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Receptors involved in the antinociception of intrathecal melatonin in formalin test of rats
Neuroscience Letters 494 (2011) 207–210
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Receptors involved in the antinociception of intrathecal melatonin in formalin test of rats Dong Jin Shin a , Chul Won Jeong a , Seong Heon Lee a , Myung Ha Yoon a,b,c,∗ a b c
Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, 8 Hakdong, Donggu, Gwangju 501-757, Republic of Korea Brain Korea 21 Project, Center for Biomedical Human Resources at Chonnam National University, 8 Hakdong, Donggu, Gwangju 501-757, Republic of Korea Chonnam National University, Research Institute of Medical Sciences, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 December 2010 Received in revised form 3 March 2011 Accepted 3 March 2011 Keywords: Adrenergic or cholinergic receptors Antinociception Formalin test Melatonin Spinal cord Sprague–Dawley rat
a b s t r a c t The authors examined the antinocicepotive effect of melatonin in a nociceptive state and investigated a possible interaction with adrenergic or cholinergic receptors underlying this effect at the spinal level. Nociception was induced by a subcutaneous injection of 50 l of a 5% formalin solution to the hindpaw of male Sprague–Dawley rats. The reversal effects of alpha-1 adrenoceptor antagonist (prazosin), alpha-2 adrenoceptor antagonist (yohimbine), muscarinic receptor antagonist (atropine) and nicotinic receptor antagonist (mecamylamine) on the activity of melatonin were assessed. Intrathecal melatonin reduced the flinching response during phase 1 and phase 2 in the formalin test. Intrathecal prazosin, yohimbine, atropine and mecamylamine increased the attenuating flinching response in both phases observed by intrathecal melatonin. Collectively, the present data suggest that intrathecal melatonin attenuates the facilitated state and acute pain evoked by formalin injection. Furthermore, the antinociception of melatonin is mediated through the alpha-1 adrenoceptor, alpha-2 adrenoceptor, muscarinic and nicotinic receptors in the spinal cord. © 2011 Elsevier Ireland Ltd. All rights reserved.
The pineal gland synthesizes and secrets melatonin (N-acetyl5-methoxytryptamine) into the bloodstream [25]. Melatonin is an indolamine that is derived from the amino acid precursor l-tryptophan and plays an important role in the regulation of physiological functions, including sleep and circadian rhythms [14,25]. Previous studies suggest that melatonin also has an antinociceptive property, especially at the spinal level [1,29]. Although melatonin receptors are primarily responsible for the activity of melatonin at the spinal level, the contribution of other receptors by which melatonin induces antinociception has not yet clarified [20]. The activities of adrenergic and cholinergic receptors involved in nociceptive modulation have been studied extensively [5,8,9]. Both clonidine, an alpha-2 adrenoceptor antagonist, and neostigmine, an anticholinesterase, have an antinociceptive effect [4,28,30]. The antinociceptive effect of intraperitoneal and intracerebroventricular melatonin was antagonized by an alpha-2 adrenoceptor antagonist, but was not affected by an alpha-1 adrenoceptor antagonist [13]. In contrast, the effect of intrathecal melatonin was antagonized by an alpha-1 adrenoceptor antagonist [29]. These findings suggest a link between adrenergic receptors and the activ-
∗ Corresponding author at: Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, 8 Hakdong, Donggu, Gwangju 501757, Republic of Korea. Tel.: +82 62 220 6893; fax: +82 62 232 6294. E-mail addresses: [email protected], [email protected] (M.H. Yoon). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.014
ity of melatonin. The role that cholinergic receptors in the spinal cord play in the antinociceptive effect of melatonin has not been determined. The authors investigated the antinociceptive effect of intrathecal melatonin in a rat behavioral model of nociception and clarified the role of adrenergic and cholinergic receptors on the activity of melatonin at the spinal level. All experiments were approved by the Institutional Animal Care and Use Committee of Chonnam National University. Adult male Sprague–Dawley rats (weighing 250–300 g) were housed in a standard animal facility (22 ± 0.5 ◦ C with a 12-h light/dark cycle) and allowed free access to food and water. An intrathecal catheter was implanted after the rats were anesthetized with sevoflurane [27]. A polyethylene-10 catheter was inserted into the intrathecal space through an incision in the atlantooccipital membrane. The catheter tip was located at the lumbar enlargement of the spinal cord. The external portion of the catheter was tunneled subcutaneously and exited from the top of the head. The catheter was capped with 28-gauge stainless steel wire, and the skin was sutured with 3-0 silk. Rats that showed neurologic deficits following intrathecal catheterization were euthanized immediately with an overdose of an inhalant anesthetic. The behavioral component of the study began 5 days after the intrathecal catheterization was performed. The drugs used in the study were as follows: melatonin (Nacetyl-5-methoxytryptamine, Sigma–Aldrich Co., St. Louis, MO,
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USA), prazosin (Tocris Cookson Ltd., Bristol, Avon, UK), yohimbine (Tocris), atropine (Sigma–Aldrich), and mecamylamine (Tocris). Melatonin and prazosin were dissolved in dimethylsulfoxide (DMSO). Yohimbine, atropine, and mecamylamine were dissolved in distilled water. All drugs were administered intrathecally by a hand-driven, gear-operated syringe pump, using a total volume of 10 l. The catheter was flushed with 10 l of normal saline immediately after drug administration. The formalin test was used to examine nociceptive behavior [29]. The formalin test involved administering a 50-l subcutaneous injection of 5% formalin solution into the plantar surface of the hind paw of each rat. After the formalin injection, the rats were observed for a flinching response, which is defined as rapid and brief withdrawal of the affected paw. The flinching response was periodically counted in order to quantify the nociceptive response. The flinching response was counted for 1-min periods at 1 and 5 min post-injection and at 5-min intervals from 10 to 60 min post-injection. As previously described [29], the flinching response had two distinct phases. Therefore, the flinching response data were examined separately for phase 1 (0–9 min) and phase 2 (10–60 min). Before each test, the rats were placed in a restraint cylinder and allowed to adapt to the apparatus for 20 min. The experiments were carried out between 13:00 and 17:00 h. Intrathecal DMSO or distilled water was used as a control agent, and each rat was tested only once. After the 1-h observation period, the rats were euthanized with an overdose of an inhalant anesthetic. During the initial phase of the experiment, the antinociceptive effect of intrathecal melatonin (3, 10, and 30 g; n = 27) was examined using the formalin test. Melatonin was administered intrathecally 10 min prior to the formalin injection, and the flinching response was evaluated. In the second phase of the experiment, the contribution of spinal adrenergic and cholinergic receptors to the effect of melatonin was determined. Rats were pretreated with different adrenergic and cholinergic receptor antagonists administered intrathecally 10 min prior to the administration of intrathecal melatonin (30 g). The formalin test was performed 10 min later. Prazosin (3 g), yohimbine (10 g), atropine (10 g), and mecamylamine (10 g) were used as antagonists of alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors, respectively (n = 6 for each group). The dose of each antagonist was based on a previous study [10]. In a separate experiment (n = 5), the effects of intrathecal melatonin on general behaviors such as the consciousness and motor functions were observed for 60 min after intrathecal administration of melatonin (30 g). All data are expressed as the mean ± S.E.M. The time–response data and dose–response data of the formalin test are presented as the number of flinching responses or as the percentage of control in each phase. The number of flinching responses was converted to percentage of control as follows:
is shown in Fig. 1A. Intrathecal melatonin dose-dependently suppressed the flinching response during phase 1 (Fig. 1B) and phase 2 (Fig. 1C) in the formalin test. Intrathecal prazosin (alpha-1 adrenoceptor antagonist), yohimbine (alpha-2 adrenoceptor antagonist), atropine (muscarinic receptor antagonist), and mecamylamine (nicotinic receptor antagonist) had no statistically significant effect on the flinching response in control rats after formalin injection. However, all four antagonists increased the flinching response as a percentage of control in both phases of the formalin test as compared with melatonin alone (Figs. 2 and 3). Neither the consciousness nor motor functions were affected after intrathecal melatonin.
% of control =
Total flinching response number with drug in phase 1 (2) Total flinching response number of control in phase 1 (2) × 100
The dose–response data were analyzed by one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. An unpaired t-test was used to compare the antagonism of the melatonin effect. Statistical significance was considered at P < 0.05. The subcutaneous injection of formalin produced a reliable flinching response of the injected hind paw, in a biphasic pattern (Fig. 1). The time course of the flinching response when intrathecal melatonin was administered 10 min prior to the formalin injection
Fig. 1. Time (A) and dose–response curves (B and C) for flinching responses in the formalin test performed after intrathecal melatonin (M) administration. M was administered intrathecally at T = −10 min and formalin (F) was injected subcutaneously at T = 0. Data are presented as the number of flinching response or as a percentage of control. Each line represents the mean ± S.E.M. of 6–7 rats. Intrathecal melatonin resulted in a dose-dependent suppression of the flinching response during phase 1 and phase 2 in the formalin test. *P < 0.01, compared with control.
D.J. Shin et al. / Neuroscience Letters 494 (2011) 207–210
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Fig. 2. The effects of intrathecal prazosin (3 g) and yohimbine (10 g) on the antinociceptive action of intrathecal melatonin (30 g) in the formalin test. One of the two antagonists and melatonin were administered 20 and 10 min before the formalin test, respectively. Data are presented as a percentage of control. Both intrathecal prazosin and yohimbine reversed the effect of melatonin during phase 1 (A) and phase 2 (B) in the formalin test. Each bar represents the mean ± S.E.M. of 6 rats. *P < 0.05, † P < 0.01, compared with melatonin.
Fig. 3. The effects of intrathecal atropine (10 g) and mecamylamine (10 g) on the antinociceptive action of intrathecal melatonin (30 g) in the formalin test. One of the two antagonists and melatonin were administered 20 and 10 min before the formalin test, respectively. Data are presented as a percentage of control. Both intrathecal atropine and mecamylamine reversed the effect of melatonin during phase 1 (A) and phase 2 (B) in the formalin test. Each bar represents the mean ± S.E.M. of 6 rats. *P < 0.05, † P < 0.01, compared with melatonin.
The biphasic pattern of the flinching response during the formalin test implies that the response mechanism is different in each phase. The response during phase 1 is the result of direct activation of primary afferent nerve fibers. However, the response during phase 2 appears to result from the activation of a wider range of neurons with a continuously low level of activity in the primary afferent neurons. Therefore, phase 1 reflects acute pain and phase 2 reflects the facilitated state. In this study, intrathecal administration of melatonin inhibited the flinching response during phases 1 and 2 in the formalin test. This suggests that melatonin may be effective for the management of nociception in the facilitated state as well as acute pain at the spinal level. Melatonin plays a pivotal role in the processing of nociception in the spinal cord. The intrathecal administration of melatonin decreased formalin-induced nociception [29]. Furthermore, melatonin depressed nociceptive transmission and synaptic potentiation inhibition in the spinal cord [10,18]. Autoradiographic studies demonstrated the presence of melatonin receptors in the spinal cord [20]. Therefore, it could be assumed that melatonin exerts antinociceptive effects through melatonin receptors in the spinal cord. On the other hand, the present study showed that intrathecal administration of adrenergic and cholinergic antagonists reversed the antinociceptive effect of intrathecal melatonin. These observations suggest that alpha-1 adrenergic, alpha-2 adrenergic,
muscarinic, and nicotinic receptors are involved in the activity of melatonin at the spinal level. Several lines of evidence have shown melatonin-induced antinociception and the role of adrenergic and cholinergic receptors on the antinociceptive action of some drugs. The antinociceptive effect of intraperitoneal melatonin was reversed by pretreatment with intraperitoneal alpha-2 adrenoceptor antagonist; however, no effect on melatonin was seen with alpha-1 adrenoceptor antagonist administration [13]. In contrast to intraperitoneal alpha-1 adrenoceptors, alpha-1 adrenoceptors in the spinal cord are involved in the modulation of nociception [2,3,6]. Immunohistochemical and in situ hybridization studies indicate that alpha-2 adrenoceptors and muscarinic receptors exist in lamina I and II of the dorsal horn of the spinal cord, which is an important area in the modulation of nociception [22,24,26]. Electrical stimulation of the brain produced an antinociceptive effect via the activation of muscarinic receptors, alpha-1 adrenoceptors, and alpha-2 adrenoceptors in the spinal cord; nicotinic receptors were not involved [5]. However, melatonin inhibited nicotinic currents from cultured rat cerebellar granule neurons in a dose-dependent manner, which suggests that melatonin may directly modulate nicotinic receptors [12]. Furthermore, spinally administered epibatidine, a nicotinic receptor agonist, diminished arthritic, thermal, and formalin-induced nociceptive behaviors [11,16,17]. Nicotinic binding sites were also found in the dorsal horn of the spinal cord [7]. In a previous study, intrathecal melatonin interacted additively
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with clonidine (an alpha-2 adrenoceptor antagonist) and neostigmine (an anticholinesterase) in the formalin test [29]. This indicates that the mechanism of action of melatonin is linked to the actions of clonidine and neostigmine. These findings suggest that melatonin produces antinociception by a mechanism mediated via alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors in the spinal cord. However, the mechanisms of melatonin inhibition of alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors have not been clearly determined. One possible explanation would be the second messenger system cyclic guanosine monophosphate (cGMP) [15]. Melatonin may activate guanylyl cyclase, which may lead to an increase of cGMP levels [19]. Furthermore, cholinergic receptor would be responsible for the cGMP-mediated antinociception [21] and alpha-2 adrenergic agonist induced the antinociceptive effect of through cGMP system [23]. Therefore, it is conceivable that the increased cGMP by intrathecal melatonin may act on alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors, thereby producing the antinociception. Another importance in the formalin test is that the phase 1 response may affect the phase 2 response. Thus, it is assumed that the suppression of the phase 1 response may result in the attenuation of the phase 2 response. Such explanation suggests that the antinociceptive effect of melatonin during phase 2 may be originated from the phase 1 response rather than the direct action on adrenergic or cholinergic receptors mediating neurotransmitter release. Taken together, intrathecal melatonin suppressed the facilitated state as well as the acute pain evoked by formalin injection. And, intrathecal melatonin did not cause change of the consciousness or motor functions. Then, alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors are involved in the activity of melatonin at the spinal level. Advancements in the understanding of adrenergic and cholinergic modulation of nociception in the spinal cord may lead to the development of therapeutically useful analgesics such as melatonin. References [1] M. Ambriz-Tututi, V. Granados-Soto, Oral and spinal melatonin reduces tactile allodynia in rats via activation of MT2 and opioid receptors, Pain 132 (2007) 273–280. [2] H. Baba, P.A. Goldstein, M. Okamoto, T. Kohno, T. Ataka, M. Yoshimura, K. Shimoji, Norepinephrine facilitates inhibitory transmission in substantia gelatinosa of adult rat spinal cord (part 2): effects on somatodendritic sites of GABAergic neurons, Anesthesiology 92 (2000) 485–492. [3] H. Baba, K. Shimoji, M. Yoshimura, Norepinephrine facilitates inhibitory transmission in substantia gelatinosa of adult rat spinal cord (part1): effects on axon terminals of GABAergic and glycinergic neurons, Anesthesiology 92 (2000) 473–484. [4] S.R. Chen, H.L. Pan, Spinal GABAB receptors mediate antinociceptive actions of cholinergic agents in normal and diabetic rats, Brain Res. 965 (2003) 67–74. [5] Q.M. Dias, S.F. Crespilho, J.W. Silveira, W.A. Prado, Muscarinic and alpha(1)adrenergic mechanisms contribute to the spinal mediation of stimulationinduced antinociception from the pedunculopontine tegmental nucleus in the rat, Pharmacol. Biochem. Behav. 92 (2009) 488–494.
[6] Z.J. Ge, Y.M. Zeng, Y.F. Tan, Effects of intrathecal 6-hydroxydopamine, ␣1 and ␣2 adrenergic receptor antagonists on antinociception of propofol in mice, Acta Pharmacol. Sin. 26 (2005) 186–191. [7] P.G. Gillberg, R. d’Argy, S.M. Aquilonius, Autoradiographic distribution of [3 H] acetylcholine binding sites in the cervical spinal cord of man and some other species, Neurosci. Lett. 90 (1988) 197–202. [8] M.J. Khodayar, B. Shafaghi, N. Naderi, M.R. Zarrindast, Antinociceptive effect of spinally administered cannabinergic and 2-adrenoceptor drugs on the formalin test in rat: possible interactions, J. Psychopharmacol. 20 (2006) 67–74. [9] S.Y. Kim, M.H. Yoon, H.G. Lee, W.M. Kim, J.D. Lee, Y.O. Kim, L.J. Huang, J.H. Cui, The role of adrenergic and cholinergic receptors on the antinociception of korean red ginseng in the spinal cord of rats, Korean J. Pain 21 (2008) 27–32. [10] C. Laurido, T. Pelissier, R. Soto-Moyano, L. Valladares, F. Flores, A. Hernandez, Effect of melatonin on rat spinal cord nociceptive transmission, Neuroreport 13 (2002) 89–91. [11] N.B. Lawand, Y. Lu, K.N. Westlund, Nicotinic cholinergic receptors: potential targets for inflammatory pain relief, Pain 80 (1999) 291–299. [12] P. Lax, Melatonin inhibits nicotinic currents in cultured rat cerebellar granule neurons, J. Pineal Res. 44 (2008) 70–77. [13] M. Mantovani, M.P. Kaster, R. Pertile, J.B. Calixto, A.L. Rodrigues, A.R. Santos, Mechanisms involved in the antinociception caused by melatonin in mice, J. Pineal Res. 41 (2006) 382–389. [14] P.J. Morgan, P. Barrett, H.E. Howell, R. Helliwell, Melatonin receptors: localization, molecular pharmacology and physiological significance, Neurochem. Int. 24 (1994) 101–146. [15] D.C New, S.T. Tsim, Y.H. Wong, G protein-linked effector and second messenger systems involved in melatonin signal transduction, Neurosignals 12 (2003) 59–70. [16] T. Nishiyama, Interaction between midazolam and epibatidine in spinally mediated antinociception in rats, J. Anesth. 23 (2009) 370–377. [17] T. Nishiyama, L. Gyermek, M.L. Trudell, K. Hanaoka, Spinally mediated analgesia and receptor binding affinity of epibatidine analogs, Eur. J. Pharmacol. 470 (2003) 27–31. [18] R. Noseda, A. Hernández, L. Valladares, M. Mondaca, C. Laurido, R. Soto-Moyano, Melatonin-induced inhibition of spinal cord synaptic potentiation in rats is MT2 receptor-dependent, Neurosci. Lett. 360 (2004) 41–44. [19] J. Olcese, C. Majora, A. Stephan, D. Müller, Nocturnal accumulation of cyclic 3 ,5 guanosine monophosphate (cGMP) in the chick pineal organ is dependent on activation of guanylyl cyclase-B, J. Neuroendocrinol. 14 (2002) 14–18. [20] S.F. Pang, Q. Wan, G.M. Brown, Melatonin receptors in the spinal cord, Biol. Signals 6 (1997) 272–283. [21] C.S. Patil, N.K. Jain, V.P. Singh, S.K. Kulkarni, Cholinergic-NO-cGMP mediation of sildenafil-induced antinociception, Indian J. Exp. Biol. 42 (2004) 361– 367. [22] A. Pertovaara, Noradrenergic pain modulation, Prog. Neurobiol. 80 (2006) 53–83. [23] T.R. Romero, I.D. Duarte, alpha(2)-Adrenoceptor agonist xylazine induces peripheral antinociceptive effect by activation of the l-arginine/nitric oxide/cyclic GMP pathway in rat, Eur. J. Pharmacol. 613 (2009) 64–67. [24] T.J. Shi, U. Winzer-Serhan, F. Leslie, T. Hökfelt, Distribution of alpha2adrenoceptor mRNAs in the rat lumbar spinal cord in normal and axotomized rats, Neuroreport 10 (1999) 2835–2839. [25] J. Vanecek, Cellular mechanisms of melatonin action, Physiol. Rev. 78 (1998) 687–721. [26] J.W. Villiger, R.L. Faull, Muscarinic cholinergic receptors in the human spinal cord: differential localization of [3 H] pirenzepine and [3 H] quinuclidinylbenzilate binding sites, Brain Res. 345 (1985) 196–199. [27] T.L. Yaksh, T.A. Rudy, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. 17 (1976) 1031–1036. [28] M.H. Yoon, J.I. Choi, Pharmacologic interaction between cannabinoid and either clonidine or neostigmine in the rat formalin test, Anesthesiology 99 (2003) 701–707. [29] M.H. Yoon, H.C. Park, W.M. Kim, H.G. Lee, Y.O. Kim, L.J. Huang, Evaluation for the interaction between intrathecal melatonin and clonidine or neostigmine on formalin-induced nociception, Life Sci. 83 (2008) 845–850. [30] W. Zeng, X. Chen, S. Dohi, Antinociceptive synergistic interaction between clonidine and ouabain on thermal nociceptive tests in the rat, J. Pain 8 (2007) 983–988.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Receptors involved in the antinociception of intrathecal melatonin in formalin test of rats Dong Jin Shin a , Chul Won Jeong a , Seong Heon Lee a , Myung Ha Yoon a,b,c,∗ a b c
Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, 8 Hakdong, Donggu, Gwangju 501-757, Republic of Korea Brain Korea 21 Project, Center for Biomedical Human Resources at Chonnam National University, 8 Hakdong, Donggu, Gwangju 501-757, Republic of Korea Chonnam National University, Research Institute of Medical Sciences, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 December 2010 Received in revised form 3 March 2011 Accepted 3 March 2011 Keywords: Adrenergic or cholinergic receptors Antinociception Formalin test Melatonin Spinal cord Sprague–Dawley rat
a b s t r a c t The authors examined the antinocicepotive effect of melatonin in a nociceptive state and investigated a possible interaction with adrenergic or cholinergic receptors underlying this effect at the spinal level. Nociception was induced by a subcutaneous injection of 50 l of a 5% formalin solution to the hindpaw of male Sprague–Dawley rats. The reversal effects of alpha-1 adrenoceptor antagonist (prazosin), alpha-2 adrenoceptor antagonist (yohimbine), muscarinic receptor antagonist (atropine) and nicotinic receptor antagonist (mecamylamine) on the activity of melatonin were assessed. Intrathecal melatonin reduced the flinching response during phase 1 and phase 2 in the formalin test. Intrathecal prazosin, yohimbine, atropine and mecamylamine increased the attenuating flinching response in both phases observed by intrathecal melatonin. Collectively, the present data suggest that intrathecal melatonin attenuates the facilitated state and acute pain evoked by formalin injection. Furthermore, the antinociception of melatonin is mediated through the alpha-1 adrenoceptor, alpha-2 adrenoceptor, muscarinic and nicotinic receptors in the spinal cord. © 2011 Elsevier Ireland Ltd. All rights reserved.
The pineal gland synthesizes and secrets melatonin (N-acetyl5-methoxytryptamine) into the bloodstream [25]. Melatonin is an indolamine that is derived from the amino acid precursor l-tryptophan and plays an important role in the regulation of physiological functions, including sleep and circadian rhythms [14,25]. Previous studies suggest that melatonin also has an antinociceptive property, especially at the spinal level [1,29]. Although melatonin receptors are primarily responsible for the activity of melatonin at the spinal level, the contribution of other receptors by which melatonin induces antinociception has not yet clarified [20]. The activities of adrenergic and cholinergic receptors involved in nociceptive modulation have been studied extensively [5,8,9]. Both clonidine, an alpha-2 adrenoceptor antagonist, and neostigmine, an anticholinesterase, have an antinociceptive effect [4,28,30]. The antinociceptive effect of intraperitoneal and intracerebroventricular melatonin was antagonized by an alpha-2 adrenoceptor antagonist, but was not affected by an alpha-1 adrenoceptor antagonist [13]. In contrast, the effect of intrathecal melatonin was antagonized by an alpha-1 adrenoceptor antagonist [29]. These findings suggest a link between adrenergic receptors and the activ-
∗ Corresponding author at: Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, 8 Hakdong, Donggu, Gwangju 501757, Republic of Korea. Tel.: +82 62 220 6893; fax: +82 62 232 6294. E-mail addresses: [email protected], [email protected] (M.H. Yoon). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.014
ity of melatonin. The role that cholinergic receptors in the spinal cord play in the antinociceptive effect of melatonin has not been determined. The authors investigated the antinociceptive effect of intrathecal melatonin in a rat behavioral model of nociception and clarified the role of adrenergic and cholinergic receptors on the activity of melatonin at the spinal level. All experiments were approved by the Institutional Animal Care and Use Committee of Chonnam National University. Adult male Sprague–Dawley rats (weighing 250–300 g) were housed in a standard animal facility (22 ± 0.5 ◦ C with a 12-h light/dark cycle) and allowed free access to food and water. An intrathecal catheter was implanted after the rats were anesthetized with sevoflurane [27]. A polyethylene-10 catheter was inserted into the intrathecal space through an incision in the atlantooccipital membrane. The catheter tip was located at the lumbar enlargement of the spinal cord. The external portion of the catheter was tunneled subcutaneously and exited from the top of the head. The catheter was capped with 28-gauge stainless steel wire, and the skin was sutured with 3-0 silk. Rats that showed neurologic deficits following intrathecal catheterization were euthanized immediately with an overdose of an inhalant anesthetic. The behavioral component of the study began 5 days after the intrathecal catheterization was performed. The drugs used in the study were as follows: melatonin (Nacetyl-5-methoxytryptamine, Sigma–Aldrich Co., St. Louis, MO,
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D.J. Shin et al. / Neuroscience Letters 494 (2011) 207–210
USA), prazosin (Tocris Cookson Ltd., Bristol, Avon, UK), yohimbine (Tocris), atropine (Sigma–Aldrich), and mecamylamine (Tocris). Melatonin and prazosin were dissolved in dimethylsulfoxide (DMSO). Yohimbine, atropine, and mecamylamine were dissolved in distilled water. All drugs were administered intrathecally by a hand-driven, gear-operated syringe pump, using a total volume of 10 l. The catheter was flushed with 10 l of normal saline immediately after drug administration. The formalin test was used to examine nociceptive behavior [29]. The formalin test involved administering a 50-l subcutaneous injection of 5% formalin solution into the plantar surface of the hind paw of each rat. After the formalin injection, the rats were observed for a flinching response, which is defined as rapid and brief withdrawal of the affected paw. The flinching response was periodically counted in order to quantify the nociceptive response. The flinching response was counted for 1-min periods at 1 and 5 min post-injection and at 5-min intervals from 10 to 60 min post-injection. As previously described [29], the flinching response had two distinct phases. Therefore, the flinching response data were examined separately for phase 1 (0–9 min) and phase 2 (10–60 min). Before each test, the rats were placed in a restraint cylinder and allowed to adapt to the apparatus for 20 min. The experiments were carried out between 13:00 and 17:00 h. Intrathecal DMSO or distilled water was used as a control agent, and each rat was tested only once. After the 1-h observation period, the rats were euthanized with an overdose of an inhalant anesthetic. During the initial phase of the experiment, the antinociceptive effect of intrathecal melatonin (3, 10, and 30 g; n = 27) was examined using the formalin test. Melatonin was administered intrathecally 10 min prior to the formalin injection, and the flinching response was evaluated. In the second phase of the experiment, the contribution of spinal adrenergic and cholinergic receptors to the effect of melatonin was determined. Rats were pretreated with different adrenergic and cholinergic receptor antagonists administered intrathecally 10 min prior to the administration of intrathecal melatonin (30 g). The formalin test was performed 10 min later. Prazosin (3 g), yohimbine (10 g), atropine (10 g), and mecamylamine (10 g) were used as antagonists of alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors, respectively (n = 6 for each group). The dose of each antagonist was based on a previous study [10]. In a separate experiment (n = 5), the effects of intrathecal melatonin on general behaviors such as the consciousness and motor functions were observed for 60 min after intrathecal administration of melatonin (30 g). All data are expressed as the mean ± S.E.M. The time–response data and dose–response data of the formalin test are presented as the number of flinching responses or as the percentage of control in each phase. The number of flinching responses was converted to percentage of control as follows:
is shown in Fig. 1A. Intrathecal melatonin dose-dependently suppressed the flinching response during phase 1 (Fig. 1B) and phase 2 (Fig. 1C) in the formalin test. Intrathecal prazosin (alpha-1 adrenoceptor antagonist), yohimbine (alpha-2 adrenoceptor antagonist), atropine (muscarinic receptor antagonist), and mecamylamine (nicotinic receptor antagonist) had no statistically significant effect on the flinching response in control rats after formalin injection. However, all four antagonists increased the flinching response as a percentage of control in both phases of the formalin test as compared with melatonin alone (Figs. 2 and 3). Neither the consciousness nor motor functions were affected after intrathecal melatonin.
% of control =
Total flinching response number with drug in phase 1 (2) Total flinching response number of control in phase 1 (2) × 100
The dose–response data were analyzed by one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. An unpaired t-test was used to compare the antagonism of the melatonin effect. Statistical significance was considered at P < 0.05. The subcutaneous injection of formalin produced a reliable flinching response of the injected hind paw, in a biphasic pattern (Fig. 1). The time course of the flinching response when intrathecal melatonin was administered 10 min prior to the formalin injection
Fig. 1. Time (A) and dose–response curves (B and C) for flinching responses in the formalin test performed after intrathecal melatonin (M) administration. M was administered intrathecally at T = −10 min and formalin (F) was injected subcutaneously at T = 0. Data are presented as the number of flinching response or as a percentage of control. Each line represents the mean ± S.E.M. of 6–7 rats. Intrathecal melatonin resulted in a dose-dependent suppression of the flinching response during phase 1 and phase 2 in the formalin test. *P < 0.01, compared with control.
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Fig. 2. The effects of intrathecal prazosin (3 g) and yohimbine (10 g) on the antinociceptive action of intrathecal melatonin (30 g) in the formalin test. One of the two antagonists and melatonin were administered 20 and 10 min before the formalin test, respectively. Data are presented as a percentage of control. Both intrathecal prazosin and yohimbine reversed the effect of melatonin during phase 1 (A) and phase 2 (B) in the formalin test. Each bar represents the mean ± S.E.M. of 6 rats. *P < 0.05, † P < 0.01, compared with melatonin.
Fig. 3. The effects of intrathecal atropine (10 g) and mecamylamine (10 g) on the antinociceptive action of intrathecal melatonin (30 g) in the formalin test. One of the two antagonists and melatonin were administered 20 and 10 min before the formalin test, respectively. Data are presented as a percentage of control. Both intrathecal atropine and mecamylamine reversed the effect of melatonin during phase 1 (A) and phase 2 (B) in the formalin test. Each bar represents the mean ± S.E.M. of 6 rats. *P < 0.05, † P < 0.01, compared with melatonin.
The biphasic pattern of the flinching response during the formalin test implies that the response mechanism is different in each phase. The response during phase 1 is the result of direct activation of primary afferent nerve fibers. However, the response during phase 2 appears to result from the activation of a wider range of neurons with a continuously low level of activity in the primary afferent neurons. Therefore, phase 1 reflects acute pain and phase 2 reflects the facilitated state. In this study, intrathecal administration of melatonin inhibited the flinching response during phases 1 and 2 in the formalin test. This suggests that melatonin may be effective for the management of nociception in the facilitated state as well as acute pain at the spinal level. Melatonin plays a pivotal role in the processing of nociception in the spinal cord. The intrathecal administration of melatonin decreased formalin-induced nociception [29]. Furthermore, melatonin depressed nociceptive transmission and synaptic potentiation inhibition in the spinal cord [10,18]. Autoradiographic studies demonstrated the presence of melatonin receptors in the spinal cord [20]. Therefore, it could be assumed that melatonin exerts antinociceptive effects through melatonin receptors in the spinal cord. On the other hand, the present study showed that intrathecal administration of adrenergic and cholinergic antagonists reversed the antinociceptive effect of intrathecal melatonin. These observations suggest that alpha-1 adrenergic, alpha-2 adrenergic,
muscarinic, and nicotinic receptors are involved in the activity of melatonin at the spinal level. Several lines of evidence have shown melatonin-induced antinociception and the role of adrenergic and cholinergic receptors on the antinociceptive action of some drugs. The antinociceptive effect of intraperitoneal melatonin was reversed by pretreatment with intraperitoneal alpha-2 adrenoceptor antagonist; however, no effect on melatonin was seen with alpha-1 adrenoceptor antagonist administration [13]. In contrast to intraperitoneal alpha-1 adrenoceptors, alpha-1 adrenoceptors in the spinal cord are involved in the modulation of nociception [2,3,6]. Immunohistochemical and in situ hybridization studies indicate that alpha-2 adrenoceptors and muscarinic receptors exist in lamina I and II of the dorsal horn of the spinal cord, which is an important area in the modulation of nociception [22,24,26]. Electrical stimulation of the brain produced an antinociceptive effect via the activation of muscarinic receptors, alpha-1 adrenoceptors, and alpha-2 adrenoceptors in the spinal cord; nicotinic receptors were not involved [5]. However, melatonin inhibited nicotinic currents from cultured rat cerebellar granule neurons in a dose-dependent manner, which suggests that melatonin may directly modulate nicotinic receptors [12]. Furthermore, spinally administered epibatidine, a nicotinic receptor agonist, diminished arthritic, thermal, and formalin-induced nociceptive behaviors [11,16,17]. Nicotinic binding sites were also found in the dorsal horn of the spinal cord [7]. In a previous study, intrathecal melatonin interacted additively
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with clonidine (an alpha-2 adrenoceptor antagonist) and neostigmine (an anticholinesterase) in the formalin test [29]. This indicates that the mechanism of action of melatonin is linked to the actions of clonidine and neostigmine. These findings suggest that melatonin produces antinociception by a mechanism mediated via alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors in the spinal cord. However, the mechanisms of melatonin inhibition of alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors have not been clearly determined. One possible explanation would be the second messenger system cyclic guanosine monophosphate (cGMP) [15]. Melatonin may activate guanylyl cyclase, which may lead to an increase of cGMP levels [19]. Furthermore, cholinergic receptor would be responsible for the cGMP-mediated antinociception [21] and alpha-2 adrenergic agonist induced the antinociceptive effect of through cGMP system [23]. Therefore, it is conceivable that the increased cGMP by intrathecal melatonin may act on alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors, thereby producing the antinociception. Another importance in the formalin test is that the phase 1 response may affect the phase 2 response. Thus, it is assumed that the suppression of the phase 1 response may result in the attenuation of the phase 2 response. Such explanation suggests that the antinociceptive effect of melatonin during phase 2 may be originated from the phase 1 response rather than the direct action on adrenergic or cholinergic receptors mediating neurotransmitter release. Taken together, intrathecal melatonin suppressed the facilitated state as well as the acute pain evoked by formalin injection. And, intrathecal melatonin did not cause change of the consciousness or motor functions. Then, alpha-1 adrenergic, alpha-2 adrenergic, muscarinic, and nicotinic receptors are involved in the activity of melatonin at the spinal level. Advancements in the understanding of adrenergic and cholinergic modulation of nociception in the spinal cord may lead to the development of therapeutically useful analgesics such as melatonin. References [1] M. Ambriz-Tututi, V. Granados-Soto, Oral and spinal melatonin reduces tactile allodynia in rats via activation of MT2 and opioid receptors, Pain 132 (2007) 273–280. [2] H. Baba, P.A. Goldstein, M. Okamoto, T. Kohno, T. Ataka, M. Yoshimura, K. Shimoji, Norepinephrine facilitates inhibitory transmission in substantia gelatinosa of adult rat spinal cord (part 2): effects on somatodendritic sites of GABAergic neurons, Anesthesiology 92 (2000) 485–492. [3] H. Baba, K. Shimoji, M. Yoshimura, Norepinephrine facilitates inhibitory transmission in substantia gelatinosa of adult rat spinal cord (part1): effects on axon terminals of GABAergic and glycinergic neurons, Anesthesiology 92 (2000) 473–484. [4] S.R. Chen, H.L. Pan, Spinal GABAB receptors mediate antinociceptive actions of cholinergic agents in normal and diabetic rats, Brain Res. 965 (2003) 67–74. [5] Q.M. Dias, S.F. Crespilho, J.W. Silveira, W.A. Prado, Muscarinic and alpha(1)adrenergic mechanisms contribute to the spinal mediation of stimulationinduced antinociception from the pedunculopontine tegmental nucleus in the rat, Pharmacol. Biochem. Behav. 92 (2009) 488–494.
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