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Involvement of M3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice
Brain Research 859 Ž2000. 38–44 www.elsevier.comrlocaterbres
Research report
Involvement of M 3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice Kenji Honda ) , Akitsugu Harada, Yukio Takano, Hiro-o Kamiya Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka UniÕersity, Fukuoka 814-0180, Japan Accepted 7 December 1999
Abstract Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase Ž1–5 min. and the second phase Ž7–30 min. after the injection. The muscarinic antagonist atropine Ž0.1–10 ng, i.t.. and the M 3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide Ž4-DAMP. Ž0.1–20 ng, i.t.. inhibited the second phase of this response, whereas higher doses of atropine Ž20–100 ng, i.t.. did not cause inhibition. The M 1 muscarinic receptor antagonist pirenzepine Ž10–100 ng, i.t.. did not inhibit either the first or the second phase response, but a high dose of pirenzepine Ž1000 ng, i.t.. tended to inhibit the second phase response. On the other hand, the M 2 muscarinic receptor antagonist 11-Ž2-wŽdiethylamino.methylx-1-piperidinyl4acetyl.-5,11-dihydro-6 HpyridoŽ2,3-b .Ž1,4.benzodiazepine-6-one ŽAF-DX116; 10–1000 ng, i.t.. had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine Ž25 ng. significantly inhibited only the second phase. The acetylcholine ŽACh. depletor hemicholinium-3 ŽHC-3. Ž1 mg, i.t.. completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP Ž10 ng, i.t... These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M 3 muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Formalin-induced nociception; Spinal cord; M 3 muscarinic receptor; Acetylcholine content
1. Introduction Pharmacological and ligand binding studies have led to the classification of muscarinic acetylcholine ŽACh. receptors in the central and the peripheral tissues into several subtypes w4,18x. Moreover, molecular cloning studies have demonstrated the existence of five distinct muscarinic receptor subtypes: M 1 , M 2 , M 3 , M 4 and M 5 muscarinic receptors w18,36x. It is known that M 1 muscarinic receptors are mainly involved in neuronal activity, M 2 muscarinic receptors in the inhibition of myocardium contraction, and M 3 muscarinic receptors in contraction of smooth muscle and in salivation. However, the functions of M 4 and M 5 muscarinic receptors are unknown. There are reports that injections of muscarinic receptor agonists into the spinal cord have anti-nociceptive effects on a noxious heat stimulus via M 1 muscarinic receptors ) Corresponding author. [email protected]
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w28x, M 2 muscarinic receptors w22x or M 3 muscarinic receptors w28x. Studies of radioligand binding and analysis of mRNA of muscarinic receptors showed the existence of M 1 , M 2 , M 3 and M 4 muscarinic receptors in the spinal cord w16,35x, and immunohistochemical studies demonstrated the presence of choline acetyltransferase in the spinal cord w34x. These findings suggest the possibility that muscarinic receptors in the spinal cord are involved in the nociceptive mechanism. Dubuisson and Dennis w10x reported that subcutaneous injection of formalin produced a biphasic nociceptive response in rats. Since formalin-induced nociception is thought to be caused by inflammation of tissues, studies on the mechanism of formalin-induced nociception is a useful model for studies on clinical pain w3,32,37x. However, the involvement of muscarinic receptors in inflammatory pain is unclear. In the present study, we attempted to elucidate how endogenous acetylcholine and muscarinic receptor subtypes in the spinal cord are involved in formalin-induced nociception in mice.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 4 5 6 - 7
K. Honda et al.r Brain Research 859 (2000) 38–44
2. Materials and methods Male ddy mice weighing 25–30 g were purchased from Kyudo ŽKumamoto, Japan.. They were kept in a room at 24 " 28C with a 12r12 h lightrdark cycle Žlight on at 0700 h. and were given free access to commercial food and tap water. Experimental procedures were based on the Guidelines of The Committee for Animal Care and Use of Fukuoka University. Each mouse was placed in a plastic cage more than 30 min before formalin injection to allow it to adapt to the new environment. Then 10 ml of 5% formalin Ž5% formalin solution in saline. was injected subcutaneously into the right hind paw, and licking and biting of the paw were recorded for 30 min as an indicator of pain. 2.1. Intrathecal injections of drugs For intrathecal injections Ž5 ml, i.t.. of drugs, a 1r2-in. needle Ž30 gauge. of a microsyringe Ž10 ml. was inserted between the lumbar 5 ŽL5. and lumbar 6 ŽL6. regions of the spinal cord in conscious mice w19x. The accurate placement of the needle was evidenced by a quick ‘‘flick’’ of the mouse’s tail. Drugs were dissolved in artificial cerebrospinal fluid ŽACSF.. Control mice received only ACSF. Cholinergic drugs were injected i.t. 10 min before formalin injection. In the preliminary experiment for confirmation of the injection site, 5 ml of malachite green was injected i.t. The dye was observed at the surface of lumbar and thoracic regions of the spinal cord. 2.2. Assay of ACh content in the spinal cord After formalin injection into the right hind paw, animals were decapitated and the lumbar spines was removed within 20 s. Samples were subjected to microwave irradiation Ž600 W, 90 s. to inactivate acetylcholinesterase, and the spinal cord was separated from the spines on an ice-cold dish and then weighed. The spinal cord was homogenized in 0.2 M perchloric acid containing ethylhomocholine as an internal standard for 10 s with an ultrasonic cell disruptor Žsonifier celldisruptor 185, Branson.. Homogenates were centrifuged at 6700 = g for 15 min at 48C, and the supernatants were adjusted to pH 3 with 1 M sodium acetate, and then filtered through a 0.45 mm filter ŽType Millex-HV, Japan Millipore.. The ACh contents of filtrates were determined by high performance liquid chromatography with an electrochemical detection ŽHPLCECD. system ŽEP-10, ECD-100, AC-Gel, AC-Enzympak, EICOM.. ACh contents are expressed as pmolrmg wet weight. 2.3. Drugs Atropine was purchased from Nacalai Tesque ŽKyoto, Japan.. Neostigmine, naloxone and hemicholinium-3 ŽHC3. were from Sigma ŽSt. Louis, MO, USA.. The 4-diphen-
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ylacetoxy-N-methylpiperidine methiodide Ž4-DAMP. was from Research Biochemicals ŽRBI, Natick, MA, USA.. Pirenzepine and 11-Ž2-wŽdiethylamino.methylx-1-piperidinyl4 acetyl. -5,11-dihydro-6 H-pyridoŽ2,3-b . Ž1,4. benzodiazepine-6-one ŽAF-DX116. were gifts from Boehringer Ingelheim ŽMannheim, Germany.. 2.4. Statistical analyses Data are expressed as means " S.E.M. Statistical analyses of data were performed by Student’s t-test Žunpaired. for pairs of values and Dannett’s test or Bonferroni’s test for multiple comparisons. 3. Results 3.1. Effects of the muscarinic antagonist atropine on formalin-induced nociception As shown in Fig. 1, subcutaneous injection of 5% formalin into the right hind paw of a mouse caused two distinct phases of licking and biting of the injected paw after the injection. The first phase was observed for 5 min after the injection, and then the second phase appeared and persisted for about 20 min. The peak period of the second phase of response was observed 15–20 min after the injection of formalin. Pretreatment with the muscarinic receptor antagonist atropine Ž10 ng, i.t.. inhibited the second phase of the formalin-induced response, but not the first phase ŽFig. 1A.. High doses of atropine Ž20–100 ng, i.t.. did not significantly inhibit the formalin-induced nociceptive responses. The i.t. injection of atropine had no effect on the general behavior of mice, such as their locomotor activity Ždata not shown.. 3.2. Effects of muscarinic receptor antagonists on formalin-induced nociception Pretreatment with the M 3 muscarinic receptor antagonist 4-DAMP Ži.t.. significantly inhibited the second phase of the response dose-dependently ŽFig. 2A,B., but not the first phase. Pretreatment with the M 1 muscarinic receptor antagonist pirenzepine Ži.t.. did not inhibit either the first or the second phase response, but a high dose of pirenzepine Ž1000 ng, i.t.. tended to inhibit the second phase ŽFig. 3A.. On the other hand, pretreatment with the M 2 muscarinic receptor antagonist AF-DX116 Ži.t.. had no effect on either the first or the second phase response ŽFig. 3B.. The i.t. injection of 4-DAMP had no effect on the general behavior of mice, such as their locomotor activity Ždata not shown.. 3.3. Effects of the opiate receptor antagonist naloxone on 4-DAMP-induced anti-nociception Since pretreatment with the M 3 muscarinic receptor antagonist 4-DAMP Ži.t.. significantly inhibited the forma-
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effect. HC-3 Ž1 mg, i.t.. injected 4 h before the i.t. injection of 4-DAMP completely abolished the 4-DAMP Ž10 ng, i.t..-induced anti-nociceptive effect ŽFig. 6., although HC-3 Ž1 mg, i.t.. did not affect formalin-induced nociception. 3.5. Effects of stimuli induced by formalin-induced nociception on the ACh content of the spinal cord Fig. 7 shows the content of ACh in the spinal cord of mice after injection of 5% formalin into a paw of mouse. The ACh content of the spinal cord of the formalin-injected group was about 64% higher than that of the saline-injected group 14 min after injections ŽFig. 7.. ACh content in the spinal cord was also significantly higher at 14 min than at 1 min after the injection of formalin. The significant increase of ACh observed 14 min after formalin
Fig. 1. Effect of atropine on formalin-induced nociception. Times of licking and biting are expressed as summations of these times in 5 min periods ŽA.. Nociception responses of the first phase and second phase are expressed as times of paw-licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in the controls were 105.67"4.14 s in the first phase and 119.90"6.89 s in the second. All data are means"S.E.M. Ž ns8–10.. U P - 0.05 vs. corresponding control value with ACSF. UU P - 0.01 vs. corresponding control value with ACSF.
lin-induced second phase response, we next examined the effect of the opiate receptor antagonist naloxone on 4DAMP-induced anti-nociception. Pretreatment with naloxone Ž3 mgrkg, s.c.. 30 min before 4-DAMP injection Ž10 ng, i.t.. did not affect the 4-DAMP-induced anti-nociceptive response ŽFig. 4., although it completely inhibited morphine Ž10 mgrkg, s.c..-induced analgesia in a hot-plate test Ždata not shown.. 3.4. Effects of neostigmine and HC-3 on the formalin-induced nociceptiÕe response We examined the effect of the acetylcholinesterase inhibitor neostigmine on formalin-induced nociception. The i.t. injection of neostigmine Ž25 ng. significantly inhibited only the second phase of the response ŽFig. 5.. Next, we examined the effect of the choline uptake inhibitor HC-3 on the 4-DAMP-induced anti-nociceptive
Fig. 2. Effect of the muscarinic M 3 receptor antagonist 4-DAMP on formalin-induced nociception. Times of the licking and biting are expressed as summations of these times in 5 min periods ŽA.. Nociception responses in the first phase and second phase are expressed as times of paw-licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in the controls were 105.82"3.91 s in the first phase and 116.16"6.62 s in the second phase, respectively. All data are means"S.E.M. Ž ns 4–8.. U P - 0.01 vs. corresponding control value with ACSF.
K. Honda et al.r Brain Research 859 (2000) 38–44
Fig. 3. Effects of the muscarinic M 1 receptor antagonist pirenzepine ŽA. and muscarinic M 2 antagonist AF-DX116 ŽB. on formalin-induced nociception. Nociception responses in the first phase and second phase are expressed as times of paw-licking and biting 0–5 min and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t... The times of paw-licking and biting of the controls were 106.83"4.33 s in the first phase and 108.52"11.13 s in the second phase. All data are means"S.E.M. Ž ns8–13..
injection was not observed after pretreatment with 4-DAMP Ž10 ng, i.t..
4. Discussion The present study shows that inhibition of ACh release from the cholinergic neurons in the spinal cord is con-
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cerned with formalin-induced nociception, and that inhibition of ACh release is mediated via presynapic M 3 muscarinic receptors. Since the i.t. injection of muscarinic receptor agonists results in anti-nociception to a thermal nociceptive examinations by the hot plate or tail flick test, activation of muscarinic receptors in the spinal cord seems to be involved in anti-nociception in thermal nociceptive responses w11,22,28,33,38x. In the present study, the i.t. injection of the muscarinic non-selective antagonist atropine at a low dose Ž0.1–10 ng. inhibited the formalin-induced second phase response ŽFig. 1.. This also suggests that the activation of muscarinic receptors in the spinal cord may cause the second phase of the formalin-induced nociceptive response. Therefore, to determine which subtype of muscarinic receptors is involved in formalin-induced nociception, we examined the effects of i.t. injections of muscarinic antagonists on formalin-induced nociceptive responses. Three types of antagonists against M1, M2 and M3 muscarinic receptors have been developed based on pharmacological affinities w26x, namely pirenzepine ŽM 1 ., AF-DX116 ŽM 2 . and 4-DAMP ŽM 3 .. In this study, we used these drugs as selective antagonists of the respective muscarinic receptor subtypes. The i.t. injection of the selective M 3 muscarinic receptor antagonist 4DAMP inhibited the second phase of formalin-induced nociception dose-dependently ŽFig. 2.. There is a report that 4-DAMP also binds to the M 1 muscarinic receptor w8,9x. However, the i.t. injection of the M 1 muscarinic receptor antagonist pirenzepine Ž1–100 ng. did not affect formalin-induced nociception, although it tended be inhibitory at a higher dose of 1000 ng ŽFig. 3A.. An i.t. injection of the M 2 selective muscarinic receptor antagonist AF-DX116 had no effect on formalin-induced nociceptive responses ŽFig. 3B.. In this study, inhibition by the M 1 selective antagonist pirenzepine was 100-fold less than that of 4-DAMP on formalin-induced nociceptive responses ŽFig. 3A.. These results are consistent with reports that the affinity of pirenzepine for the M 3 muscarinic
Fig. 4. Effect of the opiate receptor antagonist naloxone on 4-DAMP-induced anti-nociception. Naloxone Ž3 mgrkg. or saline was administered subcutaneously 30 min before the 4-DAMP injection Ž10 ng, i.t... The nociception responses in the first phase and second phase are expressed as times of paw-licking and biting of 0–5 and 7–30 min after the formalin injection. Times are expressed as percentages of the control Žsaline, s.c.q ACSF, i.t... The times of paw-licking and biting of controls were 111.10 " 9.77 s in the first phase and 114.30 " 11.81 s in the second phase, respectively. All data are means " S.E.M. Ž n s 5–8.. U P - 0.05 and UU P - 0.01 vs. corresponding control value with combination of saline and ACSF.
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Next, we examined the effects of the acetylcholinesterase inhibitor neostigmine on nociceptive responses induced by formalin. The i.t. injection of neostigmine inhibited only the second phase response induced by formalin ŽFig. 5.. The anti-nociceptive effect of neostigmine may be due to increases of endogenous ACh in the cholinergic neurons in the spinal cord. Similarly, injection of acetylcholinesterase inhibitors into the spinal cord of rats produced analgesia via muscarinic receptors w5,11,28x as judged by hot plate and tail flick tests. However, a problem is why the M 3 muscarinic receptor antagonist 4-DAMP inhibited the second phase response induced by formalin. To explain this, we used the choline uptake inhibitor HC-3, which is known to inhibit high-affinity choline uptake w15x and cause depletion of ACh w20x. Pretreatment with HC-3 Ž1 mg, i.t.. abolished the 4-DAMP-induced anti-nociceptive response ŽFig. 6., indicating that antinociception by 4-DAMP is weak in the condition of
Fig. 5. Effects of the acetylcholinesterase inhibitor neostigmine on formalin-induced nociception. Times of licking and biting are expressed as summations of these times in 5 min periods ŽA.. The nociception responses of the first phase and second phase are expressed as times of paw-licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in the controls were 108.31"5.25 s in the first phase and 111.93"7.40 s in the second phase. All data are means" S.E.M. Ž ns 7–13.. U P - 0.05 vs. corresponding control value with ACSF.
receptor is 100-fold weaker than that of 4-DAMP in the rat submandibular gland w8,25x, guinea pig ileal smooth muscle w17x and cloned human M 3 muscarinic receptors w8x. Therefore, our findings indicate that the anti-nociceptive effect of 4-DAMP in the second phase of formalin-induced nociception is mediated via M 3 muscarinic receptors in the spinal cord. In fact, the existence of M 3 muscarinic receptors in the spinal cord of rats has been reported both from binding w16x and molecular biological experiments w35x. Next, we examined the involvement of the opioid system in 4-DAMP-induced anti-nociception. Pretreatment with the opioid antagonist naloxone Ž3 mgrkg, s.c.. did not inhibit 4-DAMP-induced anti-nociception ŽFig. 4., although it completely inhibited morphine-induced analgesia Ždata not shown.. Thus, 4-DAMP-induced anti-nociception seems to be independent of the opioid system in the spinal cord.
Fig. 6. Effect of choline uptake inhibitor HC-3 on formalin-induced nociception. Times of licking and biting are expressed as summations of these times in 5 min periods ŽA.. The nociception responses of the first phase and second phase are expressed as times of paw licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in controls were 86.40"7.03 s in the first phase and 115.00"18.42 s in the second phase. All data are means"S.E.M. Ž ns 5–6..
K. Honda et al.r Brain Research 859 (2000) 38–44
Fig. 7. The ACh content of the spinal cord after injection of 5% formalin ŽF. or saline ŽS. into the right hind paw of mice. All data are means" S.E.M. Ž ns 5–6.. U P - 0.05 vs. the corresponding value on saline ŽS. injection, aP - 0.05 vs. the corresponding value 1 min after formalin ŽF. injection.
endogenous ACh depletion. Presynaptic muscarinic receptors are known to regulate ACh release. In fact, presynaptic M 3 muscarinic autoreceptors are present in the brain w6,21,27x and sympathetic ganglions w24x. Thus, M 3 muscarinic receptors may modulate ACh release from cholinergic neurons as the presynaptic autoreceptors in the spinal cord. In the present study, therefore, ACh release seemed to be increased by 4-DAMP via blockade of presynaptic M 3 autoreceptors in the spinal cholinergic neurons, and the increased ACh may have caused anti-nociception. In the present study, low doses of atropine counteracted nociception, but high doses had no effect. These results may be explained by supposing that atropine at a low dose acts only on presynaptic M 3 muscarinic receptors, but a high dose acts not only at presynaptic M 3 muscarinic receptors but also at postsynaptic muscarinic receptors in the spinal cord. Similar results were reported by Bartolini et al. w2x and Ghelardini et al. w13x. Naguib and Yaksh w28x reported that i.t. injection of muscarinic agonists caused anti-nociception in thermal nociceptive responses via M 1 or M 3 muscarinic receptors. On the other hand, Iwamoto and Marion w22x reported that M 2 muscarinic agonists cause anti-nociception. Recently, M 4 muscarinic receptors in the spinal cord were reported to be involved in muscarinic anti-nociception to thermal nociceptive responses in mice w12x. In the present study, however, we could not clarify the subtype of postsynaptic muscarinic receptors in anti-nociception to formalin-induced nociception. We examined the effect of formalin-induced nociception on the ACh content in the mouse spinal cord. The content in the formalin-treated group was higher than that in the saline-treated group 14 min after injections ŽFig. 7., a time corresponding with the formalin-induced second phase response. Significant increase of the ACh content was inhibited by i.t. injection of M 3 antagonist 4-DAMP ŽFig. 7.. Similarly, 4-DAMP inhibited the second phase
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response induced by formalin. Increase of the ACh content in the spinal cord is thought to be due to a decrease of ACh release w14,29–31x or an increase of ACh synthesis w7x. In the present study, the increase in ACh content induced by formalin seemed to be due to a decrease of ACh release rather than an increase of ACh synthesis, because high doses of atropine did not inhibit formalin-induced nociceptive responses, and the ACh depletor HC-3 also had no affect. These phenomena may be explained as follows, the signals caused by noxious stimulation, such as formalin injection, is transferred to the cholinergic interneurons in the spinal cord via C-fibers. In fact, Kobayashi et al. w23x reported that substance P caused ACh release in the spinal cord. In addition, it has been reported that substance P in the spinal cord may be serving in the second phase of nociception induced by formalin w39x. On the other hand, it has been reported that the cholinergic interneurons may be exited in the spinal cord w1,34x. Therefore, the continuous stimulation induced by formalin may cause negative feedback of ACh release via the presynaptic M 3 receptors in the spinal cord. Further studies are needed to establish the ACh transmission processing in the spinal cord. In conclusion, the present study suggests that endogenous ACh in the spinal cord acts as a transmitter in anti-nociception, and that ACh release regulated by the presynaptic M 3 muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.
Acknowledgements We thank Dr. R. Saito for helpful comments and suggestions. We also thank Miss S. Nagayo and Miss A. Iwashita for their technical assistance. This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, and a grant ŽNo. 996001. from the Central Research Institute of Fukuoka University.
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Research report
Involvement of M 3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice Kenji Honda ) , Akitsugu Harada, Yukio Takano, Hiro-o Kamiya Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka UniÕersity, Fukuoka 814-0180, Japan Accepted 7 December 1999
Abstract Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase Ž1–5 min. and the second phase Ž7–30 min. after the injection. The muscarinic antagonist atropine Ž0.1–10 ng, i.t.. and the M 3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide Ž4-DAMP. Ž0.1–20 ng, i.t.. inhibited the second phase of this response, whereas higher doses of atropine Ž20–100 ng, i.t.. did not cause inhibition. The M 1 muscarinic receptor antagonist pirenzepine Ž10–100 ng, i.t.. did not inhibit either the first or the second phase response, but a high dose of pirenzepine Ž1000 ng, i.t.. tended to inhibit the second phase response. On the other hand, the M 2 muscarinic receptor antagonist 11-Ž2-wŽdiethylamino.methylx-1-piperidinyl4acetyl.-5,11-dihydro-6 HpyridoŽ2,3-b .Ž1,4.benzodiazepine-6-one ŽAF-DX116; 10–1000 ng, i.t.. had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine Ž25 ng. significantly inhibited only the second phase. The acetylcholine ŽACh. depletor hemicholinium-3 ŽHC-3. Ž1 mg, i.t.. completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP Ž10 ng, i.t... These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M 3 muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Formalin-induced nociception; Spinal cord; M 3 muscarinic receptor; Acetylcholine content
1. Introduction Pharmacological and ligand binding studies have led to the classification of muscarinic acetylcholine ŽACh. receptors in the central and the peripheral tissues into several subtypes w4,18x. Moreover, molecular cloning studies have demonstrated the existence of five distinct muscarinic receptor subtypes: M 1 , M 2 , M 3 , M 4 and M 5 muscarinic receptors w18,36x. It is known that M 1 muscarinic receptors are mainly involved in neuronal activity, M 2 muscarinic receptors in the inhibition of myocardium contraction, and M 3 muscarinic receptors in contraction of smooth muscle and in salivation. However, the functions of M 4 and M 5 muscarinic receptors are unknown. There are reports that injections of muscarinic receptor agonists into the spinal cord have anti-nociceptive effects on a noxious heat stimulus via M 1 muscarinic receptors ) Corresponding author. [email protected]
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q81-92-863-0389;
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w28x, M 2 muscarinic receptors w22x or M 3 muscarinic receptors w28x. Studies of radioligand binding and analysis of mRNA of muscarinic receptors showed the existence of M 1 , M 2 , M 3 and M 4 muscarinic receptors in the spinal cord w16,35x, and immunohistochemical studies demonstrated the presence of choline acetyltransferase in the spinal cord w34x. These findings suggest the possibility that muscarinic receptors in the spinal cord are involved in the nociceptive mechanism. Dubuisson and Dennis w10x reported that subcutaneous injection of formalin produced a biphasic nociceptive response in rats. Since formalin-induced nociception is thought to be caused by inflammation of tissues, studies on the mechanism of formalin-induced nociception is a useful model for studies on clinical pain w3,32,37x. However, the involvement of muscarinic receptors in inflammatory pain is unclear. In the present study, we attempted to elucidate how endogenous acetylcholine and muscarinic receptor subtypes in the spinal cord are involved in formalin-induced nociception in mice.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 4 5 6 - 7
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2. Materials and methods Male ddy mice weighing 25–30 g were purchased from Kyudo ŽKumamoto, Japan.. They were kept in a room at 24 " 28C with a 12r12 h lightrdark cycle Žlight on at 0700 h. and were given free access to commercial food and tap water. Experimental procedures were based on the Guidelines of The Committee for Animal Care and Use of Fukuoka University. Each mouse was placed in a plastic cage more than 30 min before formalin injection to allow it to adapt to the new environment. Then 10 ml of 5% formalin Ž5% formalin solution in saline. was injected subcutaneously into the right hind paw, and licking and biting of the paw were recorded for 30 min as an indicator of pain. 2.1. Intrathecal injections of drugs For intrathecal injections Ž5 ml, i.t.. of drugs, a 1r2-in. needle Ž30 gauge. of a microsyringe Ž10 ml. was inserted between the lumbar 5 ŽL5. and lumbar 6 ŽL6. regions of the spinal cord in conscious mice w19x. The accurate placement of the needle was evidenced by a quick ‘‘flick’’ of the mouse’s tail. Drugs were dissolved in artificial cerebrospinal fluid ŽACSF.. Control mice received only ACSF. Cholinergic drugs were injected i.t. 10 min before formalin injection. In the preliminary experiment for confirmation of the injection site, 5 ml of malachite green was injected i.t. The dye was observed at the surface of lumbar and thoracic regions of the spinal cord. 2.2. Assay of ACh content in the spinal cord After formalin injection into the right hind paw, animals were decapitated and the lumbar spines was removed within 20 s. Samples were subjected to microwave irradiation Ž600 W, 90 s. to inactivate acetylcholinesterase, and the spinal cord was separated from the spines on an ice-cold dish and then weighed. The spinal cord was homogenized in 0.2 M perchloric acid containing ethylhomocholine as an internal standard for 10 s with an ultrasonic cell disruptor Žsonifier celldisruptor 185, Branson.. Homogenates were centrifuged at 6700 = g for 15 min at 48C, and the supernatants were adjusted to pH 3 with 1 M sodium acetate, and then filtered through a 0.45 mm filter ŽType Millex-HV, Japan Millipore.. The ACh contents of filtrates were determined by high performance liquid chromatography with an electrochemical detection ŽHPLCECD. system ŽEP-10, ECD-100, AC-Gel, AC-Enzympak, EICOM.. ACh contents are expressed as pmolrmg wet weight. 2.3. Drugs Atropine was purchased from Nacalai Tesque ŽKyoto, Japan.. Neostigmine, naloxone and hemicholinium-3 ŽHC3. were from Sigma ŽSt. Louis, MO, USA.. The 4-diphen-
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ylacetoxy-N-methylpiperidine methiodide Ž4-DAMP. was from Research Biochemicals ŽRBI, Natick, MA, USA.. Pirenzepine and 11-Ž2-wŽdiethylamino.methylx-1-piperidinyl4 acetyl. -5,11-dihydro-6 H-pyridoŽ2,3-b . Ž1,4. benzodiazepine-6-one ŽAF-DX116. were gifts from Boehringer Ingelheim ŽMannheim, Germany.. 2.4. Statistical analyses Data are expressed as means " S.E.M. Statistical analyses of data were performed by Student’s t-test Žunpaired. for pairs of values and Dannett’s test or Bonferroni’s test for multiple comparisons. 3. Results 3.1. Effects of the muscarinic antagonist atropine on formalin-induced nociception As shown in Fig. 1, subcutaneous injection of 5% formalin into the right hind paw of a mouse caused two distinct phases of licking and biting of the injected paw after the injection. The first phase was observed for 5 min after the injection, and then the second phase appeared and persisted for about 20 min. The peak period of the second phase of response was observed 15–20 min after the injection of formalin. Pretreatment with the muscarinic receptor antagonist atropine Ž10 ng, i.t.. inhibited the second phase of the formalin-induced response, but not the first phase ŽFig. 1A.. High doses of atropine Ž20–100 ng, i.t.. did not significantly inhibit the formalin-induced nociceptive responses. The i.t. injection of atropine had no effect on the general behavior of mice, such as their locomotor activity Ždata not shown.. 3.2. Effects of muscarinic receptor antagonists on formalin-induced nociception Pretreatment with the M 3 muscarinic receptor antagonist 4-DAMP Ži.t.. significantly inhibited the second phase of the response dose-dependently ŽFig. 2A,B., but not the first phase. Pretreatment with the M 1 muscarinic receptor antagonist pirenzepine Ži.t.. did not inhibit either the first or the second phase response, but a high dose of pirenzepine Ž1000 ng, i.t.. tended to inhibit the second phase ŽFig. 3A.. On the other hand, pretreatment with the M 2 muscarinic receptor antagonist AF-DX116 Ži.t.. had no effect on either the first or the second phase response ŽFig. 3B.. The i.t. injection of 4-DAMP had no effect on the general behavior of mice, such as their locomotor activity Ždata not shown.. 3.3. Effects of the opiate receptor antagonist naloxone on 4-DAMP-induced anti-nociception Since pretreatment with the M 3 muscarinic receptor antagonist 4-DAMP Ži.t.. significantly inhibited the forma-
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effect. HC-3 Ž1 mg, i.t.. injected 4 h before the i.t. injection of 4-DAMP completely abolished the 4-DAMP Ž10 ng, i.t..-induced anti-nociceptive effect ŽFig. 6., although HC-3 Ž1 mg, i.t.. did not affect formalin-induced nociception. 3.5. Effects of stimuli induced by formalin-induced nociception on the ACh content of the spinal cord Fig. 7 shows the content of ACh in the spinal cord of mice after injection of 5% formalin into a paw of mouse. The ACh content of the spinal cord of the formalin-injected group was about 64% higher than that of the saline-injected group 14 min after injections ŽFig. 7.. ACh content in the spinal cord was also significantly higher at 14 min than at 1 min after the injection of formalin. The significant increase of ACh observed 14 min after formalin
Fig. 1. Effect of atropine on formalin-induced nociception. Times of licking and biting are expressed as summations of these times in 5 min periods ŽA.. Nociception responses of the first phase and second phase are expressed as times of paw-licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in the controls were 105.67"4.14 s in the first phase and 119.90"6.89 s in the second. All data are means"S.E.M. Ž ns8–10.. U P - 0.05 vs. corresponding control value with ACSF. UU P - 0.01 vs. corresponding control value with ACSF.
lin-induced second phase response, we next examined the effect of the opiate receptor antagonist naloxone on 4DAMP-induced anti-nociception. Pretreatment with naloxone Ž3 mgrkg, s.c.. 30 min before 4-DAMP injection Ž10 ng, i.t.. did not affect the 4-DAMP-induced anti-nociceptive response ŽFig. 4., although it completely inhibited morphine Ž10 mgrkg, s.c..-induced analgesia in a hot-plate test Ždata not shown.. 3.4. Effects of neostigmine and HC-3 on the formalin-induced nociceptiÕe response We examined the effect of the acetylcholinesterase inhibitor neostigmine on formalin-induced nociception. The i.t. injection of neostigmine Ž25 ng. significantly inhibited only the second phase of the response ŽFig. 5.. Next, we examined the effect of the choline uptake inhibitor HC-3 on the 4-DAMP-induced anti-nociceptive
Fig. 2. Effect of the muscarinic M 3 receptor antagonist 4-DAMP on formalin-induced nociception. Times of the licking and biting are expressed as summations of these times in 5 min periods ŽA.. Nociception responses in the first phase and second phase are expressed as times of paw-licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in the controls were 105.82"3.91 s in the first phase and 116.16"6.62 s in the second phase, respectively. All data are means"S.E.M. Ž ns 4–8.. U P - 0.01 vs. corresponding control value with ACSF.
K. Honda et al.r Brain Research 859 (2000) 38–44
Fig. 3. Effects of the muscarinic M 1 receptor antagonist pirenzepine ŽA. and muscarinic M 2 antagonist AF-DX116 ŽB. on formalin-induced nociception. Nociception responses in the first phase and second phase are expressed as times of paw-licking and biting 0–5 min and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t... The times of paw-licking and biting of the controls were 106.83"4.33 s in the first phase and 108.52"11.13 s in the second phase. All data are means"S.E.M. Ž ns8–13..
injection was not observed after pretreatment with 4-DAMP Ž10 ng, i.t..
4. Discussion The present study shows that inhibition of ACh release from the cholinergic neurons in the spinal cord is con-
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cerned with formalin-induced nociception, and that inhibition of ACh release is mediated via presynapic M 3 muscarinic receptors. Since the i.t. injection of muscarinic receptor agonists results in anti-nociception to a thermal nociceptive examinations by the hot plate or tail flick test, activation of muscarinic receptors in the spinal cord seems to be involved in anti-nociception in thermal nociceptive responses w11,22,28,33,38x. In the present study, the i.t. injection of the muscarinic non-selective antagonist atropine at a low dose Ž0.1–10 ng. inhibited the formalin-induced second phase response ŽFig. 1.. This also suggests that the activation of muscarinic receptors in the spinal cord may cause the second phase of the formalin-induced nociceptive response. Therefore, to determine which subtype of muscarinic receptors is involved in formalin-induced nociception, we examined the effects of i.t. injections of muscarinic antagonists on formalin-induced nociceptive responses. Three types of antagonists against M1, M2 and M3 muscarinic receptors have been developed based on pharmacological affinities w26x, namely pirenzepine ŽM 1 ., AF-DX116 ŽM 2 . and 4-DAMP ŽM 3 .. In this study, we used these drugs as selective antagonists of the respective muscarinic receptor subtypes. The i.t. injection of the selective M 3 muscarinic receptor antagonist 4DAMP inhibited the second phase of formalin-induced nociception dose-dependently ŽFig. 2.. There is a report that 4-DAMP also binds to the M 1 muscarinic receptor w8,9x. However, the i.t. injection of the M 1 muscarinic receptor antagonist pirenzepine Ž1–100 ng. did not affect formalin-induced nociception, although it tended be inhibitory at a higher dose of 1000 ng ŽFig. 3A.. An i.t. injection of the M 2 selective muscarinic receptor antagonist AF-DX116 had no effect on formalin-induced nociceptive responses ŽFig. 3B.. In this study, inhibition by the M 1 selective antagonist pirenzepine was 100-fold less than that of 4-DAMP on formalin-induced nociceptive responses ŽFig. 3A.. These results are consistent with reports that the affinity of pirenzepine for the M 3 muscarinic
Fig. 4. Effect of the opiate receptor antagonist naloxone on 4-DAMP-induced anti-nociception. Naloxone Ž3 mgrkg. or saline was administered subcutaneously 30 min before the 4-DAMP injection Ž10 ng, i.t... The nociception responses in the first phase and second phase are expressed as times of paw-licking and biting of 0–5 and 7–30 min after the formalin injection. Times are expressed as percentages of the control Žsaline, s.c.q ACSF, i.t... The times of paw-licking and biting of controls were 111.10 " 9.77 s in the first phase and 114.30 " 11.81 s in the second phase, respectively. All data are means " S.E.M. Ž n s 5–8.. U P - 0.05 and UU P - 0.01 vs. corresponding control value with combination of saline and ACSF.
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K. Honda et al.r Brain Research 859 (2000) 38–44
Next, we examined the effects of the acetylcholinesterase inhibitor neostigmine on nociceptive responses induced by formalin. The i.t. injection of neostigmine inhibited only the second phase response induced by formalin ŽFig. 5.. The anti-nociceptive effect of neostigmine may be due to increases of endogenous ACh in the cholinergic neurons in the spinal cord. Similarly, injection of acetylcholinesterase inhibitors into the spinal cord of rats produced analgesia via muscarinic receptors w5,11,28x as judged by hot plate and tail flick tests. However, a problem is why the M 3 muscarinic receptor antagonist 4-DAMP inhibited the second phase response induced by formalin. To explain this, we used the choline uptake inhibitor HC-3, which is known to inhibit high-affinity choline uptake w15x and cause depletion of ACh w20x. Pretreatment with HC-3 Ž1 mg, i.t.. abolished the 4-DAMP-induced anti-nociceptive response ŽFig. 6., indicating that antinociception by 4-DAMP is weak in the condition of
Fig. 5. Effects of the acetylcholinesterase inhibitor neostigmine on formalin-induced nociception. Times of licking and biting are expressed as summations of these times in 5 min periods ŽA.. The nociception responses of the first phase and second phase are expressed as times of paw-licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in the controls were 108.31"5.25 s in the first phase and 111.93"7.40 s in the second phase. All data are means" S.E.M. Ž ns 7–13.. U P - 0.05 vs. corresponding control value with ACSF.
receptor is 100-fold weaker than that of 4-DAMP in the rat submandibular gland w8,25x, guinea pig ileal smooth muscle w17x and cloned human M 3 muscarinic receptors w8x. Therefore, our findings indicate that the anti-nociceptive effect of 4-DAMP in the second phase of formalin-induced nociception is mediated via M 3 muscarinic receptors in the spinal cord. In fact, the existence of M 3 muscarinic receptors in the spinal cord of rats has been reported both from binding w16x and molecular biological experiments w35x. Next, we examined the involvement of the opioid system in 4-DAMP-induced anti-nociception. Pretreatment with the opioid antagonist naloxone Ž3 mgrkg, s.c.. did not inhibit 4-DAMP-induced anti-nociception ŽFig. 4., although it completely inhibited morphine-induced analgesia Ždata not shown.. Thus, 4-DAMP-induced anti-nociception seems to be independent of the opioid system in the spinal cord.
Fig. 6. Effect of choline uptake inhibitor HC-3 on formalin-induced nociception. Times of licking and biting are expressed as summations of these times in 5 min periods ŽA.. The nociception responses of the first phase and second phase are expressed as times of paw licking and biting 0–5 and 7–30 min after formalin injection. Times are expressed as percentages of the control ŽACSF, i.t.. ŽB.. The times of paw-licking and biting in controls were 86.40"7.03 s in the first phase and 115.00"18.42 s in the second phase. All data are means"S.E.M. Ž ns 5–6..
K. Honda et al.r Brain Research 859 (2000) 38–44
Fig. 7. The ACh content of the spinal cord after injection of 5% formalin ŽF. or saline ŽS. into the right hind paw of mice. All data are means" S.E.M. Ž ns 5–6.. U P - 0.05 vs. the corresponding value on saline ŽS. injection, aP - 0.05 vs. the corresponding value 1 min after formalin ŽF. injection.
endogenous ACh depletion. Presynaptic muscarinic receptors are known to regulate ACh release. In fact, presynaptic M 3 muscarinic autoreceptors are present in the brain w6,21,27x and sympathetic ganglions w24x. Thus, M 3 muscarinic receptors may modulate ACh release from cholinergic neurons as the presynaptic autoreceptors in the spinal cord. In the present study, therefore, ACh release seemed to be increased by 4-DAMP via blockade of presynaptic M 3 autoreceptors in the spinal cholinergic neurons, and the increased ACh may have caused anti-nociception. In the present study, low doses of atropine counteracted nociception, but high doses had no effect. These results may be explained by supposing that atropine at a low dose acts only on presynaptic M 3 muscarinic receptors, but a high dose acts not only at presynaptic M 3 muscarinic receptors but also at postsynaptic muscarinic receptors in the spinal cord. Similar results were reported by Bartolini et al. w2x and Ghelardini et al. w13x. Naguib and Yaksh w28x reported that i.t. injection of muscarinic agonists caused anti-nociception in thermal nociceptive responses via M 1 or M 3 muscarinic receptors. On the other hand, Iwamoto and Marion w22x reported that M 2 muscarinic agonists cause anti-nociception. Recently, M 4 muscarinic receptors in the spinal cord were reported to be involved in muscarinic anti-nociception to thermal nociceptive responses in mice w12x. In the present study, however, we could not clarify the subtype of postsynaptic muscarinic receptors in anti-nociception to formalin-induced nociception. We examined the effect of formalin-induced nociception on the ACh content in the mouse spinal cord. The content in the formalin-treated group was higher than that in the saline-treated group 14 min after injections ŽFig. 7., a time corresponding with the formalin-induced second phase response. Significant increase of the ACh content was inhibited by i.t. injection of M 3 antagonist 4-DAMP ŽFig. 7.. Similarly, 4-DAMP inhibited the second phase
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response induced by formalin. Increase of the ACh content in the spinal cord is thought to be due to a decrease of ACh release w14,29–31x or an increase of ACh synthesis w7x. In the present study, the increase in ACh content induced by formalin seemed to be due to a decrease of ACh release rather than an increase of ACh synthesis, because high doses of atropine did not inhibit formalin-induced nociceptive responses, and the ACh depletor HC-3 also had no affect. These phenomena may be explained as follows, the signals caused by noxious stimulation, such as formalin injection, is transferred to the cholinergic interneurons in the spinal cord via C-fibers. In fact, Kobayashi et al. w23x reported that substance P caused ACh release in the spinal cord. In addition, it has been reported that substance P in the spinal cord may be serving in the second phase of nociception induced by formalin w39x. On the other hand, it has been reported that the cholinergic interneurons may be exited in the spinal cord w1,34x. Therefore, the continuous stimulation induced by formalin may cause negative feedback of ACh release via the presynaptic M 3 receptors in the spinal cord. Further studies are needed to establish the ACh transmission processing in the spinal cord. In conclusion, the present study suggests that endogenous ACh in the spinal cord acts as a transmitter in anti-nociception, and that ACh release regulated by the presynaptic M 3 muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.
Acknowledgements We thank Dr. R. Saito for helpful comments and suggestions. We also thank Miss S. Nagayo and Miss A. Iwashita for their technical assistance. This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, and a grant ŽNo. 996001. from the Central Research Institute of Fukuoka University.
References w1x R.P. Baber, P.E. Phelps, C.R. Houser, G.D. Grawford, P.M. Salvaterra, J.E. Vaughn, The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study, J. Comp. Neurol. 229 Ž1984. 329–346. w2x A. Bartolini, C. Ghelardini, L. Fantetti, M. Malcangio, P. Malmberg-Aiello, A. Giotti, Role of muscarinic receptor subtypes in central anti-nociception, Br. J. Pharmacol. 105 Ž1992. 77–82. w3x J.H. Beown, J.W. Kissel, P.M. Lish, Studies on the acute inflammatory response: I. Involvement of the central nervous system in certain models of inflammation, J. Pharmacol. Exp. Ther. 160 Ž1968. 231–242. w4x N.J.M. Birdsall, A.S.V. Burgen, R. Hummer, E.C. Hulme, J. Stockon, Pirenzepine a ligand with original binding properties to muscarinic receptors, Scand. J. Gastroenterol. Suppl. 15 Ž1980. 1–4.
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