Series of Selected Papers from President's Undergraduate Research Fellowship,Peking University(2003)
Involvement of opioid receptors in the oxytocin-induced antinociception in central nervous system of rats
Lian Gao, Long-Chuan Yu*
Laboratory of Neurobiology, College of Life Sciences
Abstract
Recent studies showed that oxytocin and opioid peptides play important roles in pain modulation at different levels in the central nervous system. The present study was performed to explore whether opioid system is involved in the oxytocin-induced antinociception in the brain of rats.The results showed that: (1) Intracerebroventricular injection of oxytocin induced dose-dependent increases in hindpaw withdrawal latencies (HWL) to noxious thermal and mechanical stimulation in rats. (2)The antinociceptive effect of oxytocin was attenuated dose-dependently by intracerebroventricular injection of naloxone, indicating an involvement of opioid system in the oxytocin-induced antinociception. (3) It is interesting that the antinociceptive effect of oxytocin was attenuated by subsequent intracerebroventricularinjection of the mu-opioid antagonist β-funaltrexamine (β-FNA) and the kappa-opioid antagonist nor-binaltorphimine (nor-BNI), but not the delta-opioid antagonist naltrindole. The results indicate that oxytocin plays an antinociceptive role in the brain of rats, mu- and kappa-opioid receptors, not delta-receptors, are involved in the oxytocin-induced antinociception in the central nervous system of rats.
Key words:Oxytocin, Oxytocin receptor, Opioid receptor, lateral ventricle,Antinociception
1. Introduction
Oxytocin (OT) is a hormone synthesized in the neurohypophysis. It has been demonstrated that oxytocin is involved in the modulation of pain at different levels of the central nervous system (CNS) [1-7]. Oxytocin exerts its actions via epynomouslynamed OT receptors [8], which localize in many parts of the CNS (cortex, olfactory system, basal ganglia, limbic system, thalamus, hypothalamus, brain stem and dorsal horn of the spinal cord) and in the peripheral nervous system.
Antinociceptive effect of oxytocin has been reported in many studies. Intracerebroventricular injection of oxytocin induced antinociception [3].Intraperitoneal or intracisternal injection of oxytocin produced antinociceptive effect in rats or in mice [9]. Madrazo et al. found that intracerebroventricular injection of oxytocin reduced suffering of a patient with intractable cancer pain [10]. There is, thus, increasing interest in the role of oxytocin in antinociception.
A network of descending pathways projecting from cerebralstructures to the dorsal horn of the spinal cord plays a complex and crucial role in the transmission of nociceptive information from the peripheryto the CNS [8,11]. Oxytocin and opioid peptides are involved in the process. Our reports demonstrated that intra-periaqueductal gray (PAG) or nucleus raphe magnus (NRM) and intracerebroventricular injection of oxytocin induced dose-dependent antinociceptive effects in rats [5,7,12].
It is well known that opioid peptides play an important role in antinociception in the brain. The efficacy of intracerebroventricular administration of morphine has been well documented [13,14]. Also, intracerebroventricular injection of mu- and delta-opioid receptor agonists produced dose-dependent antinociception in the Randall Sellito test [15] and hot-plate test [16] in rats, and in mice, the intracerebroventricular administration of opioid receptor agonist have been shown to produce antinociception tested by hot-plate or tail-flick [17-19]. Furthermore, it has been demonstrated that oxytocin-induced antinociceptive effect was attenuated by intrathecal administration of the opioid antagonist naloxone, suggesting an involvement of the endogenous opioid system in the oxytocin-induced antinociception at the spinal level in rats with inflammation [20]. Recent studies in our laboratory have shown that opioid receptors were involved in the oxytocin-induced antinociception in the PAG [5] and NRM [12] of rats. The aim of the present study was to investigate the involvement of opioid receptors in the oxytocin-induced antinociception in the central nervous system of rats.
2. Materials and methods
2.1. Animals
All experiments were performed on freely moving male SD rats (220–250 g; Experimental AnimalCenter of Henan Medical University, Henan, China). The ratswere housed in cages with free access to food and water,and maintained in a room temperature of 25±2oC with a12 h light–dark cycle. All experiments were conducted according to theguidelines of the International Association for the Study of Pain [21] and every effort was made to minimize both the animal suffering and the number of animals used.
2.2. Chemicals
Solutions for intracerebroventricular injection were prepared with sterilized saline (0.9%), each with a volume of 5 μl of: (1) 0.05, 0.1 or 0.25 nmol of oxytocin (Penisula Laboratories, USA); (2) 0.1, 0.5 or 1 μg of naloxone (naloxone hydrochloride; Sigma Chemical Company, St. Louis, MO); (3) 10 nmol ofβ-funaltrexamine (β-FNA hydrochloride; Tocris, Balwin, MO 63011, USA); (4) 10 nmol of nor-binaltorphimine (nor-BNI dihydrochloride; Tocris, Balwin, MO 63011, USA); (5) 10 nmol of naltrindole (naltrindole hydrochloride; Tocris, Balwin, MO 63011, USA).
2.3. Neuropharmacological experiment
2.3.1. Training before the operations
All rats were accustomed to the test condition for 5 days before the experiment was carried out. After the training, the latency of hindpaw withdrawal to thermal stimulation was about 4.5-5.5 s and to mechanical stimulation was about 5-7 s.
2.3.2. The hot-plate and Randall Selitto test
The response to noxious thermal stimulation was assessed by the hot-plate test [22,23].The entire ventral surface of the rat’s hindpaw was placed manually on the hot-plate, which was maintained at a temperature of 52 oC (51.7-52.3oC).The latencies to hindpaw withdrawal during thermal and mechanical stimulation were measured and expressed in seconds to be referred to as the hindpaw withdrawal latency (HWL).
The Randall Selitto Test (Ugo Basile, Type 7200, Italy) was used to assess the HWLs to noxious mechanical stimulation [22,23]. A wedge-shaped pusher at a loading rate of 30 g/s was applied to the dorsal surface of the manually handled hindpaw and the latency required to initiate the withdrawal response was assessed and expressed in seconds.
In both of the tests, 15 s was the cut-off time to prevent possible tissue damage. The average values obtainedbefore intracerebroventricular injection were regarded as the basal HWL. The HWLs recorded during subsequent experiments were expressed as percentage changes of the basal level for each rat (% changes of the HWL). Each rat was tested with both types of stimulation.
2.3.3.Intracerebroventricular injection
The animals were anaesthetized by intraperitoneal pentobarbital (45 mg/kg) and were mounted on a stereotaxicinstrument. A stainless steel guide cannular of 0.8 mmouter-diameter was directed into the lateral ventricle (AP, -0.92 mm; LR, 1.5 mm; V, 3.6 mm from the surface of the skull. AP, anterior (+) or posterior (-) to Bregma; L or R, left or right to midline; V, ventral to the surface of skull)according to Paxinos and Watson [24] and was fixed to the skull bydental acrylic. On the day of experiment, a stainless steelneedle with 0.4 mm diameter was directly inserted into theguide cannula, with 1 mm beyond the tip of the latter. Five microliter of solution was thereafter infused into the lateral ventricle over 1min.
2.3.4. Histologic verification of the injection site
At the end of the experiments the rat was killed by a high dose of pentobarbital (80 mg/kg) and the rat heads were fixed in 10% formalin for 24hours with the injecting tube in situ before section. The location of the tip of the injecting tube was verified and all the tips of the injecting tube were in lateral ventricle area of rats in the present study.
2.3.5. Statistical analysis
Data from nociceptive tests were presented as mean±S.E.M. The two-way analysis of variance (ANOVA) was used to evaluate the difference in HWLs between two groups (Fleft/left is the F value of the two groups: the left HWL of the first group compared with the left HWL of the second group). *P<0.05, **P<0.01 and***P<0.001 were considered as significant differences.
3. Results
3.1.Effects of intracerebroventricular injection of oxytocin on HWLs to noxious thermal and mechanical stimulation in rats
Rats received intracerebroventricular injection of 0.05 (n=6), 0.1 (n=6) or 0.25 nmol of oxytocin (n=6), or 5 μl of 0.9% saline as a control (n=8). As shown in Fig. 1, the HWLs to thermal and mechanical stimulation increased significantly after intracerebroventricular injection of 0.25 nmol of oxytocin (Thermal test: Fleft/left=26.17, P<0.001; Fright/right=59.26, P<0.001; Randall Selitto test: Fleft/left=38.72, P<0.001; Fright/right=14.58, P<0.01),but not 0.1 nmol of oxytocin (Thermal test: Fleft/left=4.16, P=0.06; Fright/right=0.68, P=0.43; Randall Selitto test: Fleft/left=8.03, P<0.05; Fright/right=2.09, P=0.17), or 0.05 nmol of oxytocin (Thermal test: Fleft/left=1.01, P＝0.33; Fright/right=0.26, P=0.62; Randall Selitto test: Fleft/left=0.36, P=0.56; Fright/right=0.13, P=0.72), compared with the control group. The HWLs increased and to reach the peak at 10 min after intracerebroventricular injection of oxytocin, and then recovered to the basal line at 30 min.
3.2. Blockade effects of intracerebroventricular injection of naloxone on the oxytocin-induced increase in HWLs
Rats received intracerebroventricular injection of 0.25 nmol of oxytocin, followed 5 min later by intracerebroventricular administration of 0.1 (n=6), 0.5 (n=6) or 1 μg of naloxone (n=6), or 5 μl of 0.9% saline as a control (n=6). The results are shown in Fig. 2.Compared with the control group, the increased HWLs to thermal stimulation were attenuated significantly after administration of 1 μg (Fleft/left=21.26, P <0.001; Fright/right=38.23, P<0.001) and 0.5 μg of naloxone (Fleft/left=12.23, P<0.01; Fright/right=9.50, P<0.05), but not 0.1 μg of naloxone (Fleft/left=0.01, P=0.91; Fright/right=0.76, P＝0.40) compared to the control group.And the increased HWLs to mechanical stimulation were also attenuated significantly after administration of 1 μg of naloxone (Fleft/left=15.97, P<0.01; Fright/right=15.46 P< 0.01), but not 0.5 μg (Fleft/left=1.86, P=0.20; Fright/right=1.78, P=0.21) and 0.1 μg of naloxone (Fleft/left=3.00, P＝0.11; Fright/right=1.33, P＝0.27) compared with the control group. Another group of rats (n=6) received intracerebroventricular injection of 5 μl of 0.9% saline, followed 5 min later by 1 μg of naloxone. There were no marked changes in HWLs during 30 min after the injection.
3.3. Influence of different opioid antagonists on the oxytocin-induced increase in HWLs
Rats received intracerebroventricular administration of 0.25 nmol of oxytocin, followed 5 min later by 10 nmol of β-FNA (n=6), 10 nmol of nor-BNI (n=6) or 10 nmol of naltrindole (n=6), or 5 μl of 0.9% saline as a control (n=6). The results are shown in Fig. 3. Compared with the control group, the increased HWLs to both thermal and mechanical stimulation were attenuated significantly after administration of 10nmol ofβ-FNA (Thermal test: Fleft/left=16.05, P<0.01; Fright/right=43.98, P<0.001; Randall Selitto test: Fleft/left=24.72, P<0.001; Fright/right=14.43, P<0.01) and 10 nmol of nor-BNI (Thermal test: Fleft/left=52.17, P<0.001; Fright/right=146.81, P<0.001; Randall Selitto test: Fleft/left=13.34, P<0.01; Fright/right=21.82, P<0.001), but not 10 nmol of naltrindole (Thermal test: Fleft/left=0.87, P=0.37; Fright/right=0.03, P=0.87; Randall Selitto test: Fleft/left=0.30, P=0.60; Fright/right=0.04, P=0.84).Two groups of rats received intracerebroventricular injection of 5 μl of 0.9% saline, followed 5 min later by 10 nmol of β-FNA (n=6) or nor-BNI (n=6). There were no marked changes in HWLs during 30 min after the injection.
4. Discussion
The present study showed that intracerebroventricular injection of oxytocin increased the rat’s pain threshold, and the increase in induced HWLs to both thermal and mechanical stimulation was about 40% by 0.25 nmol of oxytocin and about 20% by 0.1 nmol of oxytocin (Fig. 1). It demonstrated that oxytocin played a dose-dependent antinociceptive effect in the brain of rats. Arletti et al. found that intracerebroventricular injection of oxytocin increased the latencies of rat’s tail-flick, what could be reversed by the selective oxytocin antagonist [3]. The result demonstrated that the antinociceptive effect of oxytocin in the lateral ventricle has respect to oxytocin receptors, and indicated that oxytocin activate oxytocin receptors in the brain to modulate the nociceptive response. When high dose (10 nmol/rat) of oxytocin was used, 5-10 min after injection, rats would become paralyzed and kept barrel rotating [3]. In present study, rats were mild insane for hours after intracerebroventricular injection of 1 nmol of oxytocin, which could also increase the HWLs by about 50%. The result may be impact by both the antinociception of oxytocin and the nonspecificepilepsia, in view of that, present study use a relatively low dose, 0.25 nmol of oxytocin to minimize the influence of insanity to the thermal and mechanical test. Studies showed that in the central nervous system, the oxytocin gene is primarily expressed in magnocellular neurons in the hypothalamic paraventricular nucleus and supraoptic nucleus [25]. Action potentials in these neurosecretory cells trigger the release of oxytocin from their axon terminals in the neurohypophysis [26]. Also, studies showed that oxytocin fibers and endings have been described in nuclei around the lateral ventricle of rats [25].
It is well known that opioid peptides play a key role in antinociception in the central nervous system [8,27,28]. Intracerebroventricular injection of opioid peptides produced dose-dependent antinociception [13-19]. Previous studies showed that oxytocin-induced antinociception could be blocked by the non-selective opioid receptor antagonist naloxone in the supraspinal level [3]. In the present study, same result was found that the oxytocin-induced increases in HWLs were attenuated significantly by administration of non-selective opioid antagonist naloxone (Fig. 2), indicating an involvement of opioid system in the process of nociceptive modulation of oxytocin in the brain. It is well known that there are three types of opioid receptors in the rat central nervous system, mu-, delta- and kappa-opioid receptors [29-31]. Interestingly, the oxytocin-induced antinociceptive effect to both thermal and mechanical stimulation were blocked significantly by intracerebroventricularadministration of 10nmol of β-FNA or nor-BNI, the antagonist of mu- and kappa-opioid receptors, but not the antagonist of delta-opioid receptors naltrindole (Fig. 3), indicating that mu- and kappa-opioid receptors, not delta0opioid receptor, are involved in the oxytocin-induced antinociception in the brain.
The hot-plate and the Randall Sellito test were used to assess the effect of antinociception in the present study. According to our results, it seems that the oxytocin-induced increase of HWLs to thermal stimulation was more significant than that to mechanical stimulation. Whether oxytocin has more effect on thermal-sensor than pressure-sensor remains to be determined.
Taken together, the present study demonstrated that oxytocin plays an antinociceptive role in the brain of rats, mu- and kappa-opioid receptors, not delta-receptors, are involved in the oxytocin-induced antinociception in the central nervous system of rats.
Acknowledgements
This study was supported by the President's Undergraduate Research Fellowship (PURF),Peking University and funds from the National Natural Science Foundation of China (NSFC). Thanks to my advisor, Professor Yu, and all my workmates in my laboratory.
References
[1]Raggenbass M. Vasopressin- and oxytocin-induced activity in the central nervous system: electrophysiological studies using in-vitro systems. Prog Neurobiol 2001;64:307-26.
[2]Agren G, Uvnas-Moberg K, Lundeberg T. Olfactory cues from anoxytocin-injected male rat can induce antinociception in its cage-mates. Neuroreport 1997;8:3073–6.
[3]Arletti R, Benelli A, Bertolini A. Influence of oxytocin on nociception and morphine antinociception. Neuropeptides 1993;24:125-9.
[4]Caron RW, Leng G, Ludwig M, Russell JA. Naloxone-inducedsupersensitivity of oxytocin neurones to opioid antagonists. Neuropharmacology 1998;37:887–97.
[5]Ge Y, Lundeberg T, Yu LC. Blockade effect of mu and kappa opioid antagonists on theantinociception induced by intra-periaqueductal grey injection ofoxytocin in rats. Brain Res 2002;927:204–7.
[6]Robinson DA, Wei F, Wang GD, Li P, Kim SJ, Vogt SK, Muglia LJ, Zhuo M.Oxytocin mediates stress-induced analgesia in adult mice.J Physiol 2002;540:593-606.
[7]Lund I, Yu LC, Uvnas-Moberg K, Wang J, Yu C, Kurosawa M, Agren G, RosenA,Lekman M, Lundeberg T.Repeated massage-like stimulation induces long-term effects on nociception:contribution of oxytocinergic mechanisms.Eur J Neurosci 2002;16:330-8.
[8]Millan MJ, Descending control of pain. ProgNeurobiol 2002;66:355-474.
[9]Lundeberg T,Uvnasmoberg K, Agren G, Bruzelius G. Antinociceptive effects of oxytocin in rats and mice. Neurosci Lett 1994;170:153-7.
[10]Madrazo I, Franco-Bourland RE, Leon-Meza VM, Mena I. Intraventricular somatostatin-14, arginine casopressin, and oxytocin: analgesic effect in a patient with intractable cancer pain. Appl Neurophysiol 1987;50:427-31.
[11]Liu ZJ, Zhuang DB, Lundeberg T, Yu LC. Involvement of 5-hydroxytryptamine1A receptors in the descending anti-nociceptive pathway from periaqueductal grey to the spinal dorsal horn in intact rats, rats with mononeuropathy and rats with inflammation. Neuroscience 2002;112:399-407.
[12]Wang JW, Lundeberg T, Yu LC. Antinociceptive role of oxytocin in the nucleus raphe magnus of rats, an involvement of mu opioid receptor in the effect. Regulatory Peptides 2003;115(3):153-9.
[13]Yaksh TL, Rudy TA. Narcotic analgesics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques. Pain 1978;4:299-359.
[14]Adams JU, Tallarida RJ, Geller EB, Adler MW. Isobolographic superaddictivity between delta- and mu-opioid agonists in rat depends on the ratio of compounds, the mu-agonist and the analgesic assay used. J Pharmacol Exp Ther 1993;266:1261-7.
[15]Miaskowski C, Taiwo YO, Levine JD. Contribution of supraspinal mu- and delta-opioid receptors to antinociception in the rat. Eur J Pharmacol 1991;205:247-52.
[16]Dauge V, Petil F, Rossignol P, Roques BP. Use of mu- and delta-opioid peptides of various selectivity gives further evidence of specific involvement of mu-opioid receptors in supraspinal analgesia (tail-flick test). Pain 1987;14:171-8.
[17]Porreca F, Mosberg HI, Hurst R, Hruby VJ, Burks TF. Role of mu- delta- and kappa-opioid receptors in the spinal and supraspinal mediation of gastrointestinal transit effects and hot-plate analgesia in the mouse. J Pharmacol Exp Ther 1984;230:341-8.
[18]Heyman JS, Mulvaney SA, Mosberg HI, Porreca F. Opioid delta-receptor involvement in supraspinal and spinal antinociception in mice. Brain Res 1987;100-8.
[19]Jiang Q, Mosberg HI, Porreca F. Antinociceptive effects of [D-Ala2]deltophin II, a high selective delta-agonist in vivo. Life Sci 1990;47:PL43-7.
[20]Yu SQ, Lundeberg T, Yu LC. Involvement of oxytocin in spinal antinociception in rats withinflammation. Neuroscience 2003;983:13-22.
[21]Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animal. Pain 1983;16:109-10.
[22]Yu LC, Hansson P, Lundeberg T. The calcitonin gene-relatedpeptide antagonist CGRP8-37 increases the latency to withdrawalresponses bilaterally in rats with experimental mononeuropathy, aneffect partly reversed by naloxone. Neuroscience 1996;71:523–31.
[23]Yu LC, Lundeberg S, An H, Wang FX,Lundeberg T. Effects of intrathecal galanin on nociceptive responses in rats with mononeuropathy. Life Sci 1999;64:1145-53.
[24]Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates, 3rd Edition, Academic Press, Sydney, 1998.
[25]Gimpl G, Fahrenholz F. The Oxytocin Receptor System: Structure, Function, and Regulation. Physiological Reviews 2001;2:629-83.
[26]Poulain DA, Wakerley JB. Electrophysiology of hypothalamic magnocellular neurons secreting oxytocin and vasopressin. Neuroscience 1982;7:773-808.
[27]Fields HL, Basbaum AI. Central nervous system mechanismsof pain modulation. In: Wall, P.D., Melzack, R. (Eds.), Textbook ofPain, 4th Edition. Churchill Livingston, Edinburgh, 1999, pp. 309–29.