior. These results were consistent with previous results from our laboratory (Sheng et al., 2009), showing that quinpirole administration into the VLO resulted in raclopride-reversible antinociception in the rat tail flick test. The above-described results suggested that D2-like dopamine receptors were involved in mediating the dopamine-induced antinociceptive effect on acute physiological pain, as well as persistent inflammatory pain, in the VLO. However, in the present study, activation of VLO D2-like dopamine receptors did not influence the formalin-induced early phase nociceptive response. These results might be due to plastic changes in the descending antinociceptive pathway under a persistent inflammatory state (Millan, 2002;
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
101
Fig. 4. Effects of microinjection of D2-like dopamine receptor agonist quinpirole (Quinp, 2.0, 5.0 μg) into the VLO on formalin-evoked nociceptive behavior (agitation), and the influence of D2-like dopamine receptor antagonist raclopride (Rac, 3.0 μg) on quinpirole (5.0 μg)-evoked inhibition of the agitation response. A and B: time course curves showing difference at time points between different groups; C and D: bar graphs showing effects in phase 1 and 2 observation periods, respectively. *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05 and ###P b 0.001, compared with 2.0 μg quinpirole; + P b 0.05 and +++P b 0.001, compared with 5.0 μg quinpirole.
Pertovaara, 2000; Ren and Dubner, 2002; Vanegas and Schaible, 2004), resulting in inhibited D2-like dopamine receptor activity in the VLO. It is important to note that the D2-like receptor antagonist raclopride injected alone into the VLO significantly enhanced the early phase nociceptive response (see Fig. 3A and C), which suggested that D2-like receptors in this region and at this time could exhibit transitory tonic inhibitory activity on nociceptive behavior, thereby inducing inhibition of D2-like receptors with raclopride enhances formalin-evoked acute nociceptive behavior. However, further studies are needed to determine the possible action mechanisms involved. Mesolimbic dopaminergic brain areas receive input from ventral tegmental areas (Benes et al., 1993; Ohara et al., 2003), such as the nucleus accumbens and dorsolateral striatum, which are involved in nociception modulation in the formalin test. Treatment with dopamine D2-like receptor agonists in these brain areas significantly reduces formalin-induced nociceptive behavior (Magnusson and Fisher, 2000; Taylor et al., 2003; Koyanagi et al., 2008). The present results provide novel evidence for the involvement of D2 dopamine receptors in mediating VLO-induced antinociception in the formalin test. Results demonstrated that microinjection of the D1-like dopamine receptor agonist SKF-38393 into the VLO exhibited no effect on formalininduced nociceptive behavior. However, the antagonist SCH-23390 resulted in a dose-dependent decrease in nociceptive behavior. Although a smaller dose (1.0 μg) of SCH-23390 did not influence early phase nociceptive behavior when injected alone (Fig. 3B and D), it provoked an inhibitory effect on apomorphine during early phase (see Fig. 2B and D). These results suggested that D1-like dopamine receptors exhibited a tonic inhibitory influence on the antinociceptive pathway, an action that antagonizes D2-like dopamine receptor activation-induced antinociception, thereby blocking D1-like dopamine receptors could induce antinociception. These findings were consistent with a previous study
(Baliki et al., 2003), which showed that 6-hydroxydopamine (6-OHDA) lesions of dopaminergic terminals in the VLO temporarily attenuate spared nerve injury (SNI)-induced neuropathic pain in the rat. Results from the present study were also consistent with a previous report by Coffeen et al. (2008) showing that activation of D2 receptors and inhibition of D1 receptors in the rostral agranular insular cortex result in antinociception in a rat model of sciatic denervation. In contrast, however, results obtained from Burkey et al. (1999) demonstrated that D1 receptors are involved in rostral agranular insular cortexmediated descending antinociception in the heated paw withdrawal test, as well as in the nociceptive response of spinal wide-dynamic range neurons to peripheral noxious stimulation. Studies have suggested that D1 and D2 receptors are simultaneously involved in mediating antinociception in the anterior cingulate cortex in a rat model of sciatic denervation (López-Avila et al., 2004). These differences may be due to varying experimental conditions, procedures, animal models, and the brain areas investigated; different dopamine receptor subtypes exhibit different modulation mechanisms for mediating descending antinociception (Altier and Stewart, 1999). As mentioned above, the VLO is part of the endogenous analgesic system, which consists of an ascending pathway from the spinal cord to the VLO via the thalamic nucleus submedius (Sm), as well as a descending pathway to the spinal cord via the periaqueductal gray (PAG) (Tang et al., 2009). It is reasonable to propose that D2-like dopamine receptor activation-induced antinociception could be mediated by activation of the antinociceptive pathway, which depresses nociceptive information transmission at the spinal level. However, the D2-like dopamine receptor is an inhibitory G-protein-coupled receptor, and activation of this receptor depresses neuronal activity by inducing hyperpolarization. In contrast, D1-like dopamine receptors are coupled to excitatory G-proteins, which, when activated, produce an excitatory
102
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
Fig. 5. Effects of microinjection of D1-like dopamine receptor agonist SKF-38393 (SKF, 5.0 μg) and antagonist SCH-23390 (SCH 2.5, 5.0 and 10 μg) into the VLO on formalin-evoked nociceptive behavior (agitation). A and B: time course curves showing difference at time points between different groups; C and D: bar graphs showing effects in phase 1 and 2 observation periods, respectively. *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05, compared with 2.5 μg SCH-23390; +P b 0.05 and +++P b 0.001, compared with 5.0 μg SCH-23390.
effect on target neurons by depolarizing the cellular membrane (Missale et al., 1998; Vallone et al., 2000; Millan, 2002). As observed in μ-opioid receptors and 5-HT1A receptors (Qu et al., 2006; Huo et al., 2008), it is possible that the excitatory effect induced by D2-like receptor activation could result from blocking inhibitory GABAergic interneurons (disinhibition), which could lead to activation of projection neurons. In contrast, the inhibitory effect induced by D1-like receptor activation could result from excitation of GABAergic neurons (proinhibition), which could lead to inhibition of projection neurons. Therefore, GABAergic disinhibition and proinhibition mechanisms could be involved in VLO D1 and D2-like receptor activation-induced effects (facilitation and depression, respectively). Morphological studies have established that GABAergic neurons and GABAA receptors are distributed in the frontal cortex, including the VLO (Esclapez et al., 1987; Pirker et al., 2000; Huo et al., 2005, 2008), and GABAergic neurons in these brain areas receive dopaminergic terminal projections from the ventral tegmental area (Benes et al., 1993; Ohara et al., 2003) and express D1 and D2 dopamine receptors (Gesper et al., 1995; Vincent et al., 1995; Seamans et al., 2001). Studies have also shown that VLO neurons projecting to PAG express GABAA receptors and display symmetrical (presumably inhibitory) synapses with GABAergic terminals (Huo et al., 2009). Therefore, a local neuronal circuit consisting of dopamine afferent terminals, GABAergic interneurons, and output neurons, in addition to the receptors, exists in the VLO and provides a morphological foundation for GABAergic modulation. It is reasonable to propose that inhibitory effects of VLO-injected apomorphine on formalin-induced nociceptive behavior could be the result of interactions between D1 and D2-like receptors. However, further behavioral, electrophysiological and molecular biological studies are needed to determine the GABAergic modulation mechanisms influencing D1- and D2-like dopamine receptors in VLO-evoked antinociception in inflammatory pain model.
5. Conclusion Results from the present study suggested that the role of dopamine receptors in the VLO is different under conditions of persistent inflammatory pain. D2-like dopamine receptors mediated VLO-induced antinociception, but D1-like dopamine receptors exhibited a tonic facilitatory effect on nociceptive behavior. GABAergic modulation could be implicated in D1- and D2-like dopamine receptor activation-induced effects. Acknowledgments This project was supported by grants from the National Natural Science Foundation of China (No. 30700222; 30800334) and the Ministry of Science and Technology of China (No. 2009DFA31080). There was no conflict of interest in relation to this article. References Abbott, F.V., Franklin, K.B., Westbrook, R.F., 1995. The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 60, 91–102. Altier, N., Stewart, J., 1998. Dopamine receptor antagonists in the nucleus accumbens attenuate analgesia induced by ventral tegmental area substance P or morphine and by nucleus accumbens amphetamine. J. Pharmacol. Exp. Ther. 285, 208–215. Altier, N., Stewart, J., 1999. The role of dopamine in the nucleus accumbens in analgesia. Life Sci. 22, 2269–2287. Baliki, M., Al-Amin, H.A., Atweh, S.F., Jaber, M., Hawwa, N., Jabbur, S.J., Apkarian, A.V., Saadé, N.E., 2003. Attenuation of neuropathic manifestations by local block of the activities of the ventrolateral orbito-frontal area in the rat. Neuroscience 120, 1093–1104. Benes, F.M., Vincent, S.L., Molloy, R., 1993. Dopamine-immunoreactive varicosities form nonrandom contacts with GABA-immunoreactive neurons of rat medial prefrontal cortex. Synapse 15, 285–295. Burkey, A.R., Carstens, E., Jasmin, L., 1999. Dopamine reuptake inhibition in the rostral agranular insular cortex produces antinociception. J. Neurosci. 19, 4169–4179.
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103 Coderre, T.J., Fundytus, M.E., McKenna, J.E., Dalal, S., Melzack, R., 1993. The formalin test: a validation of the weighted-scores method of behavioural pain rating. Pain 54, 43–50. Coffeen, U., López-Avila, A., Ortega-Legaspi, J.M., del Ángel, R., López-Muñoz, F.J., Pellicer, F., 2008. Dopamine receptors in the anterior insular cortex modulate long-term nociception in the rat. Eur. J. Pain 12, 535–543. Dubuisson, D., Dennis, S.G., 1977. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4, 161–174. 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. 77, 131–136. Gesper, P., Bloch, B., Le Moine, C., 1995. D1 and D2 receptor gene expression in the rat frontal cortex: cellular localization in different classes of efferent neurons. Eur. J. Neurosci. 7, 1050–1063. Huo, F.Q., Wang, J., Li, Y.Q., Chen, T., Han, F., Tang, J.S., 2005. GABAergic neurons express μ-opioid receptors in the ventrolateral orbital cortex of the rat. Neurosci. Lett. 382, 265–268. Huo, F.Q., Qu, C.L., Li, Y.Q., Tang, J.S., Jia, H., 2008. GABAergic modulation is involved in the ventrolateral orbital cortex 5-HT1A receptor activation-induced antinociception in the rat. Pain 139, 398–405. Huo, F.Q., Chen, T., Lv, B.C., Wang, J., Zhang, T., Qu, C.L., Li, Y.Q., Tang, J.S., 2009. Synaptic connections between GABAergic elements and serotonergic terminals or projecting neurons in the ventrolateral orbital cortex. Cereb. Cortex 19, 1263–1272. Koyanagi, S., Himukashi, S., Mukaida, K., Shichino, T., Fukuda, K., 2008. Dopamine D2-like receptor in the nucleus accumbens is involved in the antinociceptive effect of nitrous oxide. Aneth. Analg. 106, 1904–1909. Li, Y., Yuan, B., Tang, J.S., 2007. Effect of stimulation and lesion of the thalamic nucleus submedius on formalin-evoked nociceptive behavior in rats. Acta Physiol. Sin. 59, 777–783. López-Avila, A., Coffeen, U., Ortaga-Legaspi, J., del Angel, R., Pellicer, F., 2004. Dopamine and NMDA system modulate long-term nociception in the rat anterior cingulate cortex. Pain 111, 136–143. Magnusson, J., Fisher, K., 2000. The involvement of dopamine in nociception: the role of D(1) and D(2) receptors in the dorsolateral striatus. Brain Res. 855, 260–266. Meyer, P.J., Morgan, M.M., Kozell, L.B., Ingram, S.L., 2009. Contribution of dopamine receptors to periaqueductal gray-mediated antinociception. Psychopharmacol. Berl. 204, 531–540. Millan, M.J., 2002. Descending control of pain. Prog. Neurobiol. 66, 355–474. Missale, C., Nash, S.R., Robinson, S.W., Jaber, M., Caron, M.G., 1998. Dopamine receptors: from structure to function. Physiol. Rev. 78, 189–225. Ohara, P.T., Granato, A., Moallem, T.M., Wang, B.R., Tillet, Y., Lasmin, L., 2003. Dopaminergic input to GABAergic neurons in the rostral agranular insular cortex of the rat. J. Neurocytol. 32, 131–141. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, Sydney. Pertovaara, A., 2000. Plasticity in descending pain modulatory systems. Prog. Brain Res. 129, 231–242.
103
Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., Sperk, G., 2000. GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815–850. Qu, C.L., Tang, J.S., Jia, H., 2006. Involvement of GABAergic modulation of antinociception induced by morphine microinjection into the ventrolateral orbital cortex. Brain Res. 1073–1074, 281–289. Ren, K., Dubner, R., 2002. Descending modulation in persistent pain: an update. Pain 100, 1–6. Seamans, J.K., Gorelova, N., Durstewitz, D., Yang, C.R., 2001. Bidirectional dopamine modulation of GABAergic inhibition in prefrontal cortical pyramidal neurons. J. Neurosci. 21, 3628–3638. Sheng, H.Y., Qu, C.L., Huo, F.Q., Du, J.Q., Tang, J.S., 2009. D2-like but not D1-like dopamine receptors are involved in the ventrolateral orbital cortex-induced antinociception: a GABAergic modulation mechanism. Exp. Neurol. 215, 128–134. Tang, J.S., Qu, C.L., Huo, F.Q., 2009. The thalamic nucleus submedius and ventrolateral orbital cortex are involved in nociceptive modulation: a novel pain modulation pathway. Prog. Neurobiol. 89, 383–389. Taylor, B.K., Joshi, C., Uppal, H., 2003. Stimulation of dopamine D2 receptors in the nucleus accumbens inhibits inflammatory pain. Brain Res. 987, 135–143. Vallone, D., Oicetti, R., Borrelli, E., 2000. Stucture and function of dopamine receptors. Neurosci. Biobehav. Res. 24, 125–132. Vanegas, H., Schaible, H.G., 2004. Descending control of persistent pain: inhibitory or facilitatory? Brain Res. Rev. 46, 295–309. Vincent, S.L., Khan, Y., Benes, F.M., 1995. Cellular colocalization of dopamine D1 and D2 receptors in rat medial prefrontal cortex. Synapse 19, 112–120. Watson, G.S., Sufka, K.J., Coderre, T.J., 1997. Optimal scoring strategies and weights for the formalin test in rats. Pain 70, 53–58. Wheeler-Aceto, H., Cowan, A., 1991. Standardization of the rat paw formalin test for the evaluation of analgesics. Psychopharmacol. Berl. 104, 35–44. Zhang, Y.Q., Tang, J.S., 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, Y.Q., Tang, J.S., 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, J.S., 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, J.S., 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, J.S., 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, J.S., 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. Zimmermann, M., 1983. Ethical guideline for investigation of experimental pain in conscious animals. Pain 16, 109–110.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
The role of dopamine receptors in ventrolateral orbital cortex-evoked antinociception in a rat formalin test model Yong-Hui Dang b, Bo Xing b, Yan Zhao b, Xin-Jie Zhao b, Fu-Quan Huo a, Jing-Shi Tang a,⁎, Chao-Ling Qu a,⁎, Teng Chen b a
Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Xi'an Jiaotong University School of Medicine, Yanta Road West 76#, Xi'an, Shaanxi 710061, China Department of Forensic Medicine, Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Xi'an Jiaotong University School of Medicine, Yanta Road West 76#, Xi'an, Shaanxi 710061, China
b
a r t i c l e
i n f o
Article history: Received 2 August 2010 Received in revised form 29 December 2010 Accepted 27 January 2011 Available online 17 February 2011 Keywords: Ventrolateral orbital cortex Dopamine receptor Antinociception Persistent inflammatory pain Formalin test (Rat)
a b s t r a c t The present study examined the roles of dopamine and D1- and D2-like dopamine receptors in ventrolateral orbital cortex (VLO)-evoked antinociception in rats with persistent inflammatory pain. Following formalin injection into the rat unilateral hindpaw pad, the effects of dopamine receptor agonist and antagonist microinjections into the VLO on nociceptive behavior were observed. Results demonstrated that VLO microinjection of the non-selective dopamine receptor agonist apomorphine (R(−)-apomorphine hydrochloride, 1.0, 2.5 and 5.0 μg) depressed later-phase nociceptive behavior induced by formalin injection; this effect was attenuated by the D2-like dopamine receptor antagonist S(−)-raclopride(+)-tartrate salt (raclopride, 3.0 μg), but not by the D1-like dopamine receptor antagonist R(+)-SCH-23390 hydrochloride (SCH-23390, 1.0 μg). Apomorphine-induced antinociception was mimicked by microinjection of the D2-like dopamine receptor agonist (−)-quinpirole hydrochloride (2.0 and 5.0 μg) into the same VLO site, and this effect was antagonized by raclopride (3.0 μg). In addition, microinjection of the D1-like dopamine receptor agonist R(+)-SKF-38393 hydrochloride (5.0 μg) had no effect on formalin-induced nociceptive behavior during the later phase. However, the D1-like dopamine receptor antagonist SCH-23390 (2.5, 5.0 and 10 μg) depressed nociceptive behavior in a dose-dependent manner. These results suggested that dopamine mediated VLO-induced antinociception via different mechanisms in the persistent inflammatory pain model; D2-like receptors mediated dopamine-induced antinociception, while D1-like dopamine receptors exhibited tonic facilitatory action on nociceptive behavior, thereby blocking D1-like dopamine receptors could induce antinociception. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Previous studies have shown that the ventrolateral orbital cortex (VLO) is part of an endogenous analgesic system, consisting of the spinal cord–thalamic nucleus submedius (Sm)–ventrolateral orbital cortex (VLO)–periaqueductal gray (PAG)–spinal cord loop (Tang et al., 2009). Electrical stimulation of the VLO or microinjection of excitatory amino acid glutamate into the VLO depresses tail flick and jaw opening reflexes, and these effects are eliminated by electrolytic lesions of the PAG or microinjection of inhibitory neurotransmitter γ-aminobutyric acid (GABA) into the PAG (Zhang et al., 1997a,b, 1998a). However,
⁎ Corresponding authors at: Tel.: + 86 29 82655172; fax: + 86 29 82656364. E-mail addresses: [email protected] (J.-S. Tang), [email protected] (C.-L. Qu). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.064
lesions or chemical inhibition of the VLO eliminates the antinociceptive effect induced by Sm activation (Zhang et al., 1995, 1998b, 1999). Anatomic studies have demonstrated that the limbic forebrain area, which includes the VLO, receives input from dopaminergic terminals originating from the ventral tegmental area (VTA) (Benes et al., 1993; Ohara et al., 2003). Behavioral studies have shown that the nucleus accumbens, dorsolateral striatum, rostral agranular insular cortex, and anterior cingulate cortex are involved in dopamine-mediated antinociception (Magnusson and Fisher, 2000; Taylor et al., 2003; Koyanagi et al., 2008; Coffeen et al., 2008; Burkey et al., 1999; López-Avila et al., 2004). A recent study from our laboratory demonstrated that D2-like (D2/D3) dopamine receptors are involved in VLO-evoked descending antinociception in the rat tail flick test (Sheng et al., 2009). However, it remains unknown whether dopamine and its receptors are involved in modulation of persistent inflammatory pain. Formalin injection to the rat unilateral hindpaw pad, which evokes biphasic nociceptive behavior, has been widely used to study persistent inflammatory nociceptive processes and the
98
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
efficacy of analgesic drugs (Dubuisson and Dennis, 1977; Coderre et al., 1993; Abbott et al., 1995; Watson et al., 1997). The present study examined the effects of nonselective dopamine receptor agonist R(−)apomorphine hydrochloride (apomorphine) microinjection into the VLO on formalin-induced nociceptive behavior. In addition, the influence of D2-like and D1-like dopamine receptor antagonists S(−)-raclopride (+)-tartrate salt (raclopride) and R(+)-SCH-23390 hydrochloride (SCH-23390) microinjections on apomorphine-induced effects was analyzed. Furthermore, the effects of the D2-like dopamine receptor agonist (−)-quinpirole hydrochloride (quinpirole), D1 like dopamine receptor agonist R(+)-SKF-38393 hydrochloride (SKF-38393), and antagonist SCH-23390 on formalin-induced nociceptive behavior were examined in the rat. 2. Materials and Methods 2.1. Animals Experiments were performed on male Sprague–Dawley rats (220– 250 g), which were provided by the Experimental Animal Center of Shaanxi Province, China. Experimental protocols were approved by the Institutional Animal Care Committee of Xi'an Jiaotong University and were in accordance with ethical guidelines from the International Association for the Study of Pain (Zimmermann, 1983). All efforts were made to minimize the number of animals used, as well as distress to the animals. 2.2. Intracerebral Guide Cannula Placement The rats were intraperitoneally anesthetized with sodium pentobarbital (50 mg kg−1), and the head was immobilized in a stereotaxic frame. A small craniotomy was performed just above the VLO. A stainless steel guide cannula (0.8 mm in diameter) was stereotaxically inserted, with the tip 2.0 mm dorsal to the VLO, at the following coordinates: 3.2 mm anterior to bregma, 2.5 mm lateral, and 4.5 mm below cortical surface (Paxinos and Watson, 1986). The cannula was then attached to the skull with three microscrews and dental cement. Once the animals recovered from anesthesia, sodium penicillin was administered (0.2 million units/day for 4 days, intraperitoneally) to prevent wounds and intracerebral infections. The animals were carefully nursed and fed in clean cages. 2.3. Formalin Test The formalin test was performed as previously described (Dubuisson and Dennis, 1977; Wheeler-Aceto and Cowan, 1991; Coderre et al., 1993; Abbott et al., 1995; Watson et al., 1997). Each rat was placed in a plastic chamber with a mirror placed under the glass plane at a 45° angle to observe spontaneous activity of the affected paw. The rat received a 50-μl subcutaneous injection of diluted (5%) formalin to the unilateral hindpaw pad, contralateral to the intracerebral cannula, and was immediately returned to the chamber. The number of flinches or shakes of the affected paw from the glass plane, as well as the duration (s) of licking/lifting of the affected paw within a 5-min period, were recorded for 60 min by two experimenters blind to grouping. Formalin was injected 1–2 times in each rat over a 1-week interval to assure disappearance of local inflammation and experimental repeating (second injection site was different from the first injection site) (Li et al., 2007). 2.4. Intracerebral Drug Microinjection The rats were lightly anesthetized with enflurane (Baxter Caribe, Guayama, Puerto Rico, USA), a very short-acting anesthetic, and a 1.0-μl microsyringe, with the tip extending 2 mm beyond the end of the guide cannula, was inserted into the VLO through the guide cannula. The drugs
were dissolved in saline (0.5 μl) and slowly infused through the microsyringe at a constant speed over a 60-s period to observe the effect on formalin-induced nociceptive behavior (agitation). Drugs used in the present study, including the nonselective dopamine receptor agonist apomorphine [R(−)-apomorphine hydrochloride], D2-like (D2/D3) dopamine receptor agonist quinpirole [(−)quinpirole hydrochloride], D1-like (D1/D5) dopamine receptor agonist SKF-38393 [R(+)-SKF-38393 hydrochloride], antagonist SCH-23390 [R (+)-SCH-23390 hydrochloride], and D2-like (D2/D3) dopamine receptor antagonist raclopride [S(−)-raclopride(+)-tartrate salt] were purchased from RBI/Sigma, St. Louis, MO, USA. The drugs were freshly prepared in saline. Agonist was injected into the VLO contralateral to the affected hindpaw, and antagonist was administered 5 min prior to the agonist injection. Drug doses were chosen according to previous studies (Altier and Stewart, 1998; Magnusson and Fisher, 2000; Taylor et al., 2003; Meyer et al., 2009), where they were reported to be effective. Equal volumes of saline were injected into the VLO as vehicle controls. 2.5. Histology At the end of the experiment, the drug injection sites were marked by injection of Pontamine Sky Blue dye (0.5 μl, 2% in 0.5 M sodium acetate solution). Under deep anesthesia, the rats were transcardially perfused with 0.9% normal saline, followed by 10% formalin. The brains were then removed and fixed in 10% formalin solution for 7 days. The brains were cut into 30-μm thick sections using a freezing microtome, and then the slices were stained with cresyl violet. The injecting sites were histologically identified within the VLO, with an example shown in Fig. 1. The drug did not spread N0.5 mm from the injection sites, as previously reported (Qu et al., 2006). 2.6. Data Analysis All values were expressed as mean± S.E.M. Due to a correlation between flinching/shaking and licking/lifting responses during the formalin test, i.e., when duration of licking/lifting paw increased, the amount of flinching/shaking decreased and vice versa, previous reports were references (Wheeler-Aceto and Cowan, 1991; Coderre et al., 1993; Watson et al., 1997). In addition, a new method for determining combined scores according to saline control experiments was utilized: the maximal number of flinching/shaking during a 5-min period (throughout a 60-min observation period) was scored as 5; maximal duration (s) of licking/lifting during an additional 5-min period was also scored as 5. The sum (10 scores) of both values represented maximal nociceptive behavior (agitation). Therefore, the agitation score at each time point was accounted for throughout the 60-min observation period. One-way analysis of variance (ANOVA) was used to analyze differences between various groups during the early phase (0–5-min) of formalin-induced nociceptive behavior. Two-way ANOVA, followed by a post-hoc multiple comparison (Fisher LSD test), was used to analyze differences during the late-phase (20–60-min) observation time and at each time point among different groups. P values b 0.05 were considered to be statistically significant. 3. Results 3.1. Effect of Apomorphine Microinjection into the VLO on Formalin-Induced Agitation Responses Microinjection of apomorphine, a nonselective dopamine receptor agonist (1.0, 2.0, and 5.0 μg, in 0.5 μl, respectively) into the VLO had no effect on formalin-induced agitation responses during the early phase (Fig. 2A and C, P N 0.05). However, during the later phase, formalininduced nociceptive behavior was significantly depressed. As shown in Fig. 2A, time course curves (saline, and different doses of apomorphinetreated groups) were different between treatments (F(3,243) = 18.378,
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
Fig. 1. Photomicrograph showing a location example of an injection site in the ventrolateral orbital cortex (VLO). Arrow points to injection site within VLO. Acb, accumbens nucleus; CI, claustrum; CPu, caudate putamen; fmi, forceps minor corpus callosum; Fr, frontal cortex; VLO, ventrolateral orbital cortex. Scale bar = 1000 μm.
P b 0.001) and across times (F(8,243) = 14.911, P b 0.001). However, there was no interaction between treatment and time (F(24,243) = 1.248, P = 0.202). Further analyses indicated that formalin-induced later phase (20–60 min) nociceptive behavior (agitation score) was significantly reduced with increasing apomorphine doses (Fig. 2C). Detailed comparisons at individual time points and between various groups are shown in Fig. 2A. Microinjection of raclopride, a D2-like dopamine receptor antagonist (3.0 μg), into the VLO at 5-min prior to apomorphine (5.0 μg) injection, significantly reduced apomorphine-induced inhibition of nociceptive
99
behavior during the later phase (F(2,207) = 30.631, P b 0.001), and the agitation score in the raclopride plus apomorphine group was significantly greater (P b 0.01) than the apomorphine group (Fig. 2B and D). Raclopride (3.0 μg) injection alone into the VLO did not influence later phase nociceptive behavior, but rather enhanced early phase nociceptive behavior (Fig. 3A and C). Pretreatment with D1-like receptor antagonist SCH-23390 (1.0 μg) did not influence the inhibitory effect of apomorphine during the later phase. However, during the early phase, it provoked an inhibitory effect on apomorphine; the agitation score in the SCH-23390 plus apomorphine group was significantly less than in the apomorphine group (Fig. 2B and D). It is important to note, however, that 1.0 μg of SCH-23390 had little to no effect on formalin-induced nociceptive behavior when injected alone (Fig. 3B and D). 3.2. Effect of Quinpirole Microinjection into the VLO on Formalin-Induced Agitation Responses The inhibitory effect of apomorphine on formalin-induced nociceptive behavior was mimicked by a microinjection of quinpirole, a D2-like dopamine receptor agonist, into the same VLO site. Quinpirole (2.0 and 5.0 μg) significantly depressed the agitation response during the later phase, but not during the early phase. As shown in Fig. 4A, time course curves (saline and different doses of quinpirole-treated groups) were different between treatments (F(2,144) = 39.416, P b 0.001) and across times (F(8,144) = 13.042, P b 0.001), but displayed no interaction between treatment and time (F(16,144) = 0.936, P = 0.530). Further analyses indicated that different doses of quinpirole had no effect on formalin-
Fig. 2. Effects of microinjection of apomorphine (Apo) into the VLO on formalin-evoked nociceptive behavior (agitation) and the influence of D2- and D1-like dopamine receptor antagonists on apomorphine effects. A and C: effects of different doses (1, 2, and 5 μg) of apomorphine on agitation at those time points (A) and during phase 1 and 2 observation periods, respectively (C). *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05, compared with 1.0 μg apomorphine; +P b 0.05, compared with 2.0 μg apomorphine. B and D: effects of D2- and D1-like dopamine receptor antagonists, raclopride (Rac, 3 μg) and SCH-23390 (SCH, 1.0 μg), on apomorphine (5 μg)-induced inhibition of agitation at those time points (C) and during phase 1 and 2 observation periods, respectively (D). *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05 and ##P b 0.01, compared with 5.0 μg apomorphine.
100
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
Fig. 3. Effects of microinjection of D2- and D1-like dopamine receptor antagonists, raclopride (Rac, 3.0 μg) and SCH-23390 (SCH, 1.0 μg), alone into the VLO on formalin-evoked nociceptive behavior (agitation), respectively. A and B: time course curves showing no effects in all but 20-min time point; C and D: bar graphs showing that raclopride enhances agitation responses in phase 1, but not in phase 2 observation period, while SCH-23390 reduces agitation response in phase 2, but not in phase 1 observation period. *P b 0.05 and **P b 0.01, compared with saline.
induced nociceptive behavior during the early phase, and the agitation score was not significantly different from the saline group. However, during the later phase, agitation scores significantly decreased following increasing quinpirole doses (P b 0.001, Fig. 4B). Detailed comparisons at individual time points and between various groups are shown in Fig. 4A. Microinjection of raclopride, a D2-like dopamine receptor antagonist (3.0 μg), into the VLO at 5 min prior to quinpirole (5.0 μg) injection, significantly reduced quinpirole-induced inhibition of nociceptive behavior during the later phase, and agitation scores in the raclopride plus quinpirole group were significantly larger (P b 0.001) than in the quinpirole group (Fig. 4B and D). 3.3. Effect of Microinjection of SKF-38393 and SCH-23390 into the VLO on Formalin-Induced Agitation Responses Microinjection of SKF-38393 (5.0 μg), a D1-like dopamine receptor agonist, into the VLO had no effect on formalin-induced nociceptive behavior during the later phase. Agitation scores were not significantly different from the saline group during the 20–60 min observation period, as well as at all but the 20-min time point (P N 0.05, Fig. 5A and C). However, during the early phase, SKF-38393 significantly increased nociceptive behavior, and agitation scores were significantly larger than in the saline group (P b 0.001, Fig. 5A and C). Microinjection of SCH-23390, a D1-like dopamine receptor antagonist (2.5, 5.0, and 10 μg), into the VLO significantly decreased the agitation response during the early and later phases. As shown in Fig. 5B, time course curves (saline and different doses of SCH-23390treated groups) were different between treatments (F(3,234) = 49.256, P b 0.001), across times (F(8,243) = 8.282, P b 0.001), and between interactions (F(24,243) = 1.694, P = 0.026). Further analyses indicated that 5.0 and 10 μg SCH-23390 resulted in significantly decreased
agitation scores during the early phase compared with the saline group (P b 0.001). However, agitation scores decreased with increasing SCH-23390 doses during the later phase (P b 0.05 and P b 0.001, Fig. 5D). Detailed comparisons at individual time points and between various groups are shown in Fig. 5B. 4. Discussion Results from the present study demonstrated that microinjection of the nonselective dopamine receptor agonist apomorphine into the VLO significantly depressed formalin-induced nociceptive behavior during the later phase, but not during the early phase. This effect was attenuated by the D2-like dopamine receptor antagonist raclopride, but not by the D1-like dopamine receptor antagonist SCH-23390. Furthermore, the apomorphine effect was mimicked by microinjection of the D2-like dopamine receptor agonist quinpirole into the same VLO site, and this effect was antagonized by raclopride. In addition, raclopride microinjected alone into the VLO did not influence formalin-evoked nociceptive behavior during the later phase, which suggested that the antagonizing effect of raclopride on quinpirole and apomorphineinduced inhibition was not due to a result of a direct raclopride-induced increase of nociceptive behavior. These results were consistent with previous results from our laboratory (Sheng et al., 2009), showing that quinpirole administration into the VLO resulted in raclopride-reversible antinociception in the rat tail flick test. The above-described results suggested that D2-like dopamine receptors were involved in mediating the dopamine-induced antinociceptive effect on acute physiological pain, as well as persistent inflammatory pain, in the VLO. However, in the present study, activation of VLO D2-like dopamine receptors did not influence the formalin-induced early phase nociceptive response. These results might be due to plastic changes in the descending antinociceptive pathway under a persistent inflammatory state (Millan, 2002;
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
101
Fig. 4. Effects of microinjection of D2-like dopamine receptor agonist quinpirole (Quinp, 2.0, 5.0 μg) into the VLO on formalin-evoked nociceptive behavior (agitation), and the influence of D2-like dopamine receptor antagonist raclopride (Rac, 3.0 μg) on quinpirole (5.0 μg)-evoked inhibition of the agitation response. A and B: time course curves showing difference at time points between different groups; C and D: bar graphs showing effects in phase 1 and 2 observation periods, respectively. *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05 and ###P b 0.001, compared with 2.0 μg quinpirole; + P b 0.05 and +++P b 0.001, compared with 5.0 μg quinpirole.
Pertovaara, 2000; Ren and Dubner, 2002; Vanegas and Schaible, 2004), resulting in inhibited D2-like dopamine receptor activity in the VLO. It is important to note that the D2-like receptor antagonist raclopride injected alone into the VLO significantly enhanced the early phase nociceptive response (see Fig. 3A and C), which suggested that D2-like receptors in this region and at this time could exhibit transitory tonic inhibitory activity on nociceptive behavior, thereby inducing inhibition of D2-like receptors with raclopride enhances formalin-evoked acute nociceptive behavior. However, further studies are needed to determine the possible action mechanisms involved. Mesolimbic dopaminergic brain areas receive input from ventral tegmental areas (Benes et al., 1993; Ohara et al., 2003), such as the nucleus accumbens and dorsolateral striatum, which are involved in nociception modulation in the formalin test. Treatment with dopamine D2-like receptor agonists in these brain areas significantly reduces formalin-induced nociceptive behavior (Magnusson and Fisher, 2000; Taylor et al., 2003; Koyanagi et al., 2008). The present results provide novel evidence for the involvement of D2 dopamine receptors in mediating VLO-induced antinociception in the formalin test. Results demonstrated that microinjection of the D1-like dopamine receptor agonist SKF-38393 into the VLO exhibited no effect on formalininduced nociceptive behavior. However, the antagonist SCH-23390 resulted in a dose-dependent decrease in nociceptive behavior. Although a smaller dose (1.0 μg) of SCH-23390 did not influence early phase nociceptive behavior when injected alone (Fig. 3B and D), it provoked an inhibitory effect on apomorphine during early phase (see Fig. 2B and D). These results suggested that D1-like dopamine receptors exhibited a tonic inhibitory influence on the antinociceptive pathway, an action that antagonizes D2-like dopamine receptor activation-induced antinociception, thereby blocking D1-like dopamine receptors could induce antinociception. These findings were consistent with a previous study
(Baliki et al., 2003), which showed that 6-hydroxydopamine (6-OHDA) lesions of dopaminergic terminals in the VLO temporarily attenuate spared nerve injury (SNI)-induced neuropathic pain in the rat. Results from the present study were also consistent with a previous report by Coffeen et al. (2008) showing that activation of D2 receptors and inhibition of D1 receptors in the rostral agranular insular cortex result in antinociception in a rat model of sciatic denervation. In contrast, however, results obtained from Burkey et al. (1999) demonstrated that D1 receptors are involved in rostral agranular insular cortexmediated descending antinociception in the heated paw withdrawal test, as well as in the nociceptive response of spinal wide-dynamic range neurons to peripheral noxious stimulation. Studies have suggested that D1 and D2 receptors are simultaneously involved in mediating antinociception in the anterior cingulate cortex in a rat model of sciatic denervation (López-Avila et al., 2004). These differences may be due to varying experimental conditions, procedures, animal models, and the brain areas investigated; different dopamine receptor subtypes exhibit different modulation mechanisms for mediating descending antinociception (Altier and Stewart, 1999). As mentioned above, the VLO is part of the endogenous analgesic system, which consists of an ascending pathway from the spinal cord to the VLO via the thalamic nucleus submedius (Sm), as well as a descending pathway to the spinal cord via the periaqueductal gray (PAG) (Tang et al., 2009). It is reasonable to propose that D2-like dopamine receptor activation-induced antinociception could be mediated by activation of the antinociceptive pathway, which depresses nociceptive information transmission at the spinal level. However, the D2-like dopamine receptor is an inhibitory G-protein-coupled receptor, and activation of this receptor depresses neuronal activity by inducing hyperpolarization. In contrast, D1-like dopamine receptors are coupled to excitatory G-proteins, which, when activated, produce an excitatory
102
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103
Fig. 5. Effects of microinjection of D1-like dopamine receptor agonist SKF-38393 (SKF, 5.0 μg) and antagonist SCH-23390 (SCH 2.5, 5.0 and 10 μg) into the VLO on formalin-evoked nociceptive behavior (agitation). A and B: time course curves showing difference at time points between different groups; C and D: bar graphs showing effects in phase 1 and 2 observation periods, respectively. *P b 0.05 and ***P b 0.001, compared with saline; #P b 0.05, compared with 2.5 μg SCH-23390; +P b 0.05 and +++P b 0.001, compared with 5.0 μg SCH-23390.
effect on target neurons by depolarizing the cellular membrane (Missale et al., 1998; Vallone et al., 2000; Millan, 2002). As observed in μ-opioid receptors and 5-HT1A receptors (Qu et al., 2006; Huo et al., 2008), it is possible that the excitatory effect induced by D2-like receptor activation could result from blocking inhibitory GABAergic interneurons (disinhibition), which could lead to activation of projection neurons. In contrast, the inhibitory effect induced by D1-like receptor activation could result from excitation of GABAergic neurons (proinhibition), which could lead to inhibition of projection neurons. Therefore, GABAergic disinhibition and proinhibition mechanisms could be involved in VLO D1 and D2-like receptor activation-induced effects (facilitation and depression, respectively). Morphological studies have established that GABAergic neurons and GABAA receptors are distributed in the frontal cortex, including the VLO (Esclapez et al., 1987; Pirker et al., 2000; Huo et al., 2005, 2008), and GABAergic neurons in these brain areas receive dopaminergic terminal projections from the ventral tegmental area (Benes et al., 1993; Ohara et al., 2003) and express D1 and D2 dopamine receptors (Gesper et al., 1995; Vincent et al., 1995; Seamans et al., 2001). Studies have also shown that VLO neurons projecting to PAG express GABAA receptors and display symmetrical (presumably inhibitory) synapses with GABAergic terminals (Huo et al., 2009). Therefore, a local neuronal circuit consisting of dopamine afferent terminals, GABAergic interneurons, and output neurons, in addition to the receptors, exists in the VLO and provides a morphological foundation for GABAergic modulation. It is reasonable to propose that inhibitory effects of VLO-injected apomorphine on formalin-induced nociceptive behavior could be the result of interactions between D1 and D2-like receptors. However, further behavioral, electrophysiological and molecular biological studies are needed to determine the GABAergic modulation mechanisms influencing D1- and D2-like dopamine receptors in VLO-evoked antinociception in inflammatory pain model.
5. Conclusion Results from the present study suggested that the role of dopamine receptors in the VLO is different under conditions of persistent inflammatory pain. D2-like dopamine receptors mediated VLO-induced antinociception, but D1-like dopamine receptors exhibited a tonic facilitatory effect on nociceptive behavior. GABAergic modulation could be implicated in D1- and D2-like dopamine receptor activation-induced effects. Acknowledgments This project was supported by grants from the National Natural Science Foundation of China (No. 30700222; 30800334) and the Ministry of Science and Technology of China (No. 2009DFA31080). There was no conflict of interest in relation to this article. References Abbott, F.V., Franklin, K.B., Westbrook, R.F., 1995. The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 60, 91–102. Altier, N., Stewart, J., 1998. Dopamine receptor antagonists in the nucleus accumbens attenuate analgesia induced by ventral tegmental area substance P or morphine and by nucleus accumbens amphetamine. J. Pharmacol. Exp. Ther. 285, 208–215. Altier, N., Stewart, J., 1999. The role of dopamine in the nucleus accumbens in analgesia. Life Sci. 22, 2269–2287. Baliki, M., Al-Amin, H.A., Atweh, S.F., Jaber, M., Hawwa, N., Jabbur, S.J., Apkarian, A.V., Saadé, N.E., 2003. Attenuation of neuropathic manifestations by local block of the activities of the ventrolateral orbito-frontal area in the rat. Neuroscience 120, 1093–1104. Benes, F.M., Vincent, S.L., Molloy, R., 1993. Dopamine-immunoreactive varicosities form nonrandom contacts with GABA-immunoreactive neurons of rat medial prefrontal cortex. Synapse 15, 285–295. Burkey, A.R., Carstens, E., Jasmin, L., 1999. Dopamine reuptake inhibition in the rostral agranular insular cortex produces antinociception. J. Neurosci. 19, 4169–4179.
Y.-H. Dang et al. / European Journal of Pharmacology 657 (2011) 97–103 Coderre, T.J., Fundytus, M.E., McKenna, J.E., Dalal, S., Melzack, R., 1993. The formalin test: a validation of the weighted-scores method of behavioural pain rating. Pain 54, 43–50. Coffeen, U., López-Avila, A., Ortega-Legaspi, J.M., del Ángel, R., López-Muñoz, F.J., Pellicer, F., 2008. Dopamine receptors in the anterior insular cortex modulate long-term nociception in the rat. Eur. J. Pain 12, 535–543. Dubuisson, D., Dennis, S.G., 1977. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4, 161–174. 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. 77, 131–136. Gesper, P., Bloch, B., Le Moine, C., 1995. D1 and D2 receptor gene expression in the rat frontal cortex: cellular localization in different classes of efferent neurons. Eur. J. Neurosci. 7, 1050–1063. Huo, F.Q., Wang, J., Li, Y.Q., Chen, T., Han, F., Tang, J.S., 2005. GABAergic neurons express μ-opioid receptors in the ventrolateral orbital cortex of the rat. Neurosci. Lett. 382, 265–268. Huo, F.Q., Qu, C.L., Li, Y.Q., Tang, J.S., Jia, H., 2008. GABAergic modulation is involved in the ventrolateral orbital cortex 5-HT1A receptor activation-induced antinociception in the rat. Pain 139, 398–405. Huo, F.Q., Chen, T., Lv, B.C., Wang, J., Zhang, T., Qu, C.L., Li, Y.Q., Tang, J.S., 2009. Synaptic connections between GABAergic elements and serotonergic terminals or projecting neurons in the ventrolateral orbital cortex. Cereb. Cortex 19, 1263–1272. Koyanagi, S., Himukashi, S., Mukaida, K., Shichino, T., Fukuda, K., 2008. Dopamine D2-like receptor in the nucleus accumbens is involved in the antinociceptive effect of nitrous oxide. Aneth. Analg. 106, 1904–1909. Li, Y., Yuan, B., Tang, J.S., 2007. Effect of stimulation and lesion of the thalamic nucleus submedius on formalin-evoked nociceptive behavior in rats. Acta Physiol. Sin. 59, 777–783. López-Avila, A., Coffeen, U., Ortaga-Legaspi, J., del Angel, R., Pellicer, F., 2004. Dopamine and NMDA system modulate long-term nociception in the rat anterior cingulate cortex. Pain 111, 136–143. Magnusson, J., Fisher, K., 2000. The involvement of dopamine in nociception: the role of D(1) and D(2) receptors in the dorsolateral striatus. Brain Res. 855, 260–266. Meyer, P.J., Morgan, M.M., Kozell, L.B., Ingram, S.L., 2009. Contribution of dopamine receptors to periaqueductal gray-mediated antinociception. Psychopharmacol. Berl. 204, 531–540. Millan, M.J., 2002. Descending control of pain. Prog. Neurobiol. 66, 355–474. Missale, C., Nash, S.R., Robinson, S.W., Jaber, M., Caron, M.G., 1998. Dopamine receptors: from structure to function. Physiol. Rev. 78, 189–225. Ohara, P.T., Granato, A., Moallem, T.M., Wang, B.R., Tillet, Y., Lasmin, L., 2003. Dopaminergic input to GABAergic neurons in the rostral agranular insular cortex of the rat. J. Neurocytol. 32, 131–141. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, Sydney. Pertovaara, A., 2000. Plasticity in descending pain modulatory systems. Prog. Brain Res. 129, 231–242.
103
Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., Sperk, G., 2000. GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815–850. Qu, C.L., Tang, J.S., Jia, H., 2006. Involvement of GABAergic modulation of antinociception induced by morphine microinjection into the ventrolateral orbital cortex. Brain Res. 1073–1074, 281–289. Ren, K., Dubner, R., 2002. Descending modulation in persistent pain: an update. Pain 100, 1–6. Seamans, J.K., Gorelova, N., Durstewitz, D., Yang, C.R., 2001. Bidirectional dopamine modulation of GABAergic inhibition in prefrontal cortical pyramidal neurons. J. Neurosci. 21, 3628–3638. Sheng, H.Y., Qu, C.L., Huo, F.Q., Du, J.Q., Tang, J.S., 2009. D2-like but not D1-like dopamine receptors are involved in the ventrolateral orbital cortex-induced antinociception: a GABAergic modulation mechanism. Exp. Neurol. 215, 128–134. Tang, J.S., Qu, C.L., Huo, F.Q., 2009. The thalamic nucleus submedius and ventrolateral orbital cortex are involved in nociceptive modulation: a novel pain modulation pathway. Prog. Neurobiol. 89, 383–389. Taylor, B.K., Joshi, C., Uppal, H., 2003. Stimulation of dopamine D2 receptors in the nucleus accumbens inhibits inflammatory pain. Brain Res. 987, 135–143. Vallone, D., Oicetti, R., Borrelli, E., 2000. Stucture and function of dopamine receptors. Neurosci. Biobehav. Res. 24, 125–132. Vanegas, H., Schaible, H.G., 2004. Descending control of persistent pain: inhibitory or facilitatory? Brain Res. Rev. 46, 295–309. Vincent, S.L., Khan, Y., Benes, F.M., 1995. Cellular colocalization of dopamine D1 and D2 receptors in rat medial prefrontal cortex. Synapse 19, 112–120. Watson, G.S., Sufka, K.J., Coderre, T.J., 1997. Optimal scoring strategies and weights for the formalin test in rats. Pain 70, 53–58. Wheeler-Aceto, H., Cowan, A., 1991. Standardization of the rat paw formalin test for the evaluation of analgesics. Psychopharmacol. Berl. 104, 35–44. Zhang, Y.Q., Tang, J.S., 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, Y.Q., Tang, J.S., 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, J.S., 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, J.S., 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, J.S., 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, J.S., 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. Zimmermann, M., 1983. Ethical guideline for investigation of experimental pain in conscious animals. Pain 16, 109–110.