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Role of dorsal hippocampal orexin-1 receptors in modulation of antinociception induced by chemical stimulation of the lateral hypothalamus
Physiology & Behavior 185 (2018) 79–86
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Role of dorsal hippocampal orexin-1 receptors in modulation of antinociception induced by chemical stimulation of the lateral hypothalamus Pooya Pourrezaa, Vahab Babapourb, Abbas Haghparastc,
T
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a
Department of Basic Sciences, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad university, Tehran, Iran Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Iran c Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: Inflammatory pain Orexin-1 receptors Lateral hypothalamus CA1 region of hippocampus Rat
The Hippocampus has a role not only in nociception but also in modulation of pain perception. In addition, orexinergic neurons present in the lateral hypothalamus (LH) have a recognized role in pain modulation. The presence of orexinergic projections from the lateral hypothalamus (LH) to the dorsal hippocampal Cornu Ammonis 1 (CA1) region raises the question of whether pain modulatory role of LH is mediated through the CA1. To elucidate the interactions between the LH and neural substrates involved in modulation of formalin-induced nociception, the study aimed to test the pain modulatory role of CA1 orexin receptors in the formalin test. Seventy-one male Wistar rats were unilaterally implanted with two cannulae above the LH and CA1. In the treatment groups, intra-CA1 administration of SB-334867, as an orexin-1 receptor antagonist, was performed 5 min before intra-LH microinjection of carbachol, as a cholinergic receptor agonist. In dimethyl sulfoxide (DMSO)-control group, DMSO and saline as well as in carbachol-control group, DMSO and carbachol were microinjected into the CA1 and LH, respectively. In all rats, the procedure was followed by subcutaneous injection of formalin after 5-min time interval. Carbachol reduced both phases of formalin-induced nociception. Intra-CA1 administration of SB-334867 antagonized the LH-induced analgesia during both phases in a dosedependent manner. It seems that the blockade of orexin-1 receptors has more effects on reduction of antinociception during the late phase compared to the early phase. Pain modulatory role of orexinergic system in the formalin test through a neural pathway from the LH to CA1 provides the evidence that orexins can be useful therapeutic agents for chronic pain treatment.
1. Introduction Orexin-A and -B are two neuropeptides made from a common precursor, prepro-orexin. They are also known as hypocretin-1 and -2 and two G protein-coupled receptors, i.e. orexin-1 and -2 receptors are differentially recruited by orexins [39]. A few number of neurons within the perifornical area, lateral and posterior hypothalamus produces orexin-A and -B [45]. The orexinergic system is involved in the regulation of feeding behavior, sleep/wakefulness, thermogenesis, homeostasis of energy as well as reward processing [19,45]. It has been demonstrated that electrical and chemical stimulation of the lateral hypothalamus (LH) for instance by glutamate or morphine relieves nociceptive responses in different pain models [1,6,11]. In this respect, administration of carbachol, a cholinergic receptor agonist, into the LH reduces nociceptive-related behaviors in acute pain models
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like foot withdrawal, tail-flick tests [22,37,38] and formalin-induced pain [16]. Carbachol is a potent muscarinic cholinergic receptor agonist, which activates LH orexin neurons [40]. Numerous supra-spinal and spinal sites integrated in pain modulation are innervated by orexinergic projections [28,31,33,46]. Previous studies have shown that modulation of the nociceptive responses through carbachol administration into the LH which is at least partially mediated through the orexin receptors into the ventral tegmental area (VTA) [17], nucleus accumbens (NAc) [51], periaqueductal gray (PAG) [14,15] and pons [21]. A body of evidence suggests that hippocampal formation is involved in nociception [26,42], and some neural populations in the dorsal hippocampal Cornu Ammonis 1 (CA1) region respond to persistent noxious stimuli [24]; so that, partial hippocampectomy has been used as a treatment for chronic pain [18]. Furthermore, the role of
Corresponding author at: Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, P.O.Box: 19615-1178, Tehran, Iran. E-mail address: [email protected] (A. Haghparast).
https://doi.org/10.1016/j.physbeh.2017.12.036 Received 11 July 2017; Received in revised form 28 December 2017; Accepted 29 December 2017 Available online 30 December 2017 0031-9384/ © 2017 Published by Elsevier Inc.
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hippocampus in the control of cognitive and emotional aspects of pain has been established [20,27]. Orexinergic projections are also found in the hippocampal formation including CA1 and Dentate gyrus [25,33]. In the current study, to elucidate the interactions between the LH and neural substrates involved in modulation of formalin-induced nociception, efforts were made to examine the pain modulatory role of the LH through the CA1 orexin-1 receptors in the formalin test.
surrounding the LH (n = 4) or the effect of carbachol administration into the LH in combination with SB-334867 (10 nM) into some brain regions surrounding the CA1 (n = 7) were also examined in order to distinguish between the results of drug injections into the LH and CA1, and those obtained from drug injections into the neighboring regions. During the experiments, the experimenter was blinded to the experimental conditions.
2. Materials and methods
2.4. Experimental procedure
2.1. Animals
Experiments were carried out in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were confirmed by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Adult male albino Wistar rats (n = 71) weighing 220–250 g were used in this project (Pasteur Institute, Tehran, Iran). During the whole period of experiments, animals were provided with free access to food and water. Animals were kept on a 12:12 h light:dark cycle (lights off at 7 PM).
2.4.1. Stereotaxic surgery A mixture of xylazine 2% (10 mg/kg) and ketamine 10% (100 mg/ kg) were intraperitoneally injected for anesthetizing the rats. According to the rat brain atlas [32], the coordinates for perifornical part of LH (AP = 2.65 mm caudal to the bregma, Lat = ± 1.3 mm lateral to the midline and DV = 8.6 mm ventral to the skull surface) and CA1 (AP = 3.50 mm caudal to the bregma, Lat = ± 2.2 mm lateral to the midline and DV = 2.7 mm ventral to the skull surface) were determined. A mixture of lidocaine/epinephrine (0.2 ml) was injected around the surgery site to reduce the nociceptive responses and bleeding, respectively. In each rat, two 23-gauge, 12 and 8 mm-long stainless steel guide cannulae were unilaterally (based on the guidelines of our institute for pain management in animal study) inserted into the left or right side of the skull 1 mm above the LH and CA1, respectively. Formalin was injected into the right or left hind paw contralateral to the surgery side. The guide cannulae were unilaterally implanted and fixed in place using two stainless steel screws which anchored to the skull and dental acrylic cement. After the cement was completely dried and hardened, two stainless steel obturators were used to occlude the guide cannulae during the recovery period to prevent clogging. Penicillin-G 200,000 IU/ml (0.2–0.3 ml/rat, single dose, intramuscular) was immediately administered after surgery to prevent infection. Animals were individually housed and allowed to recover for 5–7 days before the experiments.
2.2. Drug and drug administration In this study, carbachol (Carbamylcholine chloride; Sigma-Aldrich, USA) was dissolved in the sterile normal saline. 0.5 μl of a 250 nM solution of carbachol was administered into the LH as a cholinergic agonist. 0.5 μl of 0.3, 1, 3 and 10 nM solutions of SB-334867 (Tocris Bioscience, Bristol, UK) were used into the CA1 as the orexin-1 receptor antagonist. Dimethyl sulfoxide (DMSO) 12% was used as a vehicle for SB-334867. Formalin 2.5% was prepared from formaldehyde 37% (Merck, Germany) diluted with normal saline. A stainless steel injector (a 30 gauge needle) with a length of 1 mm longer than the guide cannula was used for injecting the drug into the LH and CA1. The injector was connected to a 1-μl Hamilton syringe using a polyethylene tube (PE-20). Intra-CA1 microinjection of SB334867 or DMSO was carried out 5 min prior to microinjection of carbachol or saline into the LH. Following the second microinjection (5 min later), formalin test was performed. Each microinjection lasted about 60 s and the injector was left for an extra 60 s to facilitate the drug diffusion in order to prevent the drug backflow. 2.3. Experimental design In the first set of experiments, the effect of drug (carbachol + different doses of SB-334867), vehicle and DMSO administration on locomotor activity were studied and compared to the Naïve group. Locomotor activity of animals was recorded by video tracking system and Ethovision software for 60 min. In the second set of designed experiments, three control groups including intact, sham-operated and vehicle (DMSO-control) groups were defined (7–8 in each group; n = 23). The effect of formalin test was examined on the intact group. Sham-operated group underwent the surgery and after 5 to 7 days, formalin test was done. DMSO-control group underwent the surgery and after 5 to 7 days, animals received 0.5 μl DMSO as drug vehicle into the CA1, 5 min before intra-LH administration of 0.5 μl saline (n = 7). Then, 5 min after intra-LH microinjection of saline, 50 μl of formalin 2.5% was subcutaneously injected into the plantar surface of hind paw. In the third part of study, in order to investigate the role of CA1 orexin-1 receptors in mediation of analgesia induced by LH stimulation, SB-334867 (0.3, 1, 3 and 10 nM) was microinjected into the CA1, 5 min prior to microinjection of effective dose of carbachol (250 nM) [16] into the LH (6 in each group; n = 24). DMSO-control group received 0.5 μl DMSO as drug vehicle into the CA1, 5 min before intra-LH administration of 0.5 μl saline (n = 7). Carbachol-control group received DMSO and carbachol (250 nM) into the CA1 and LH, respectively (n = 6). In all rats, 5 min after intra-LH microinjection of carbachol or saline, formalin test was performed. The effect of carbachol injection into some brain regions
2.4.2. Formalin test A Plexiglas chamber (35 × 35 × 35 cm) with a mirror angled at 45° below the surface of chamber was used as the apparatus to observe animal's behavior during the formalin test. In all experiments, 5 min after carbachol or saline microinjection (second microinjection), 50 μl of formalin 2.5% was injected into the plantar surface of right or left hind paw contralateral to the surgery side. Then, animals were immediately placed in the apparatus. Formalin induces biphasic nociceptive responses. The first phase immediately starts after formalin injection and lasts for 3–5 min. Over the next 10–15 min, pain responses decrease because of activation of the descending pain inhibitory system. The second phase starts 15–20 min after formalin administration and lasts for 20–40 min [12,44]. Nociceptive behaviors were recorded and quantified as following: 0, when the distinction between the posture of injected paw and other hind paw was difficult; 1, when putting weight on the injected paw reduced; 2, the injected paw was elevated; 3, the injected paw was licked, shacked or bitten [12]. The time spent in each type of behavior was recorded in 5-min blocks for 60-min test period. For each 5-min block of time, the weighted nociceptive score with a range from 0 to 3 was calculated:
Nociceptive score = (t0 ×0) + (t1 ×1) + (t2 ×2) + (t3×3)/t0 + t1 + t2 + t3 Following the formalin test, histological verification of samples and data analysis was performed. 80
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Fig. 1. Schematic illustrations of coronal sections of the rat brain showing the approximate location of the intra-CA1 microinjection sites [○ = vehicle (saline or DMSO), ● = Treatment (carbachol or SB-334867) and ▲ = Misplacement]. All microinjections were unilaterally performed on the left or right side of skull midline.
comparison to the naïve group, drug administration had no effect on locomotor activity [F (6,44) = 0.2322, P < 3.9634; Electronic Supplementary Fig. 1].
2.5. Histological verification To verify the accuracy of injection sites, animals were deeply anesthetized with ketamine and xylazine and sacrificed after the test. Then, the brains were removed and 50-μm transverse brain sections were cut. The rat brain atlas [32] was used to trace the cannula tracts and they were compared with drawings of the LH and CA1 position in the atlas (Fig. 1). Sites of cannula placement in the LH and CA1 have been schematically shown in Fig. 1A and B, respectively. In the data analysis, only the animals with correct cannulae placements were included.
3.1. Comparison of nociceptive responses in control groups Two-way ANOVA showed no significant differences in nociceptive scores among intact, sham-operated and vehicle (DMSO-control) groups (Treatment effect: F (2,220) = 0.3419, P < 0.7145; Time effect: F (11,220) = 15.51, P < 0.0001; Treatment and time interaction effect: F (22,220) = 0.4767, P = 0.978; Fig. 2A). Similarly, there were no marked differences among total AUC values calculated for control groups (F (2,22) = 0.3536, P = 0.7064; Fig. 2B). The percentage decrease of AUC values compared to intact group is illustrated in Fig. 2C. As can be seen in Fig. 2C, the percentage decrease of AUC values in sham-operated and vehicle groups was not significant compared to that in the intact group during the early [F (2,23) = 1.903, P = 0.1739; Fig. 2C left] and late [F (2,23) = 0.1828, P = 0.8343; Fig. 2C right] phases.
2.6. Statistics The data analyzed using GraphPad Prism 6 (GraphPad Software, CA). Here, the results were expressed as mean ± SEM (standard error of mean) and the differences with P-values < 0.05 were statistically considered significant. In order to show the effects of treatment and time on mean nociceptive scores in different groups (DMSO-control, carbachol-control and experimental groups), two-way ANOVA followed by Bonferroni's test was used. In order to evaluate the nociceptive responses during the early or late phases, AUC (area under the curve) values were calculated as raw pain scores × time by linear trapezoidal method. Then, to make comparison between the mean AUC values of different treatments, and those of DMSO-control and carbachol-control groups, one-way ANOVA followed by Newman-Keuls test was used. Finally, to study the effects of time (early or late phase of formalininduced pain) and treatment on the AUC values of treatment groups compared to those of carbachol-control group, two-way ANOVA followed by Bonferroni's test was used.
3.2. Effects of intra-CA1 administration of SB-334867 on LH-induced analgesia In the second set of analyses, two-way ANOVA followed by Bonferroni test showed that nociceptive responses were at the highest level in the DMSO-control group (Fig. 3). Carbachol injection into the LH (carbachol-control group) significantly attenuated pain scores compared to the DMSO-control group and pretreatment of the CA1 with SB-334867 (0.3, 1, 3 and 10 nM) dose-dependently blocked the antinociception induced by carbachol injection into the LH [Treatment effect: F (5,352) = 54.35, P < 0.0001; Time effect: F (11,352) = 11.07, P < 0.0001; Treatment and time interaction effect: F (55,352) = 1.315, P = 0.0768]. One-way ANOVA followed by Newman-Keuls test was done to study
3. Results One-way ANOVA followed by Newman-Keuls test showed that in 81
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Fig. 2. Comparison of nociceptive scores among the control groups including intact, sham-operated and vehicle groups during 60-min period of the test (A), and total area under the curves (AUCs) calculated for nociceptive scores shown in A (B). The percentage decrease of AUC values of sham-operated and vehicle groups separately compared to the AUC values of intact group during the early and late phases of formalin test (C). Each point shows the mean ± SEM for 7–8 rats.
P < 0.001, respectively). Although an increase in the AUC values was observed in groups that had received 0.3 and 1 nM solutions of SB334867, this increase was not significant compared to carbachol-control group. Intra-CA1 injection of 1 nM solution of SB-334867 significantly attenuated carbachol-induced antinociception during the late phase (P < 0.05). During the late phase, 3 and 10 nM solutions of SB334867 significantly antagonized the analgesic effect of carbachol (P < 0.001). 0.3 nM solution of SB-334867 did not induce significant changes in carbachol-induced analgesia compared to carbachol-control group (Fig. 4 and Electronic Supplementary Fig. 2). 3.3. Changes in AUC values of carbachol-control and SB-334867 treated groups compared to the AUC values of DMSO-control group As shown in Fig. 5, changes in the AUC values of carbachol-control group in comparison to the AUC values of saline-control group are set as 100%. The AUC values of experimental groups were separately compared to those of carbachol-control group during the early and late phases of formalin test. The AUC values of animals which had been received 10 nM solution of SB-334867 showed the lowest similarity to those of carbachol-control group during both early and late phases of formalin-induced nociception. While, the administration of 0.3 nM solution of SB-334867 showed the highest similarity to the AUC values of carbachol-control group during both phases. The AUC values in groups which had been received 1 and 3 nM solutions of SB-334867 showed more similarity to the AUC values of carbachol-control group during the early phase compared to the late phase. In other words, the effects of 1 and 3 nM solutions of SB-334867 on reduction of LH-induced analgesia were more than those during the late phase. Here, the magnitude of 50% effective dose (ED50) of SB-334867 during the early phase (3.96 nM) was more than that during the late phase (1.37 nM). Altogether, these findings clearly prove that the contribution of orexin-1 receptors to mediation of LH-induced antinociception and the anti-analgesic effect of SB-334867 during the late phase are, to a high extent, more than those during the early phase.
Fig. 3. The effects of time and treatment on the nociceptive responses during both phases of formalin-induced nociception. Intra-CA1 administration of 0.3, 1, 3 and 10 nM solutions of SB-334867 reduced antinociception induced by intra-LH microinjection of carbachol (250 nM) in the formalin test. The average of nociceptive scores was recorded in 5min blocks for 60-min test period. Each point shows the mean ± SEM for 6–7 rats in each group. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the DMSO-control group.
the effect of different doses of SB-334867 on the carbachol-induced analgesia during the early [F (5,36) = 13.95, P < 0.0001; Fig. 4A] and late [F (5,36) = 20.81, P < 0.0001; Fig. 4B] phases. The results showed that carbachol administration reduces the AUC values in comparison to the DMSO-control group (P < 0.001) and intra-CA1 application of SB-334867 dose-dependently diminished LH-induced antinociception during both phases. These changes can be seen as an increase in the AUC values. In comparison to the carbachol-control group, during the early phase, 3 and 10 nM solutions of SB-334867 significantly diminished antinociceptive effects of carbachol which is represented as an increase in the AUC values (P < 0.05 and
3.4. Effect of drug administration into some brain regions next to the LH and CA1 The results of last part of study demonstrated no antinociceptive effects after carbachol injection in some areas close to the LH. Furthermore, the administration of SB-334867(10 nM) into some 82
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Fig. 4. Area under the curves (AUCs) separately calculated for the nociceptive scores during the early (A) and late (B) phases of formalin-induced nociception. DMSO-control and carbachol-control groups showed the highest and lowest AUC values. Intra-CA1 administration of SB-334867 (0.3, 1, 3 and 10 nM) before intra-LH administration of carbachol (250 nM) increased AUC values in a dose-dependent manner. ** P < 0.01 and *** P < 0.001 compared to the DMSO-control group. †P < 0.05and ††† P < 0.001 compared to the carbachol-control group.
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administration of orexin-1 receptor antagonist SB-334867 into the CA1 region of dorsal hippocampus reduced carbachol-evoked analgesia during both phases of formalin-induced nociception. This provides the evidence that VTA, NAc and hippocampal orexin receptors are involved in mediation of LH-induced analgesia during the early and late phases of formalin test. In other words, it seems that the LH-induced analgesia observed in this study is not only mediated through the hippocampal orexin-1 receptors but also via the VTA and NAc orexin receptors. The prominent role of activation of mesolimbic dopamine pathway, which originates from the VTA, is determined in the modulation of tonic pain [2,49]. The pain modulatory role of VTA is mostly mediated through the NAc [2]. In turn, NAc modulates the tonic pain through the spinal and supra-spinal sites such as the medial part of the thalamus and amygdala [2]. However, more studies are needed to shed light on the mechanisms involved in the mediation of antinociception via hippocampal CA1 orexin-1 receptors and disclosing the neural connection between CA1 and descending pain modulatory circuitry. The role of rostroventral medulla (RVM) and PAG in mediation of orexin-induced analgesia in the formalin test should not be neglected; so that, in two separate studies intra-RVM and -PAG administration of orexin-A reduced formalin-induced nociceptive responses [3,5,36,47]. Holden and Pizzi [22] have shown that chemical stimulation of the LH by carbachol reduced pain responses in acute pain model. They demonstrated that this effect was related to activation of substance Pimmunoreactive (SP-ir) neurons in the LH, which have cholinergic input. Anatomical evidence demonstrates that there is co-localization between substance P and orexin in the LH. The SP-ir neurons have been recognized in the LH [23]. More studies are suggested to reveal the possible role of LH substance P neurons in mediation of LH-induced analgesia in the formalin test. According to the results of the present study, intra-CA1 administration of SB-334867 attenuated LH-induced analgesia. The reduction of antinociception was more, during the late phase, than the early phase. It may be attributed to different mechanisms underlying the development of both phases of formalin-induced nociception [44,48]. In other words, it seems that the orexinergic system plays a more important role in the control of mechanisms of central sensitization, which is important in the development of tonic pain during the second phase. The role of CA1 in modification of nociception is not only mediated through the orexin receptors, but also through the N-methyl-D-aspartate glutamate [43], cholinergic [10] and serotonergic receptors [42]. Interestingly, the role of all these receptors in the dorsal hippocampal CA1 in modification of nociception was more during the late phase. The last set of experiments showed that there is no antinociceptive effect after carbachol injection in some areas close to the LH. On the other hand, the administration of SB-334867(10 nM) into some regions near the CA1 before intra-LH administration of carbachol did not bring about any changes on the antinociceptive effects of carbachol. Accordingly, what can be assumed is that the results are most likely due to the effect of carbachol and SB-334867administration into the LH and CA1, and not drug injection into the neighboring areas. The phenomenon of “pain memory” or “pain plasticity” which may occur at primary afferent nociceptors or at other parts of central nervous system - like hippocampus - receiving nociceptive input, mostly results in an exacerbation of nociception signaling and lead to allodynia, hyperalgesia or spontaneous pain. It is claimed that all these features can occur following an injury, especially in chronic pain disorders [34]. On the other hand, it has been demonstrated that the blockade of orexin-1 receptors into the CA1 attenuated the development of conditioned place preference induced by chemical stimulation of the LH [35], and orexin A injection regulates synaptic plasticity in the hippocampus [41]. Accordingly, it seems that activation of orexin-1 receptors in the CA1 region is involved in modulation of formalin-induced nociception at the level of hippocampus through induction of changes in synaptic plasticity. Altogether, the results of the present study revealed that CA1
Fig. 5. A log dose-response curve of the effect of intra-CA1 administration of 0.3, 1, 3 and 10 nM solutions of SB-334867 on carbachol-induced antinociception during the early phase compared to late phase. The effective dose 50% of SB-334867 in the late phase (1.37) was less than that in the early phase (3.96).
regions near the CA1before intra-LH administration of carbachol did not alter the antinociceptive effects of carbachol (Fig.1).
4. Discussion The following describes the findings of the present study: a) There is a neural pathway from the lateral hypothalamus (LH) to the CA1 which modulates both phases of formalin-induced nociception; b) Blockade of CA1 orexin-1 receptors using SB-334867 diminishes LH-induced analgesia during both phases in a dose-dependent manner; and finally c) Anti-analgesic effect of orexin-1 receptor antagonist is more during the late phase. Previous studies have been shown that orexin signaling in some spinal and supra-spinal sites that are known as parts of descending pain modulatory circuitry involve in modulation of nociception. In this respect, it has been demonstrated that the administration of orexin-A and -B into these brain areas reduces nociceptive-related behaviors induced in the model of formalin test [13,29,50], acute pain models [13] and carrageenan-induced hyperalgesia [7]. Orexin neurons in the LH have cholinergic input, and carbachol increases the activity of these neurons [30,40]. In light of the fact that lateral hypothalamic orexin neurons have neuroanatomical connections with the spinal and supra-spinal sites implicated in modification of nociception [28,31,33,46], the role of orexinergic system in modulation of nociception through these neural substrates has been examined. In this regard, in acute pain models, the microinjection of orexin-1 and -2 receptor antagonists (SB-334867 or TCS OX2 29, respectively) into the VTA, NAc, PAG and pons before carbachol injection into the LH, reduced LH-induced analgesia [4,14,15,21,37]. It shows that antinociceptive responses evoked by intra-LH injection of carbachol are at least partially mediated through the orexin receptors into the VTA, NAc, PAG and pons in acute pain model. Also, in tail-flick test, LHinduced analgesia was reversed by lidocaine injection into the locus coeruleus [38]. It has been demonstrated that in animal model of formalin test, carbachol injection into the LH attenuated both phases of formalin-induced nociceptive responses [16]; and intra-VTA [17] and intra-NAc [51] administration of orexin receptor antagonists attenuated carbachol-induced analgesia. Based on the findings of the present study, the 84
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orexin-1 receptors are engaged in mediation of LH-induced analgesia in the formalin test, and the effect of orexin-1 receptor antagonist administration on blockade of LH-induced analgesia is more, during the late phase, than the early phase. In this respect, due to the similarity between the mechanisms underlying the persistent inflammatory pain during the late phase of formalin test and chronic pain, human studies are highly recommended in order to reveal whether the orexins can be considered as therapeutic agents for treatment of chronic pain or not. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.physbeh.2017.12.036.
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Author contributions Substantial contributions to conception and design: Abbas Haghparast; Acquisition, Analysis and interpretation of data: Pooya Pourreza, Abbas Haghparast, Vahab Babapour; Drafting the article: Vahab Babapour, Pooya Pourreza; Revising the article critically for important intellectual content: Pooya Pourreza, Abbas Haghparast; Final approval of the version to be published: Abbas Haghparast. Acknowledgements The authors would like to thank Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran, for their cooperation in conducting this study. References [1] L.D. Aimone, G.F. Gebhart, Spinal monoamine mediation of stimulation-produced antinociception from the lateral hypothalamus, Brain Res. 403 (1987) 290–300. [2] N. Altier, J. Stewart, The role of dopamine in the nucleus accumbens in analgesia, Life Sci. 65 (1999) 2269–2287. [3] H. Azhdari-Zarmehri, S. Semnanian, Y. Fathollahi, E. Erami, R. Khakpay, H. Azizi, K. Rohampour, Intra-periaqueductal gray matter microinjection of orexin-A decreases formalin-induced nociceptive behaviors in adult male rats, J. Pain 12 (2011) 280–287. [4] H. Azhdari-Zarmehri, Z. Reisi, A. Vaziri, A. Haghparast, P. Shaigani, A. Haghparast, 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. [5] H. Azhdari-Zarmehri, S. Semnanian, Y. Fathollahi, Orexin-a microinjection into the rostral ventromedial medulla causes antinociception on formalin test, Pharmacol. Biochem. Behav. 122 (2014) 286–290. [6] M.M. Behbehani, M.R. Park, M.E. Clement, Interactions between the lateral hypothalamus and the periaqueductal gray, J. Neurosci. 8 (1988) 2780–2787. [7] S. Bingham, P.T. Davey, A.J. Babbs, E.A. Irving, M.J. Sammons, M. Wyles, P. Jeffrey, L. Cutler, I. Riba, A. Johns, R.A. Porter, N. Upton, A.J. Hunter, A.A. Parsons, Orexin-A, an hypothalamic peptide with analgesic properties, Pain 92 (2001) 81–90. [10] I. Ceccarelli, F. Casamenti, C. Massafra, G. Pepeu, C. Scali, A. Aloisi, Effects of novelty and pain on behavior and hippocampal extracellular ACh levels in male and female rats, Brain Res. 815 (1999) 169–176. [11] N. Dafny, W.Q. Dong, C. Prieto-Gomez, C. Reyes-Vazquez, J. Stanford, J.T. Qiao, Lateral hypothalamus: site involved in pain modulation, Neuroscience 70 (1996) 449–460. [12] D. Dubuisson, S.G. Dennis, The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats, Pain 4 (1977) 161–174. [13] E. Erami, H. Azhdari-Zarmehri, E. Ghasemi-Dashkhasan, M.H. Esmaeili, S. Semnanian, Intra-paragigantocellularis lateralis injection of orexin-A has an antinociceptive effect on hot plate and formalin tests in rat, Brain Res. 1478 (2012) 16–23. [14] M.H. Esmaeili, Z. Reisi, S. Ezzatpanah, A. Haghparast, Functional interaction between orexin-1 and CB1 receptors in the periaqueductal gray matter during antinociception induced by chemical stimulation of the lateral hypothalamus in rats, Eur. J. Pain (2016), http://dx.doi.org/10.1002/ejp.899. [15] M.H. Esmaeili, Z. Reisi, S. Ezzatpanah, A. Haghparast, Role of orexin-2 and CB1 receptors within the periaqueductal gray matter in lateral hypothalamic-induced antinociception in rats, Behav. Pharmacol. 28 (1) (2017) 83–89. [16] S. Ezzatpanah, V. Babapour, B. Sadeghi, A. Haghparast, Chemical stimulation of the lateral hypothalamus by carbachol attenuated the formalin-induced pain behaviors in rats, Pharmacol. Biochem. Behav. 129 (2015) 105–110. [17] S. Ezzatpanah, V. Babapour, A. Haghparast, Differential contribution of orexin receptors within the ventral tegmental area in modulation of persistent inflammatory pain, Eur. J. Pain 20 (2016) 1090–1101. [18] A. Gol, G.M. Faibish, Effects of human hippocampal ablation, J. Neurosurg. 26 (1967) 390–398.
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[51] F. Yazdi, M. Jahangirvand, S. Ezzatpanaha, A. Haghparast, Role of orexin-2 receptors in the nucleus accumbens in antinociception induced by carbachol stimulation of the lateral hypothalamus in formalin test, Behav. Pharmacol. 27 (2016) 431–438.
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Role of dorsal hippocampal orexin-1 receptors in modulation of antinociception induced by chemical stimulation of the lateral hypothalamus Pooya Pourrezaa, Vahab Babapourb, Abbas Haghparastc,
T
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a
Department of Basic Sciences, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad university, Tehran, Iran Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Iran c Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran b
A R T I C L E I N F O
A B S T R A C T
Keywords: Inflammatory pain Orexin-1 receptors Lateral hypothalamus CA1 region of hippocampus Rat
The Hippocampus has a role not only in nociception but also in modulation of pain perception. In addition, orexinergic neurons present in the lateral hypothalamus (LH) have a recognized role in pain modulation. The presence of orexinergic projections from the lateral hypothalamus (LH) to the dorsal hippocampal Cornu Ammonis 1 (CA1) region raises the question of whether pain modulatory role of LH is mediated through the CA1. To elucidate the interactions between the LH and neural substrates involved in modulation of formalin-induced nociception, the study aimed to test the pain modulatory role of CA1 orexin receptors in the formalin test. Seventy-one male Wistar rats were unilaterally implanted with two cannulae above the LH and CA1. In the treatment groups, intra-CA1 administration of SB-334867, as an orexin-1 receptor antagonist, was performed 5 min before intra-LH microinjection of carbachol, as a cholinergic receptor agonist. In dimethyl sulfoxide (DMSO)-control group, DMSO and saline as well as in carbachol-control group, DMSO and carbachol were microinjected into the CA1 and LH, respectively. In all rats, the procedure was followed by subcutaneous injection of formalin after 5-min time interval. Carbachol reduced both phases of formalin-induced nociception. Intra-CA1 administration of SB-334867 antagonized the LH-induced analgesia during both phases in a dosedependent manner. It seems that the blockade of orexin-1 receptors has more effects on reduction of antinociception during the late phase compared to the early phase. Pain modulatory role of orexinergic system in the formalin test through a neural pathway from the LH to CA1 provides the evidence that orexins can be useful therapeutic agents for chronic pain treatment.
1. Introduction Orexin-A and -B are two neuropeptides made from a common precursor, prepro-orexin. They are also known as hypocretin-1 and -2 and two G protein-coupled receptors, i.e. orexin-1 and -2 receptors are differentially recruited by orexins [39]. A few number of neurons within the perifornical area, lateral and posterior hypothalamus produces orexin-A and -B [45]. The orexinergic system is involved in the regulation of feeding behavior, sleep/wakefulness, thermogenesis, homeostasis of energy as well as reward processing [19,45]. It has been demonstrated that electrical and chemical stimulation of the lateral hypothalamus (LH) for instance by glutamate or morphine relieves nociceptive responses in different pain models [1,6,11]. In this respect, administration of carbachol, a cholinergic receptor agonist, into the LH reduces nociceptive-related behaviors in acute pain models
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like foot withdrawal, tail-flick tests [22,37,38] and formalin-induced pain [16]. Carbachol is a potent muscarinic cholinergic receptor agonist, which activates LH orexin neurons [40]. Numerous supra-spinal and spinal sites integrated in pain modulation are innervated by orexinergic projections [28,31,33,46]. Previous studies have shown that modulation of the nociceptive responses through carbachol administration into the LH which is at least partially mediated through the orexin receptors into the ventral tegmental area (VTA) [17], nucleus accumbens (NAc) [51], periaqueductal gray (PAG) [14,15] and pons [21]. A body of evidence suggests that hippocampal formation is involved in nociception [26,42], and some neural populations in the dorsal hippocampal Cornu Ammonis 1 (CA1) region respond to persistent noxious stimuli [24]; so that, partial hippocampectomy has been used as a treatment for chronic pain [18]. Furthermore, the role of
Corresponding author at: Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, P.O.Box: 19615-1178, Tehran, Iran. E-mail address: [email protected] (A. Haghparast).
https://doi.org/10.1016/j.physbeh.2017.12.036 Received 11 July 2017; Received in revised form 28 December 2017; Accepted 29 December 2017 Available online 30 December 2017 0031-9384/ © 2017 Published by Elsevier Inc.
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hippocampus in the control of cognitive and emotional aspects of pain has been established [20,27]. Orexinergic projections are also found in the hippocampal formation including CA1 and Dentate gyrus [25,33]. In the current study, to elucidate the interactions between the LH and neural substrates involved in modulation of formalin-induced nociception, efforts were made to examine the pain modulatory role of the LH through the CA1 orexin-1 receptors in the formalin test.
surrounding the LH (n = 4) or the effect of carbachol administration into the LH in combination with SB-334867 (10 nM) into some brain regions surrounding the CA1 (n = 7) were also examined in order to distinguish between the results of drug injections into the LH and CA1, and those obtained from drug injections into the neighboring regions. During the experiments, the experimenter was blinded to the experimental conditions.
2. Materials and methods
2.4. Experimental procedure
2.1. Animals
Experiments were carried out in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were confirmed by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Adult male albino Wistar rats (n = 71) weighing 220–250 g were used in this project (Pasteur Institute, Tehran, Iran). During the whole period of experiments, animals were provided with free access to food and water. Animals were kept on a 12:12 h light:dark cycle (lights off at 7 PM).
2.4.1. Stereotaxic surgery A mixture of xylazine 2% (10 mg/kg) and ketamine 10% (100 mg/ kg) were intraperitoneally injected for anesthetizing the rats. According to the rat brain atlas [32], the coordinates for perifornical part of LH (AP = 2.65 mm caudal to the bregma, Lat = ± 1.3 mm lateral to the midline and DV = 8.6 mm ventral to the skull surface) and CA1 (AP = 3.50 mm caudal to the bregma, Lat = ± 2.2 mm lateral to the midline and DV = 2.7 mm ventral to the skull surface) were determined. A mixture of lidocaine/epinephrine (0.2 ml) was injected around the surgery site to reduce the nociceptive responses and bleeding, respectively. In each rat, two 23-gauge, 12 and 8 mm-long stainless steel guide cannulae were unilaterally (based on the guidelines of our institute for pain management in animal study) inserted into the left or right side of the skull 1 mm above the LH and CA1, respectively. Formalin was injected into the right or left hind paw contralateral to the surgery side. The guide cannulae were unilaterally implanted and fixed in place using two stainless steel screws which anchored to the skull and dental acrylic cement. After the cement was completely dried and hardened, two stainless steel obturators were used to occlude the guide cannulae during the recovery period to prevent clogging. Penicillin-G 200,000 IU/ml (0.2–0.3 ml/rat, single dose, intramuscular) was immediately administered after surgery to prevent infection. Animals were individually housed and allowed to recover for 5–7 days before the experiments.
2.2. Drug and drug administration In this study, carbachol (Carbamylcholine chloride; Sigma-Aldrich, USA) was dissolved in the sterile normal saline. 0.5 μl of a 250 nM solution of carbachol was administered into the LH as a cholinergic agonist. 0.5 μl of 0.3, 1, 3 and 10 nM solutions of SB-334867 (Tocris Bioscience, Bristol, UK) were used into the CA1 as the orexin-1 receptor antagonist. Dimethyl sulfoxide (DMSO) 12% was used as a vehicle for SB-334867. Formalin 2.5% was prepared from formaldehyde 37% (Merck, Germany) diluted with normal saline. A stainless steel injector (a 30 gauge needle) with a length of 1 mm longer than the guide cannula was used for injecting the drug into the LH and CA1. The injector was connected to a 1-μl Hamilton syringe using a polyethylene tube (PE-20). Intra-CA1 microinjection of SB334867 or DMSO was carried out 5 min prior to microinjection of carbachol or saline into the LH. Following the second microinjection (5 min later), formalin test was performed. Each microinjection lasted about 60 s and the injector was left for an extra 60 s to facilitate the drug diffusion in order to prevent the drug backflow. 2.3. Experimental design In the first set of experiments, the effect of drug (carbachol + different doses of SB-334867), vehicle and DMSO administration on locomotor activity were studied and compared to the Naïve group. Locomotor activity of animals was recorded by video tracking system and Ethovision software for 60 min. In the second set of designed experiments, three control groups including intact, sham-operated and vehicle (DMSO-control) groups were defined (7–8 in each group; n = 23). The effect of formalin test was examined on the intact group. Sham-operated group underwent the surgery and after 5 to 7 days, formalin test was done. DMSO-control group underwent the surgery and after 5 to 7 days, animals received 0.5 μl DMSO as drug vehicle into the CA1, 5 min before intra-LH administration of 0.5 μl saline (n = 7). Then, 5 min after intra-LH microinjection of saline, 50 μl of formalin 2.5% was subcutaneously injected into the plantar surface of hind paw. In the third part of study, in order to investigate the role of CA1 orexin-1 receptors in mediation of analgesia induced by LH stimulation, SB-334867 (0.3, 1, 3 and 10 nM) was microinjected into the CA1, 5 min prior to microinjection of effective dose of carbachol (250 nM) [16] into the LH (6 in each group; n = 24). DMSO-control group received 0.5 μl DMSO as drug vehicle into the CA1, 5 min before intra-LH administration of 0.5 μl saline (n = 7). Carbachol-control group received DMSO and carbachol (250 nM) into the CA1 and LH, respectively (n = 6). In all rats, 5 min after intra-LH microinjection of carbachol or saline, formalin test was performed. The effect of carbachol injection into some brain regions
2.4.2. Formalin test A Plexiglas chamber (35 × 35 × 35 cm) with a mirror angled at 45° below the surface of chamber was used as the apparatus to observe animal's behavior during the formalin test. In all experiments, 5 min after carbachol or saline microinjection (second microinjection), 50 μl of formalin 2.5% was injected into the plantar surface of right or left hind paw contralateral to the surgery side. Then, animals were immediately placed in the apparatus. Formalin induces biphasic nociceptive responses. The first phase immediately starts after formalin injection and lasts for 3–5 min. Over the next 10–15 min, pain responses decrease because of activation of the descending pain inhibitory system. The second phase starts 15–20 min after formalin administration and lasts for 20–40 min [12,44]. Nociceptive behaviors were recorded and quantified as following: 0, when the distinction between the posture of injected paw and other hind paw was difficult; 1, when putting weight on the injected paw reduced; 2, the injected paw was elevated; 3, the injected paw was licked, shacked or bitten [12]. The time spent in each type of behavior was recorded in 5-min blocks for 60-min test period. For each 5-min block of time, the weighted nociceptive score with a range from 0 to 3 was calculated:
Nociceptive score = (t0 ×0) + (t1 ×1) + (t2 ×2) + (t3×3)/t0 + t1 + t2 + t3 Following the formalin test, histological verification of samples and data analysis was performed. 80
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Fig. 1. Schematic illustrations of coronal sections of the rat brain showing the approximate location of the intra-CA1 microinjection sites [○ = vehicle (saline or DMSO), ● = Treatment (carbachol or SB-334867) and ▲ = Misplacement]. All microinjections were unilaterally performed on the left or right side of skull midline.
comparison to the naïve group, drug administration had no effect on locomotor activity [F (6,44) = 0.2322, P < 3.9634; Electronic Supplementary Fig. 1].
2.5. Histological verification To verify the accuracy of injection sites, animals were deeply anesthetized with ketamine and xylazine and sacrificed after the test. Then, the brains were removed and 50-μm transverse brain sections were cut. The rat brain atlas [32] was used to trace the cannula tracts and they were compared with drawings of the LH and CA1 position in the atlas (Fig. 1). Sites of cannula placement in the LH and CA1 have been schematically shown in Fig. 1A and B, respectively. In the data analysis, only the animals with correct cannulae placements were included.
3.1. Comparison of nociceptive responses in control groups Two-way ANOVA showed no significant differences in nociceptive scores among intact, sham-operated and vehicle (DMSO-control) groups (Treatment effect: F (2,220) = 0.3419, P < 0.7145; Time effect: F (11,220) = 15.51, P < 0.0001; Treatment and time interaction effect: F (22,220) = 0.4767, P = 0.978; Fig. 2A). Similarly, there were no marked differences among total AUC values calculated for control groups (F (2,22) = 0.3536, P = 0.7064; Fig. 2B). The percentage decrease of AUC values compared to intact group is illustrated in Fig. 2C. As can be seen in Fig. 2C, the percentage decrease of AUC values in sham-operated and vehicle groups was not significant compared to that in the intact group during the early [F (2,23) = 1.903, P = 0.1739; Fig. 2C left] and late [F (2,23) = 0.1828, P = 0.8343; Fig. 2C right] phases.
2.6. Statistics The data analyzed using GraphPad Prism 6 (GraphPad Software, CA). Here, the results were expressed as mean ± SEM (standard error of mean) and the differences with P-values < 0.05 were statistically considered significant. In order to show the effects of treatment and time on mean nociceptive scores in different groups (DMSO-control, carbachol-control and experimental groups), two-way ANOVA followed by Bonferroni's test was used. In order to evaluate the nociceptive responses during the early or late phases, AUC (area under the curve) values were calculated as raw pain scores × time by linear trapezoidal method. Then, to make comparison between the mean AUC values of different treatments, and those of DMSO-control and carbachol-control groups, one-way ANOVA followed by Newman-Keuls test was used. Finally, to study the effects of time (early or late phase of formalininduced pain) and treatment on the AUC values of treatment groups compared to those of carbachol-control group, two-way ANOVA followed by Bonferroni's test was used.
3.2. Effects of intra-CA1 administration of SB-334867 on LH-induced analgesia In the second set of analyses, two-way ANOVA followed by Bonferroni test showed that nociceptive responses were at the highest level in the DMSO-control group (Fig. 3). Carbachol injection into the LH (carbachol-control group) significantly attenuated pain scores compared to the DMSO-control group and pretreatment of the CA1 with SB-334867 (0.3, 1, 3 and 10 nM) dose-dependently blocked the antinociception induced by carbachol injection into the LH [Treatment effect: F (5,352) = 54.35, P < 0.0001; Time effect: F (11,352) = 11.07, P < 0.0001; Treatment and time interaction effect: F (55,352) = 1.315, P = 0.0768]. One-way ANOVA followed by Newman-Keuls test was done to study
3. Results One-way ANOVA followed by Newman-Keuls test showed that in 81
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Fig. 2. Comparison of nociceptive scores among the control groups including intact, sham-operated and vehicle groups during 60-min period of the test (A), and total area under the curves (AUCs) calculated for nociceptive scores shown in A (B). The percentage decrease of AUC values of sham-operated and vehicle groups separately compared to the AUC values of intact group during the early and late phases of formalin test (C). Each point shows the mean ± SEM for 7–8 rats.
P < 0.001, respectively). Although an increase in the AUC values was observed in groups that had received 0.3 and 1 nM solutions of SB334867, this increase was not significant compared to carbachol-control group. Intra-CA1 injection of 1 nM solution of SB-334867 significantly attenuated carbachol-induced antinociception during the late phase (P < 0.05). During the late phase, 3 and 10 nM solutions of SB334867 significantly antagonized the analgesic effect of carbachol (P < 0.001). 0.3 nM solution of SB-334867 did not induce significant changes in carbachol-induced analgesia compared to carbachol-control group (Fig. 4 and Electronic Supplementary Fig. 2). 3.3. Changes in AUC values of carbachol-control and SB-334867 treated groups compared to the AUC values of DMSO-control group As shown in Fig. 5, changes in the AUC values of carbachol-control group in comparison to the AUC values of saline-control group are set as 100%. The AUC values of experimental groups were separately compared to those of carbachol-control group during the early and late phases of formalin test. The AUC values of animals which had been received 10 nM solution of SB-334867 showed the lowest similarity to those of carbachol-control group during both early and late phases of formalin-induced nociception. While, the administration of 0.3 nM solution of SB-334867 showed the highest similarity to the AUC values of carbachol-control group during both phases. The AUC values in groups which had been received 1 and 3 nM solutions of SB-334867 showed more similarity to the AUC values of carbachol-control group during the early phase compared to the late phase. In other words, the effects of 1 and 3 nM solutions of SB-334867 on reduction of LH-induced analgesia were more than those during the late phase. Here, the magnitude of 50% effective dose (ED50) of SB-334867 during the early phase (3.96 nM) was more than that during the late phase (1.37 nM). Altogether, these findings clearly prove that the contribution of orexin-1 receptors to mediation of LH-induced antinociception and the anti-analgesic effect of SB-334867 during the late phase are, to a high extent, more than those during the early phase.
Fig. 3. The effects of time and treatment on the nociceptive responses during both phases of formalin-induced nociception. Intra-CA1 administration of 0.3, 1, 3 and 10 nM solutions of SB-334867 reduced antinociception induced by intra-LH microinjection of carbachol (250 nM) in the formalin test. The average of nociceptive scores was recorded in 5min blocks for 60-min test period. Each point shows the mean ± SEM for 6–7 rats in each group. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the DMSO-control group.
the effect of different doses of SB-334867 on the carbachol-induced analgesia during the early [F (5,36) = 13.95, P < 0.0001; Fig. 4A] and late [F (5,36) = 20.81, P < 0.0001; Fig. 4B] phases. The results showed that carbachol administration reduces the AUC values in comparison to the DMSO-control group (P < 0.001) and intra-CA1 application of SB-334867 dose-dependently diminished LH-induced antinociception during both phases. These changes can be seen as an increase in the AUC values. In comparison to the carbachol-control group, during the early phase, 3 and 10 nM solutions of SB-334867 significantly diminished antinociceptive effects of carbachol which is represented as an increase in the AUC values (P < 0.05 and
3.4. Effect of drug administration into some brain regions next to the LH and CA1 The results of last part of study demonstrated no antinociceptive effects after carbachol injection in some areas close to the LH. Furthermore, the administration of SB-334867(10 nM) into some 82
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Fig. 4. Area under the curves (AUCs) separately calculated for the nociceptive scores during the early (A) and late (B) phases of formalin-induced nociception. DMSO-control and carbachol-control groups showed the highest and lowest AUC values. Intra-CA1 administration of SB-334867 (0.3, 1, 3 and 10 nM) before intra-LH administration of carbachol (250 nM) increased AUC values in a dose-dependent manner. ** P < 0.01 and *** P < 0.001 compared to the DMSO-control group. †P < 0.05and ††† P < 0.001 compared to the carbachol-control group.
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administration of orexin-1 receptor antagonist SB-334867 into the CA1 region of dorsal hippocampus reduced carbachol-evoked analgesia during both phases of formalin-induced nociception. This provides the evidence that VTA, NAc and hippocampal orexin receptors are involved in mediation of LH-induced analgesia during the early and late phases of formalin test. In other words, it seems that the LH-induced analgesia observed in this study is not only mediated through the hippocampal orexin-1 receptors but also via the VTA and NAc orexin receptors. The prominent role of activation of mesolimbic dopamine pathway, which originates from the VTA, is determined in the modulation of tonic pain [2,49]. The pain modulatory role of VTA is mostly mediated through the NAc [2]. In turn, NAc modulates the tonic pain through the spinal and supra-spinal sites such as the medial part of the thalamus and amygdala [2]. However, more studies are needed to shed light on the mechanisms involved in the mediation of antinociception via hippocampal CA1 orexin-1 receptors and disclosing the neural connection between CA1 and descending pain modulatory circuitry. The role of rostroventral medulla (RVM) and PAG in mediation of orexin-induced analgesia in the formalin test should not be neglected; so that, in two separate studies intra-RVM and -PAG administration of orexin-A reduced formalin-induced nociceptive responses [3,5,36,47]. Holden and Pizzi [22] have shown that chemical stimulation of the LH by carbachol reduced pain responses in acute pain model. They demonstrated that this effect was related to activation of substance Pimmunoreactive (SP-ir) neurons in the LH, which have cholinergic input. Anatomical evidence demonstrates that there is co-localization between substance P and orexin in the LH. The SP-ir neurons have been recognized in the LH [23]. More studies are suggested to reveal the possible role of LH substance P neurons in mediation of LH-induced analgesia in the formalin test. According to the results of the present study, intra-CA1 administration of SB-334867 attenuated LH-induced analgesia. The reduction of antinociception was more, during the late phase, than the early phase. It may be attributed to different mechanisms underlying the development of both phases of formalin-induced nociception [44,48]. In other words, it seems that the orexinergic system plays a more important role in the control of mechanisms of central sensitization, which is important in the development of tonic pain during the second phase. The role of CA1 in modification of nociception is not only mediated through the orexin receptors, but also through the N-methyl-D-aspartate glutamate [43], cholinergic [10] and serotonergic receptors [42]. Interestingly, the role of all these receptors in the dorsal hippocampal CA1 in modification of nociception was more during the late phase. The last set of experiments showed that there is no antinociceptive effect after carbachol injection in some areas close to the LH. On the other hand, the administration of SB-334867(10 nM) into some regions near the CA1 before intra-LH administration of carbachol did not bring about any changes on the antinociceptive effects of carbachol. Accordingly, what can be assumed is that the results are most likely due to the effect of carbachol and SB-334867administration into the LH and CA1, and not drug injection into the neighboring areas. The phenomenon of “pain memory” or “pain plasticity” which may occur at primary afferent nociceptors or at other parts of central nervous system - like hippocampus - receiving nociceptive input, mostly results in an exacerbation of nociception signaling and lead to allodynia, hyperalgesia or spontaneous pain. It is claimed that all these features can occur following an injury, especially in chronic pain disorders [34]. On the other hand, it has been demonstrated that the blockade of orexin-1 receptors into the CA1 attenuated the development of conditioned place preference induced by chemical stimulation of the LH [35], and orexin A injection regulates synaptic plasticity in the hippocampus [41]. Accordingly, it seems that activation of orexin-1 receptors in the CA1 region is involved in modulation of formalin-induced nociception at the level of hippocampus through induction of changes in synaptic plasticity. Altogether, the results of the present study revealed that CA1
Fig. 5. A log dose-response curve of the effect of intra-CA1 administration of 0.3, 1, 3 and 10 nM solutions of SB-334867 on carbachol-induced antinociception during the early phase compared to late phase. The effective dose 50% of SB-334867 in the late phase (1.37) was less than that in the early phase (3.96).
regions near the CA1before intra-LH administration of carbachol did not alter the antinociceptive effects of carbachol (Fig.1).
4. Discussion The following describes the findings of the present study: a) There is a neural pathway from the lateral hypothalamus (LH) to the CA1 which modulates both phases of formalin-induced nociception; b) Blockade of CA1 orexin-1 receptors using SB-334867 diminishes LH-induced analgesia during both phases in a dose-dependent manner; and finally c) Anti-analgesic effect of orexin-1 receptor antagonist is more during the late phase. Previous studies have been shown that orexin signaling in some spinal and supra-spinal sites that are known as parts of descending pain modulatory circuitry involve in modulation of nociception. In this respect, it has been demonstrated that the administration of orexin-A and -B into these brain areas reduces nociceptive-related behaviors induced in the model of formalin test [13,29,50], acute pain models [13] and carrageenan-induced hyperalgesia [7]. Orexin neurons in the LH have cholinergic input, and carbachol increases the activity of these neurons [30,40]. In light of the fact that lateral hypothalamic orexin neurons have neuroanatomical connections with the spinal and supra-spinal sites implicated in modification of nociception [28,31,33,46], the role of orexinergic system in modulation of nociception through these neural substrates has been examined. In this regard, in acute pain models, the microinjection of orexin-1 and -2 receptor antagonists (SB-334867 or TCS OX2 29, respectively) into the VTA, NAc, PAG and pons before carbachol injection into the LH, reduced LH-induced analgesia [4,14,15,21,37]. It shows that antinociceptive responses evoked by intra-LH injection of carbachol are at least partially mediated through the orexin receptors into the VTA, NAc, PAG and pons in acute pain model. Also, in tail-flick test, LHinduced analgesia was reversed by lidocaine injection into the locus coeruleus [38]. It has been demonstrated that in animal model of formalin test, carbachol injection into the LH attenuated both phases of formalin-induced nociceptive responses [16]; and intra-VTA [17] and intra-NAc [51] administration of orexin receptor antagonists attenuated carbachol-induced analgesia. Based on the findings of the present study, the 84
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orexin-1 receptors are engaged in mediation of LH-induced analgesia in the formalin test, and the effect of orexin-1 receptor antagonist administration on blockade of LH-induced analgesia is more, during the late phase, than the early phase. In this respect, due to the similarity between the mechanisms underlying the persistent inflammatory pain during the late phase of formalin test and chronic pain, human studies are highly recommended in order to reveal whether the orexins can be considered as therapeutic agents for treatment of chronic pain or not. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.physbeh.2017.12.036.
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