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Involvement of orexin-2 receptors in the ventral tegmental area and nucleus accumbens in the antinociception induced by the lateral hypothalamus stimulation in rats
Peptides 47 (2013) 94–98
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Involvement of orexin-2 receptors in the ventral tegmental area and nucleus accumbens in the antinociception induced by the lateral hypothalamus stimulation in rats Hassan Azhdari-Zarmehri a,b , Zahra Reisi c , Anoumid Vaziri c , Amir Haghparast c,d , Pariya Shaigani c , Abbas Haghparast c,∗ a
Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran Department of Physiology, Qazvin University of Medical Sciences, Qazvin, Iran c Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, P.O. Box 19615-1178, Tehran, Iran d Faculty of Dentistry, International Branch of Shahid Beheshti University of Medical Sciences, Qeshm, Iran b
a r t i c l e
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Article history: Received 1 June 2013 Received in revised form 12 July 2013 Accepted 15 July 2013 Available online 26 July 2013 Keywords: Orexin-2 receptor Lateral hypothalamus Ventral tegmental area Nucleus accumbens Antinociception Rat
a b s t r a c t Orexin, which is mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), plays an important role in pain modulation. Both kinds of orexin-1 (Ox1) and orexin-2 (Ox2) receptors have been found at high density in the ventral tegmental area (VTA) and nucleus accumbens (NAc). However, the quantity of Ox1 receptors in the VTA is more than that in the NAc. Additionally, it seems that the functional interaction between the LH, VTA and NAc implicates pain processing and modulation. In this study, we tried to examine the involvement of Ox2 receptors in the NAc and VTA using tail-flick test as an animal model of acute pain following microinjection of effective dose of carbachol (125 nmol/0.5 l saline) into the LH. In this set of experiments, different doses of TCS OX2 29 as an Ox2 receptor antagonist were microinjected into the VTA (1, 7 and 20 nmol/0.3 l DMSO) and the NAc (2, 10, 20 and 40 nmol/0.5 l DMSO) 5 min prior to carbachol administration. Administration of TCS OX2 29 into the VTA and NAc dosedependently blocked intra-LH carbachol-induced antinociception. However, the inhibitory effect of TCS OX2 29 as an Ox2 receptor antagonist was more potent in the VTA than that in the NAc. It seems that VTA orexinergic receptors are more effective on LH stimulation-induced antinociception and the modulation of pain descending inhibitory system originated from the LH than those of the same receptors in the nucleus accumbens in rats. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Pain is a multidimensional experience that circumscribes physical sensation, affect, and cognition. Pain is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage,” or described in terms of such damage. Thus pain is an experience characterized by clear physiological and psychological elements [8]. The orexinergic system is most strongly associated with feeding, arousal, sleep, reward, addiction, stress, and pain processing [2,11,25,28,29]. The orexins (also known as hypocretin) are neuropeptide transmitters that comprise of two distinct peptides called orexin-A and orexin-B. These neuropeptides result from prepro-orexin molecule during the proteolytic process [4,26]. Orexin-1 receptor (Ox1r) is selective for orexin A
∗ Corresponding author. Tel.: +98 21 2243 1624; fax: +98 21 2243 1624. E-mail addresses: a [email protected], [email protected] (A. Haghparast). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.07.012
whereas Orexin-2 receptor (Ox2r) is willing for both orexins A and B [26]. The orexin produced by orexin-expressing neurons are generally located in the posterior hypothalamus and extend to the dorsomedial and lateral hypothalamus (LH) [23]. Both kinds of orexin receptors (Ox1r and Ox2r) have been found at high density in the ventral tegmental area (VTA) [19,21]. The VTA is a group of neurons existing close to the midline on the floor of the midbrain and is the root of the dopaminergic cell bodies of the mesocorticolimbic dopamine (DA) system which is widely implicated in the drug and natural reward circuitry of the brain. In the VTA, orexins stimulate both dopaminergic and non-dopaminergic neurons via a direct postsynaptic effect. Orexin receptor activation in the VTA could increase DA release via projecting neurons in the nucleus accumbens (NAc) [18,20]. The NAc is a collection of neurons that forms the main part of the ventral striatum. Some studies suggest that this area is an important neuronal substrate of pain modulation [3]. It seems that the functional interaction between the NAc and VTA implicates pain processing. In our last study, we evaluated the involvement of orexin-1 receptors
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within the VTA and NAc in antinociception induced by LH orexinergic neurons stimulation by the tail-flick test as an animal model of acute pain [24]. Findings in the study mentioned above suggest that Ox1 receptors in the VTA and NAc are involved in the intra-LH carbachol-induced antinociception, and LH orexinergic projections to these areas act, in part, through Ox1 receptors within the VTA and NAc in pain modulation. On the other hand, it has been shown that the distribution of orexin receptors in these areas are not the same, and the quantity of Ox1 receptor in the VTA is more than that in the NAc [27]. Hence, in this study, we tried to examine the involvement of Ox2 receptors in these areas in the antinociception induced by chemical stimulation of the LH in rats.
2. Materials and methods 2.1. Animals Seventy six adult male albino Wistar rats (Pasteur Institute, Tehran, Iran) weighing 210–280 g were used in these experiments. The rats were kept in a vivarium maintained at a 12:12 h light/dark cycle at room controlled temperature (23 ± 1 ◦ C) with free access to chow and tap water. Rats were habituated to their new environment and handled for a week before the experimental procedure began. The animals were randomly allocated to different experimental groups. Each animal was used only once. All experiments were executed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were approved by the Research and Ethics Committee of Shahid Beheshti and Qazvin Universities of Medical Sciences.
2.4. Drug administration Microinjections were performed by lowering a stainless steel injector cannula (30-gauge needle) with a length of 1 mm longer than the guide cannulae into the VTA or NAc and the LH. The injector cannula was attached to a 1-l Hamilton syringe by polyethylene tubing (PE-20), subsequently the drug solution or vehicle was infused unilaterally over 60 s and was left for an extra 60 s. Different doses of carbachol were administered slowly in a total volume 0.5 l/rat over a period of 60 s into the LH. TCS OX2 29 solution or 10% DMSO as a vehicle was administered over a period of 60 s in a total volume of 0.3 and 0.5 l into the VTA and NAc, respectively. Injection needles were left in place for an additional 60 s to facilitate the diffusion of the drugs, and then the stylets were reinserted into the guide cannulae. 2.5. Tail-flick test The nociceptive threshold was measured by the tail-flick analgesiometer apparatus (Harvard Apparatus, USA). Heat was applied in succession, 3, 5 and 7 cm from the caudal tip of the tail as a model of acute pain. The value of each tail-flick latency (TFL) time was calculated on the average of three consecutive TFL tests in each time point. The reaction time between the onset of heat stimulus and the movement of tail was determined by an automatic sensor as TFL. The light source was set at an intensity that yields baseline TFL values in the range of 3–4 s (about 45% of maximal light intensity). If at any time the animal failed to flick its tail within 10 s (cut-off point), the tail was removed from the coil in order to prevent damage to the skin [5,6,10]. TFLs (s) are expressed either as raw data or percentage of maximal possible effect (%MPE) which was calculated from the following formula:
2.2. Stereotaxic surgery %MPE = Rats were anesthetized with intraperitoneal (i.p.) injection of 10% Ketamine (100 mg/kg) and 2% Xylazine (10 mg/kg). Cannulae were stereotaxically (Stoelting, USA) implanted into the VTA, NAc and the LH unilaterally. The coordinates for these regions were determined by the rat brain atlas [22], AP = 4.8 mm caudal to bregma, Lat = ±0.9 mm lateral to midline, DV = 8.3 mm ventral from the skull surface for the VTA (cannula 23-gauge, 11 mm, guide cannula was 1 mm above the appropriate injection place) and for the NAc (cannula 23-gauge, 11 mm) was AP = 1.7 ± 0.5 mm to bregma, Lat = ±1.6 mm lateral to midline, DV = 7.8 mm ventral from the skull surface and for the LH (cannula 23-gauge, 12 mm) was AP = 3 mm caudal to bregma, Lat = ±1.6 mm and DV = 8.8 mm ventral from the skull surface. The guide cannulae were secured in their places using two stainless steel and Jewelers’ screws anchored to the skull with dental acryl cement. After the cement was completely dried and hardened, two stainless steel stylets were used to keep the guide cannulae from moving during recovery period. PenicillinG 200,000 IU/ml (0.2–0.3 ml/rat, single dose, intramusclar) was administered immediately after surgery. Animals were individually housed and allowed to recover for a period of 5–7 days before the experiments. 2.3. Drugs The drugs we used are: carbachol (Carbamoylcholine chloride; Tocris Bioscience, Bristol, UK) as a cholinergic agonist and which was dissolved in physiological saline, and TCS OX2 29 (Tocris Bioscience, Bristol, UK); Ox2 receptor antagonist which was dissolved in 10% dimethyl sulfoxide (DMSO; Sigma–Aldrich, Germany). Control animals received either normal saline or 10% DMSO as vehicles. All drugs were freshly prepared on the day of the experiment.
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Post-drug latency (s) − Baseline latency (s) × 100 Cut-off value (s) − Baseline latency (s)
2.6. Experimental design In this study, there were three control groups including intact, sham-operated and saline groups (n = 6 in each group) for determining the baseline TFLs, surgical manipulation and microinjection volume effects, respectively. To evaluate the involvement of Ox2 receptors within the VTA and NAc in antinociceptive responses induced by LH stimulation, tail-flick test was performed as a model of acute pain. In all of the above control and experimental groups, TFLs were recorded at 5, 15, 30, 45 and 60 min after drugs/vehicles administrations. 2.6.1. Dose–response effects of carbachol microinjected into the LH on the tail-flick test The dose–response effects of intra-LH administration of different doses of carbachol have been established in our previous work [24]. However, in this study, a dose–response relationship for carbachol as a LH chemical stimulation agent on tail-flick test was re-established. After intra-LH administration of different doses of carbachol (125 and 250 nmol/0.5 l saline; n = 6 in each group), TFLs were tested at the time set intervals. Control animals received saline (n = 6) instead of carbachol in the LH. 2.6.2. Effects of intra-VTA administration of Ox2 receptor antagonist, TCS OX2 29, on antinociception induced by carbachol into the lateral hypothalamus In order to test the role of Ox2 receptors located within the VTA during the LH stimulation-induced antinociception, different doses of TCS OX2 29 (1, 7 and 20 nmol/0.3 l DMSO; n = 5–6 in each group)
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were unilaterally injected into the VTA, 5 min prior to administration of effective dose of intra-LH carbachol. It was followed by the tail-flick test at the time set intervals. In the vehicle group (n = 6), 10% DMSO was microinjected into the VTA and 2 min later animals received saline instead of carbachol in the LH. 2.6.3. Effects of intra-NAc administration of Ox2 receptor antagonist, TCS OX2 29, on antinociception induced by carbachol into the lateral hypothalamus To examine the role of intra-accumbal OX2 receptors in the LH stimulation-induced antinociception, different doses of TCS OX2 29 (2, 10, 20 and 40 nmol/0.5 l DMSO; n = 5–7 in each group) were unilaterally injected into the NAc, 5 min prior to effective dose of intra-LH carbachol administration. In the vehicle group (n = 6), 10% DMSO was microinjected into the NAc and 2 min later animals received saline (0.5 l) in the LH. 2.7. Histology The animals were deeply anesthetized with Ketamine and Xylazine after the experiments were accomplished. Transcardial perfusion was carried out with 0.9% saline and 10% formalin solution. The brains were removed, blocked and cut coronally in 50 m sections through the cannulae placements. The neuroanatomical locations of cannulae tips were confirmed using rat brain atlas [22] .Only the results from animals with correct cannulae placements were included in the data analysis. 2.8. Statistics The obtained data are expressed as mean ± SEM (standard error of mean). The mean TFLs or the mean maximal possible effect (%MPE) of drugs, as an analgesic index in all control groups were subjected to one-way and/or two-way ANOVA followed by protected Newman–Keuls’s or Bonferroni’s test for multiple comparisons, respectively as needed. In order to evaluate the nociceptive responses, area under the curve (AUC) was calculated as raw %MPE × time by linear trapezoidal method and a single value was used in statistical analyses. The calculated AUC values in all experimental groups were subjected to one-way ANOVA followed by protected Newman–Keuls test for multiple comparisons. P-values less than 0.05 were considered to be statistically significant. 3. Results 3.1. Dose–response effects of chemical stimulation of the lateral hypothalamus by carbachol on tail-flick test as an animal model of acute pain In this study, two-way ANOVA for repeated measures over time followed by Bonferroni’s test for %MPEs confirmed a significant difference in antinociceptive responses to carbachol microinjected into the LH alone, compared to that of respective salinetreated group [Treatment effect: F(2, 75) = 26.05, P < 0.0001; Time effect: F(4, 75) = 0.2197, P = 0.9267; Interaction: F(8, 75) = 0.3576, P = 0.9394; data not shown]. Moreover, one-way ANOVA followed by Dunnett multiple comparison test also showed that there are significant differences in mean AUC calculated values for %MPEs [F(2, 17) = 20.89, P < 0.0001] among the different doses of carbachol and saline control groups. 3.2. Effect of intra-VTA administration of Ox2 receptor antagonist, TCS OX2 29, on LH stimulation-induced antinociception We examined the effect of intra-VTA administration of TCS OX2 29 as a selective Ox2r antagonist on LH stimulation-induced
Fig. 1. Effects of unilateral microinjection of different doses of TCS OX2 29 (TCS), an Ox2 receptor antagonist, in the ventral tegmental area (VTA) on (A) maximal possible effect in 5, 15, 30, 45 and 60 min after microinjection as an index of antinociception induced by chemical stimulation of the lateral hypothalamus (LH) and (B) area under the curves (AUCs) calculated for %MPEs shown in (A). Ox2 receptor antagonist can significantly decrease the intra-LH carbachol-induced antinociception in a dosedependent manner. Animals received TCS OX2 29 (1, 7 and 20 nmol/0.3 l DMSO) or 10% DMSO (0.3 l) as a vehicle in the VTA, 5 min before intra-LH administration of carbachol (125 nmol/rat). Each point shows the mean ± SEM for 5–6 rats. *** P < 0.001 different from the control (vehicles) group. †† P < 0.01, ††† P < 0.001 different from the respective DMSO-carbachol group.
antinociception by carbachol in order to evaluate the receptor specific response of Ox2 receptors in the VTA. Two-way ANOVA for repeated measures over time followed by Bonferroni’s test for %MPEs indicated significant differences in antinociceptive responses to carbachol microinjected into the LH alone or when carbachol was concurrently administrated into the LH following different doses of intra-VTA TCS OX2 29 as compared to those of respective saline-treated group [Treatment effect: F(5, 145) = 38.6, P < 0.0001; Time effect: F(4, 145) = 1.032, P = 0.3927; Interaction: F(20, 145) = 0.9356, P = 0.5435]. Furthermore, as shown in Fig. 1, one-way ANOVA followed by Newman–Keuls multiple comparison test showed that there are significant differences in AUC calculated values for %MPEs [F(5, 34) = 18.46, P < 0.0001] among the experimental and control (vehicle) groups. Data gained from these experiments showed that intra-VTA administration of TCS OX2 29 as an Ox2r antagonist dose-dependently prevents intra-LH carbachol-induced antinociception in rats. Also, administration of maximal dose of TCS OX2 29 (20 nmol/rat) alone into the VTA could not affect the baseline TFLs (%MPEs) at time set intervals and/or AUC calculated values compared to the vehicle group (Fig. 1). 3.3. Effect of intra-accumbal administration of Ox2 receptor antagonist, TCS OX2 29, on LH stimulation-induced antinociception The dose–response effects of intra-NAc administration of selective Ox2r antagonist TCS OX2 29 on the antinociceptive response of carbachol microinjected into the LH during a 60-min period was observed in the following experiments. Two-way ANOVA followed by Bonferroni’s test [Treatment effect: F(6, 174) = 44.03, P < 0.0001; Time effect F(4, 174) = 2.449, P = 0.048; Interaction F(24, 174) = 0.8744, P = 0.6364] showed that there are significant differences in %MPE values among the experimental and control groups. Although one-way ANOVA followed by Newman–Keuls multiple comparison test [F(6, 41) = 17.44, P < 0.0001; Fig. 2] showed
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Fig. 2. Effect of unilateral microinjection of different doses of TCS OX2 29, an Ox2 receptor antagonist, in the nucleus accumbens (NAc) on (A) maximal possible effect in 5, 15, 30, 45 and 60 min after microinjection as an index of antinociception induced by chemical stimulation of the lateral hypothalamus (LH) and (B) area under the curves (AUCs) calculated for %MPEs shown in (A). The high doses of Ox2 receptor antagonist can significantly decrease the intra-LH carbachol-induced antinociception in rats. Animals received TCS OX2 29 (2, 10, 20 and 40 nmol/0.5 l DMSO) or 10% DMSO as a vehicle in the NAc, 5 min before intra-LH administration of carbachol (125 nmol/rat). Each point shows the mean ± SEM for 5–7 rats. * P < 0.05, *** P < 0.001 different from the control (vehicles) group. † P < 0.05, ††† P < 0.001 different from the respective DMSO-carbachol group.
that there are significant differences in AUC calculated values for %MPEs among the experimental and control (vehicles) groups, intra-accumbal administration of TCS OX2 29 only at high doses (20 and 40 nmol/rat) could significantly reduce %MPEs in comparison with DMSO respective control group that received carbachol (125 nmol/rat) in the LH. Moreover, administration of maximal dose of TCS OX2 29 (40 nmol/rat) alone into the NAc could not affect the AUC calculated values compared to vehicle group (Fig. 2). 4. Discussion In this study we presented a detailed analysis on the antinociceptive effects caused by orexin-2 receptors in the VTA and NAc. Our results indicated that administration of different doses of carbachol into the LH caused an antinociceptive response in the tail-flick test. Nonetheless, the major findings were (I) the involvement of Ox2 receptors within the VTA and NAc in pain modulation in the rats; (II) administration of an Ox2r antagonist (TCS OX2 29) into the VTA and NAc dose dependently blocked intra-LH carbachol induced antinociception and (III) the antinociceptive effect of Ox2 receptor in the VTA was more potent than that in the NAc. In this experiment and pertaining to our laboratory’s last research on Ox1 receptors [24], both SB334867 and TCS OX2 29 as Ox1 and Ox2 receptors antagonists, respectively, were able to reduce the antinociception caused by intra-LH administration of carbachol, and we observed this attenuation by blocking the Ox2 receptors within the VTA and NAc. These responses were stronger in the VTA than those in the NAc. Stimulation of the LH using excitatory drugs like non-specific cholinergic muscarinic agonist (carbachol) can elicit marked antinociception [7,13–17]. Hence, we used two doses of carbachol, and although there was no significant difference in the antinociceptive effect, the lower dose was chosen [24]. Multiple brain regions receive orexinergic fibers input from the LH and the perifornical area [4]. Dense orexinergic innervation is observed in the VTA. This area has been shown to have a robust
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expression of Ox1 and Ox2 receptors, and orexinergic terminals synapse onto presumed dopaminergic and GABAergic neurons [21]. Narita and colleagues strongly support the idea that the activation of orexin-containing neurons in the VTA leads to the direct activation of mesolimbic dopamine neurons including the NAc at the somatodendritic level [21]. Ghalandari-Shamami et al. [9] presented that the NAc plays an important role in the modulation of pain, and the recent studies have also supported that the NAc is involved in the antinociception induced by intra-BLA administration of cannabinoid receptor agonist in the tail-flick test [9,10,12]. In addition, it has been proven that antinociception induced by intra-VTA DiMe- C7 decreases with intra-accumbal infusions of the mixed dopamine receptor antagonist flupenthixol [1]. Thus, our findings in this study suggest that Ox2 receptors in the VTA and NAc are involved in the antinociception induced by LH stimulation through activation of the LH orexinergic projecting neurons to the VTA and lesser to the NAc, and in part, through mechanisms involved in pain descending inhibitory system in these areas. On the other hand, the distribution of orexin receptors in these areas are not same, and the quantity of Ox1 receptors in the VTA is more than that in the NAc [27]. It is consistent with the results from the previous research performed on Ox1 receptors [24]; it has been shown that SB334867 as an Ox1 receptor antagonist is more effective in the VTA region. As mentioned before in this study, TCS OX2 29 as an Ox2 receptor antagonist is also more effective in the VTA in comparison with this effect in the NAc. Therefore, it seems that VTA orexinergic receptors and orexin system in this area are more effective in modulation of the pain descending inhibitory system than those of the same receptors and system in the NAc. Furthermore, we understand that Ox1 and Ox2 receptors usually act in adverse toward each other, but regarding these findings it seems that these two kinds of receptors have worked together to decrease the antinociception induced by stimulation of hypothalamus. While findings from this study and other studies are novel, we cannot say with certainty how these receptors interact with each other.
Acknowledgments The authors would like to thank Mr. Mahmoudreza Ramin for the editing of our manuscript. This study was supported by a grant from the Qazvin University of Medical Sciences, Qazvin, Iran.
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Peptides journal homepage: www.elsevier.com/locate/peptides
Involvement of orexin-2 receptors in the ventral tegmental area and nucleus accumbens in the antinociception induced by the lateral hypothalamus stimulation in rats Hassan Azhdari-Zarmehri a,b , Zahra Reisi c , Anoumid Vaziri c , Amir Haghparast c,d , Pariya Shaigani c , Abbas Haghparast c,∗ a
Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran Department of Physiology, Qazvin University of Medical Sciences, Qazvin, Iran c Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, P.O. Box 19615-1178, Tehran, Iran d Faculty of Dentistry, International Branch of Shahid Beheshti University of Medical Sciences, Qeshm, Iran b
a r t i c l e
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Article history: Received 1 June 2013 Received in revised form 12 July 2013 Accepted 15 July 2013 Available online 26 July 2013 Keywords: Orexin-2 receptor Lateral hypothalamus Ventral tegmental area Nucleus accumbens Antinociception Rat
a b s t r a c t Orexin, which is mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), plays an important role in pain modulation. Both kinds of orexin-1 (Ox1) and orexin-2 (Ox2) receptors have been found at high density in the ventral tegmental area (VTA) and nucleus accumbens (NAc). However, the quantity of Ox1 receptors in the VTA is more than that in the NAc. Additionally, it seems that the functional interaction between the LH, VTA and NAc implicates pain processing and modulation. In this study, we tried to examine the involvement of Ox2 receptors in the NAc and VTA using tail-flick test as an animal model of acute pain following microinjection of effective dose of carbachol (125 nmol/0.5 l saline) into the LH. In this set of experiments, different doses of TCS OX2 29 as an Ox2 receptor antagonist were microinjected into the VTA (1, 7 and 20 nmol/0.3 l DMSO) and the NAc (2, 10, 20 and 40 nmol/0.5 l DMSO) 5 min prior to carbachol administration. Administration of TCS OX2 29 into the VTA and NAc dosedependently blocked intra-LH carbachol-induced antinociception. However, the inhibitory effect of TCS OX2 29 as an Ox2 receptor antagonist was more potent in the VTA than that in the NAc. It seems that VTA orexinergic receptors are more effective on LH stimulation-induced antinociception and the modulation of pain descending inhibitory system originated from the LH than those of the same receptors in the nucleus accumbens in rats. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Pain is a multidimensional experience that circumscribes physical sensation, affect, and cognition. Pain is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage,” or described in terms of such damage. Thus pain is an experience characterized by clear physiological and psychological elements [8]. The orexinergic system is most strongly associated with feeding, arousal, sleep, reward, addiction, stress, and pain processing [2,11,25,28,29]. The orexins (also known as hypocretin) are neuropeptide transmitters that comprise of two distinct peptides called orexin-A and orexin-B. These neuropeptides result from prepro-orexin molecule during the proteolytic process [4,26]. Orexin-1 receptor (Ox1r) is selective for orexin A
∗ Corresponding author. Tel.: +98 21 2243 1624; fax: +98 21 2243 1624. E-mail addresses: a [email protected], [email protected] (A. Haghparast). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.07.012
whereas Orexin-2 receptor (Ox2r) is willing for both orexins A and B [26]. The orexin produced by orexin-expressing neurons are generally located in the posterior hypothalamus and extend to the dorsomedial and lateral hypothalamus (LH) [23]. Both kinds of orexin receptors (Ox1r and Ox2r) have been found at high density in the ventral tegmental area (VTA) [19,21]. The VTA is a group of neurons existing close to the midline on the floor of the midbrain and is the root of the dopaminergic cell bodies of the mesocorticolimbic dopamine (DA) system which is widely implicated in the drug and natural reward circuitry of the brain. In the VTA, orexins stimulate both dopaminergic and non-dopaminergic neurons via a direct postsynaptic effect. Orexin receptor activation in the VTA could increase DA release via projecting neurons in the nucleus accumbens (NAc) [18,20]. The NAc is a collection of neurons that forms the main part of the ventral striatum. Some studies suggest that this area is an important neuronal substrate of pain modulation [3]. It seems that the functional interaction between the NAc and VTA implicates pain processing. In our last study, we evaluated the involvement of orexin-1 receptors
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within the VTA and NAc in antinociception induced by LH orexinergic neurons stimulation by the tail-flick test as an animal model of acute pain [24]. Findings in the study mentioned above suggest that Ox1 receptors in the VTA and NAc are involved in the intra-LH carbachol-induced antinociception, and LH orexinergic projections to these areas act, in part, through Ox1 receptors within the VTA and NAc in pain modulation. On the other hand, it has been shown that the distribution of orexin receptors in these areas are not the same, and the quantity of Ox1 receptor in the VTA is more than that in the NAc [27]. Hence, in this study, we tried to examine the involvement of Ox2 receptors in these areas in the antinociception induced by chemical stimulation of the LH in rats.
2. Materials and methods 2.1. Animals Seventy six adult male albino Wistar rats (Pasteur Institute, Tehran, Iran) weighing 210–280 g were used in these experiments. The rats were kept in a vivarium maintained at a 12:12 h light/dark cycle at room controlled temperature (23 ± 1 ◦ C) with free access to chow and tap water. Rats were habituated to their new environment and handled for a week before the experimental procedure began. The animals were randomly allocated to different experimental groups. Each animal was used only once. All experiments were executed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were approved by the Research and Ethics Committee of Shahid Beheshti and Qazvin Universities of Medical Sciences.
2.4. Drug administration Microinjections were performed by lowering a stainless steel injector cannula (30-gauge needle) with a length of 1 mm longer than the guide cannulae into the VTA or NAc and the LH. The injector cannula was attached to a 1-l Hamilton syringe by polyethylene tubing (PE-20), subsequently the drug solution or vehicle was infused unilaterally over 60 s and was left for an extra 60 s. Different doses of carbachol were administered slowly in a total volume 0.5 l/rat over a period of 60 s into the LH. TCS OX2 29 solution or 10% DMSO as a vehicle was administered over a period of 60 s in a total volume of 0.3 and 0.5 l into the VTA and NAc, respectively. Injection needles were left in place for an additional 60 s to facilitate the diffusion of the drugs, and then the stylets were reinserted into the guide cannulae. 2.5. Tail-flick test The nociceptive threshold was measured by the tail-flick analgesiometer apparatus (Harvard Apparatus, USA). Heat was applied in succession, 3, 5 and 7 cm from the caudal tip of the tail as a model of acute pain. The value of each tail-flick latency (TFL) time was calculated on the average of three consecutive TFL tests in each time point. The reaction time between the onset of heat stimulus and the movement of tail was determined by an automatic sensor as TFL. The light source was set at an intensity that yields baseline TFL values in the range of 3–4 s (about 45% of maximal light intensity). If at any time the animal failed to flick its tail within 10 s (cut-off point), the tail was removed from the coil in order to prevent damage to the skin [5,6,10]. TFLs (s) are expressed either as raw data or percentage of maximal possible effect (%MPE) which was calculated from the following formula:
2.2. Stereotaxic surgery %MPE = Rats were anesthetized with intraperitoneal (i.p.) injection of 10% Ketamine (100 mg/kg) and 2% Xylazine (10 mg/kg). Cannulae were stereotaxically (Stoelting, USA) implanted into the VTA, NAc and the LH unilaterally. The coordinates for these regions were determined by the rat brain atlas [22], AP = 4.8 mm caudal to bregma, Lat = ±0.9 mm lateral to midline, DV = 8.3 mm ventral from the skull surface for the VTA (cannula 23-gauge, 11 mm, guide cannula was 1 mm above the appropriate injection place) and for the NAc (cannula 23-gauge, 11 mm) was AP = 1.7 ± 0.5 mm to bregma, Lat = ±1.6 mm lateral to midline, DV = 7.8 mm ventral from the skull surface and for the LH (cannula 23-gauge, 12 mm) was AP = 3 mm caudal to bregma, Lat = ±1.6 mm and DV = 8.8 mm ventral from the skull surface. The guide cannulae were secured in their places using two stainless steel and Jewelers’ screws anchored to the skull with dental acryl cement. After the cement was completely dried and hardened, two stainless steel stylets were used to keep the guide cannulae from moving during recovery period. PenicillinG 200,000 IU/ml (0.2–0.3 ml/rat, single dose, intramusclar) was administered immediately after surgery. Animals were individually housed and allowed to recover for a period of 5–7 days before the experiments. 2.3. Drugs The drugs we used are: carbachol (Carbamoylcholine chloride; Tocris Bioscience, Bristol, UK) as a cholinergic agonist and which was dissolved in physiological saline, and TCS OX2 29 (Tocris Bioscience, Bristol, UK); Ox2 receptor antagonist which was dissolved in 10% dimethyl sulfoxide (DMSO; Sigma–Aldrich, Germany). Control animals received either normal saline or 10% DMSO as vehicles. All drugs were freshly prepared on the day of the experiment.
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Post-drug latency (s) − Baseline latency (s) × 100 Cut-off value (s) − Baseline latency (s)
2.6. Experimental design In this study, there were three control groups including intact, sham-operated and saline groups (n = 6 in each group) for determining the baseline TFLs, surgical manipulation and microinjection volume effects, respectively. To evaluate the involvement of Ox2 receptors within the VTA and NAc in antinociceptive responses induced by LH stimulation, tail-flick test was performed as a model of acute pain. In all of the above control and experimental groups, TFLs were recorded at 5, 15, 30, 45 and 60 min after drugs/vehicles administrations. 2.6.1. Dose–response effects of carbachol microinjected into the LH on the tail-flick test The dose–response effects of intra-LH administration of different doses of carbachol have been established in our previous work [24]. However, in this study, a dose–response relationship for carbachol as a LH chemical stimulation agent on tail-flick test was re-established. After intra-LH administration of different doses of carbachol (125 and 250 nmol/0.5 l saline; n = 6 in each group), TFLs were tested at the time set intervals. Control animals received saline (n = 6) instead of carbachol in the LH. 2.6.2. Effects of intra-VTA administration of Ox2 receptor antagonist, TCS OX2 29, on antinociception induced by carbachol into the lateral hypothalamus In order to test the role of Ox2 receptors located within the VTA during the LH stimulation-induced antinociception, different doses of TCS OX2 29 (1, 7 and 20 nmol/0.3 l DMSO; n = 5–6 in each group)
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were unilaterally injected into the VTA, 5 min prior to administration of effective dose of intra-LH carbachol. It was followed by the tail-flick test at the time set intervals. In the vehicle group (n = 6), 10% DMSO was microinjected into the VTA and 2 min later animals received saline instead of carbachol in the LH. 2.6.3. Effects of intra-NAc administration of Ox2 receptor antagonist, TCS OX2 29, on antinociception induced by carbachol into the lateral hypothalamus To examine the role of intra-accumbal OX2 receptors in the LH stimulation-induced antinociception, different doses of TCS OX2 29 (2, 10, 20 and 40 nmol/0.5 l DMSO; n = 5–7 in each group) were unilaterally injected into the NAc, 5 min prior to effective dose of intra-LH carbachol administration. In the vehicle group (n = 6), 10% DMSO was microinjected into the NAc and 2 min later animals received saline (0.5 l) in the LH. 2.7. Histology The animals were deeply anesthetized with Ketamine and Xylazine after the experiments were accomplished. Transcardial perfusion was carried out with 0.9% saline and 10% formalin solution. The brains were removed, blocked and cut coronally in 50 m sections through the cannulae placements. The neuroanatomical locations of cannulae tips were confirmed using rat brain atlas [22] .Only the results from animals with correct cannulae placements were included in the data analysis. 2.8. Statistics The obtained data are expressed as mean ± SEM (standard error of mean). The mean TFLs or the mean maximal possible effect (%MPE) of drugs, as an analgesic index in all control groups were subjected to one-way and/or two-way ANOVA followed by protected Newman–Keuls’s or Bonferroni’s test for multiple comparisons, respectively as needed. In order to evaluate the nociceptive responses, area under the curve (AUC) was calculated as raw %MPE × time by linear trapezoidal method and a single value was used in statistical analyses. The calculated AUC values in all experimental groups were subjected to one-way ANOVA followed by protected Newman–Keuls test for multiple comparisons. P-values less than 0.05 were considered to be statistically significant. 3. Results 3.1. Dose–response effects of chemical stimulation of the lateral hypothalamus by carbachol on tail-flick test as an animal model of acute pain In this study, two-way ANOVA for repeated measures over time followed by Bonferroni’s test for %MPEs confirmed a significant difference in antinociceptive responses to carbachol microinjected into the LH alone, compared to that of respective salinetreated group [Treatment effect: F(2, 75) = 26.05, P < 0.0001; Time effect: F(4, 75) = 0.2197, P = 0.9267; Interaction: F(8, 75) = 0.3576, P = 0.9394; data not shown]. Moreover, one-way ANOVA followed by Dunnett multiple comparison test also showed that there are significant differences in mean AUC calculated values for %MPEs [F(2, 17) = 20.89, P < 0.0001] among the different doses of carbachol and saline control groups. 3.2. Effect of intra-VTA administration of Ox2 receptor antagonist, TCS OX2 29, on LH stimulation-induced antinociception We examined the effect of intra-VTA administration of TCS OX2 29 as a selective Ox2r antagonist on LH stimulation-induced
Fig. 1. Effects of unilateral microinjection of different doses of TCS OX2 29 (TCS), an Ox2 receptor antagonist, in the ventral tegmental area (VTA) on (A) maximal possible effect in 5, 15, 30, 45 and 60 min after microinjection as an index of antinociception induced by chemical stimulation of the lateral hypothalamus (LH) and (B) area under the curves (AUCs) calculated for %MPEs shown in (A). Ox2 receptor antagonist can significantly decrease the intra-LH carbachol-induced antinociception in a dosedependent manner. Animals received TCS OX2 29 (1, 7 and 20 nmol/0.3 l DMSO) or 10% DMSO (0.3 l) as a vehicle in the VTA, 5 min before intra-LH administration of carbachol (125 nmol/rat). Each point shows the mean ± SEM for 5–6 rats. *** P < 0.001 different from the control (vehicles) group. †† P < 0.01, ††† P < 0.001 different from the respective DMSO-carbachol group.
antinociception by carbachol in order to evaluate the receptor specific response of Ox2 receptors in the VTA. Two-way ANOVA for repeated measures over time followed by Bonferroni’s test for %MPEs indicated significant differences in antinociceptive responses to carbachol microinjected into the LH alone or when carbachol was concurrently administrated into the LH following different doses of intra-VTA TCS OX2 29 as compared to those of respective saline-treated group [Treatment effect: F(5, 145) = 38.6, P < 0.0001; Time effect: F(4, 145) = 1.032, P = 0.3927; Interaction: F(20, 145) = 0.9356, P = 0.5435]. Furthermore, as shown in Fig. 1, one-way ANOVA followed by Newman–Keuls multiple comparison test showed that there are significant differences in AUC calculated values for %MPEs [F(5, 34) = 18.46, P < 0.0001] among the experimental and control (vehicle) groups. Data gained from these experiments showed that intra-VTA administration of TCS OX2 29 as an Ox2r antagonist dose-dependently prevents intra-LH carbachol-induced antinociception in rats. Also, administration of maximal dose of TCS OX2 29 (20 nmol/rat) alone into the VTA could not affect the baseline TFLs (%MPEs) at time set intervals and/or AUC calculated values compared to the vehicle group (Fig. 1). 3.3. Effect of intra-accumbal administration of Ox2 receptor antagonist, TCS OX2 29, on LH stimulation-induced antinociception The dose–response effects of intra-NAc administration of selective Ox2r antagonist TCS OX2 29 on the antinociceptive response of carbachol microinjected into the LH during a 60-min period was observed in the following experiments. Two-way ANOVA followed by Bonferroni’s test [Treatment effect: F(6, 174) = 44.03, P < 0.0001; Time effect F(4, 174) = 2.449, P = 0.048; Interaction F(24, 174) = 0.8744, P = 0.6364] showed that there are significant differences in %MPE values among the experimental and control groups. Although one-way ANOVA followed by Newman–Keuls multiple comparison test [F(6, 41) = 17.44, P < 0.0001; Fig. 2] showed
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Fig. 2. Effect of unilateral microinjection of different doses of TCS OX2 29, an Ox2 receptor antagonist, in the nucleus accumbens (NAc) on (A) maximal possible effect in 5, 15, 30, 45 and 60 min after microinjection as an index of antinociception induced by chemical stimulation of the lateral hypothalamus (LH) and (B) area under the curves (AUCs) calculated for %MPEs shown in (A). The high doses of Ox2 receptor antagonist can significantly decrease the intra-LH carbachol-induced antinociception in rats. Animals received TCS OX2 29 (2, 10, 20 and 40 nmol/0.5 l DMSO) or 10% DMSO as a vehicle in the NAc, 5 min before intra-LH administration of carbachol (125 nmol/rat). Each point shows the mean ± SEM for 5–7 rats. * P < 0.05, *** P < 0.001 different from the control (vehicles) group. † P < 0.05, ††† P < 0.001 different from the respective DMSO-carbachol group.
that there are significant differences in AUC calculated values for %MPEs among the experimental and control (vehicles) groups, intra-accumbal administration of TCS OX2 29 only at high doses (20 and 40 nmol/rat) could significantly reduce %MPEs in comparison with DMSO respective control group that received carbachol (125 nmol/rat) in the LH. Moreover, administration of maximal dose of TCS OX2 29 (40 nmol/rat) alone into the NAc could not affect the AUC calculated values compared to vehicle group (Fig. 2). 4. Discussion In this study we presented a detailed analysis on the antinociceptive effects caused by orexin-2 receptors in the VTA and NAc. Our results indicated that administration of different doses of carbachol into the LH caused an antinociceptive response in the tail-flick test. Nonetheless, the major findings were (I) the involvement of Ox2 receptors within the VTA and NAc in pain modulation in the rats; (II) administration of an Ox2r antagonist (TCS OX2 29) into the VTA and NAc dose dependently blocked intra-LH carbachol induced antinociception and (III) the antinociceptive effect of Ox2 receptor in the VTA was more potent than that in the NAc. In this experiment and pertaining to our laboratory’s last research on Ox1 receptors [24], both SB334867 and TCS OX2 29 as Ox1 and Ox2 receptors antagonists, respectively, were able to reduce the antinociception caused by intra-LH administration of carbachol, and we observed this attenuation by blocking the Ox2 receptors within the VTA and NAc. These responses were stronger in the VTA than those in the NAc. Stimulation of the LH using excitatory drugs like non-specific cholinergic muscarinic agonist (carbachol) can elicit marked antinociception [7,13–17]. Hence, we used two doses of carbachol, and although there was no significant difference in the antinociceptive effect, the lower dose was chosen [24]. Multiple brain regions receive orexinergic fibers input from the LH and the perifornical area [4]. Dense orexinergic innervation is observed in the VTA. This area has been shown to have a robust
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expression of Ox1 and Ox2 receptors, and orexinergic terminals synapse onto presumed dopaminergic and GABAergic neurons [21]. Narita and colleagues strongly support the idea that the activation of orexin-containing neurons in the VTA leads to the direct activation of mesolimbic dopamine neurons including the NAc at the somatodendritic level [21]. Ghalandari-Shamami et al. [9] presented that the NAc plays an important role in the modulation of pain, and the recent studies have also supported that the NAc is involved in the antinociception induced by intra-BLA administration of cannabinoid receptor agonist in the tail-flick test [9,10,12]. In addition, it has been proven that antinociception induced by intra-VTA DiMe- C7 decreases with intra-accumbal infusions of the mixed dopamine receptor antagonist flupenthixol [1]. Thus, our findings in this study suggest that Ox2 receptors in the VTA and NAc are involved in the antinociception induced by LH stimulation through activation of the LH orexinergic projecting neurons to the VTA and lesser to the NAc, and in part, through mechanisms involved in pain descending inhibitory system in these areas. On the other hand, the distribution of orexin receptors in these areas are not same, and the quantity of Ox1 receptors in the VTA is more than that in the NAc [27]. It is consistent with the results from the previous research performed on Ox1 receptors [24]; it has been shown that SB334867 as an Ox1 receptor antagonist is more effective in the VTA region. As mentioned before in this study, TCS OX2 29 as an Ox2 receptor antagonist is also more effective in the VTA in comparison with this effect in the NAc. Therefore, it seems that VTA orexinergic receptors and orexin system in this area are more effective in modulation of the pain descending inhibitory system than those of the same receptors and system in the NAc. Furthermore, we understand that Ox1 and Ox2 receptors usually act in adverse toward each other, but regarding these findings it seems that these two kinds of receptors have worked together to decrease the antinociception induced by stimulation of hypothalamus. While findings from this study and other studies are novel, we cannot say with certainty how these receptors interact with each other.
Acknowledgments The authors would like to thank Mr. Mahmoudreza Ramin for the editing of our manuscript. This study was supported by a grant from the Qazvin University of Medical Sciences, Qazvin, Iran.
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