JOURNAL HOME CME HOME THIS MONTH PAST ISSUES ETOC COLLECTIONS
AUTHORS REVIEWERS EDITORIAL BOARD FEEDBACK RSS HELP
A&A International Anesthesia Research Society
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, Y.-J.
Right arrow Articles by Eisenach, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, Y.-J.
Right arrow Articles by Eisenach, J. C.
Related Collections
Right arrow Trauma
Right arrow Regional Anesthesia
Right arrow Pain

Anesth Analg 2003;96:1403-1408
© 2003 International Anesthesia Research Society

PAIN MEDICINE

Intrathecal Clonidine Reduces Hypersensitivity After Nerve Injury by a Mechanism Involving Spinal m4 Muscarinic Receptors

Yoo-Jin Kang, and James C. Eisenach

Department of Anesthesiology and Center for the Study of Pharmacologic Plasticity in the Presence of Pain, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Address correspondence and reprint requests to Professor James C. Eisenach, Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Address e-mail to eisenach{at}wfubmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{alpha}2-Adrenergic agonists reduce mechanical and thermal hypersensitivity in animals with nerve injury and effectively treat neuropathic pain in humans. Previous studies indicate a reliance of {alpha}2-adrenergic agonists in this setting on spinal cholinergic activation and stimulation of muscarinic receptors. The subtype(s) of muscarinic receptors in the spinal cord that produces antinociception in normal animals is controversial, and those involved in reducing hypersensitivity and interacting with {alpha}2-adrenergic systems after nerve injury are unstudied. To examine this, the left L5 and L6 spinal nerves were tightly ligated in rats, resulting in reduction in withdrawal threshold to punctate mechanical stimuli. Intrathecal clonidine, 15 µg, returned the withdrawal threshold to normal. Using highly specific m1 and m4 antagonists, we observed no reduction in the effect of clonidine by the m1 antagonist, but inhibition of clonidine’s effect by the m4 antagonist. Western analysis revealed no difference in quantitative expression of m1 and m4 receptor protein in the dorsal spinal cord of spinal nerve-injured animals compared with sham-operated controls, suggesting this interaction with m4 receptors does not reflect an increase in receptor expression.

IMPLICATIONS: Neuraxial clonidine is an effective adjunct in the treatment of neuropathic pain and increases acetylcholine concentrations in cerebrospinal fluid in humans. These data in animals suggest that spinal m4 type muscarinic receptors are important to the effect of clonidine in treating hypersensitivity to touch after nerve injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Surgical or traumatic injury on a peripheral nerve can be associated with spontaneous and elicited pain, both to innocuous and mildly noxious stimuli, termed neuropathic pain. This kind of pain is often very difficult to control with conventional treatments such as nonsteroidal antiinflammatory drugs and opioids (1). Intrathecal administration of the {alpha}2-adrenergic receptor agonist, clonidine, has been known to alleviate hypersensitivity in animals with peripheral nerve injury (2) and to produce analgesia in patients with cancer and neuropathic pain (3). The mechanisms underlying clonidine’s analgesic action are not fully understood, but clearly involve interactions with spinal cholinergic interneurons. For example, intrathecally administered clonidine increases concentrations of acetylcholine in cerebrospinal fluid (4) and intrathecal administration of cholinomimetic drugs produces analgesia (5). Furthermore, pharmacologic studies in rats with peripheral nerve injury demonstrate a functional role for this interaction, because blockade of spinal muscarinic receptors by atropine reduces the antihypersensitivity effect of clonidine (6).

There are five subtypes of muscarinic cholinergic receptors, and which subtype(s) is involved in spinal antinociception and interaction with {alpha}2-adrenergic agonists is unclear. In normal rats, intrathecally administered muscarinic agonists produce antinociception to acute noxious heat stimuli with a pharmacology consistent with m1 and/or m2 receptors (7) or m1 and/or m3 receptors (8). However, some behavioral studies argue against the role of m1 receptors in antinociception (9,10), and one immunohistochemical study suggests m1 receptors may not be present in rat spinal cord (11). Additionally, a role for m4 receptors in muscarinic antinociception has been suggested by use of an epibatidine analog with specificity for this subtype, and its antagonism by a highly selective m4 subtype antagonist, muscarinic toxin (MT)-3 (12).

These studies in normal rats do not address the subtype(s) of muscarinic receptors involved in reduction in tactile hypersensitivity by intrathecal clonidine after nerve injury. To investigate this, we used novel, subtype-preferring peptide antagonists. Several muscarinic antagonists have been isolated from the venom of African mamba snakes of the genus Dendroaspis angusticeps, including two that are the most selective ligands for m1 and m4 receptors known (13). MT-7 has a 10,000-fold greater affinity for the m1 subtype over all other receptors and MT-3 has a 40-fold greater affinity for m4 over m1 and approximately 500-fold greater affinity for m4 over m2, m3, and m5 receptors (13). Using these two subtype specific antagonists, we investigated which muscarinic receptor subtype is more closely related to clonidine-induced antinociception after spinal nerve root ligation in rats, a model of neuropathic pain. In addition, we quantified the amount of m1 or m4 muscarinic receptor subtype protein in the spinal cord of nerve root ligated rats using Western blot analysis to determine whether the level of receptor expression was altered by peripheral nerve injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Surgical Preparation and Behavioral Testing
Male Harlan Sprague-Dawley rats weighing 200–225 g were used, and all procedures were approved by the Animal Care and Use Committee. Rats were anesthetized with halothane, and the left L5 and L6 spinal nerves were isolated and ligated tightly with 6-0 silk suture as previously described (14).

At the same time, prestretched polyethylene-10 catheters were introduced into the intrathecal space through a 20-gauge hypodermic needle inserted into the lumbar intrathecal space as previously described (15). Correct location of the intrathecal catheter was confirmed by injection of 10 µL of 2% lidocaine, 1 day after the operation and observation of transient hindlimb motor blockade. All pharmacologic experiments were conducted 2–3 wk after spinal nerve ligation.

Withdrawal threshold to tactile stimulation was determined before and after spinal nerve ligation in all animals. After 30 min of acclimation, a series of calibrated, handmade von Frey filaments (0.9–27.9 g) were applied perpendicularly to the plantar surface of the left paw with a force to bend the filament for 5 s. Filaments of increasing force were applied until the rat withdrew or flinched its paw. Two minutes later, a filament of the next lesser force was applied, and threshold determined by an up-down method previously described (16).

To determine which subtype of muscarinic receptor is more related to the analgesic effect of intrathecally administered clonidine, m1 (MT 7; Peptide Institute, Osaka, Japan) and m4 (MT 3; Sigma, St. Louis, MO) receptor antagonists from African mamba snake venom were used. Preliminary experiments indicated that doses up to 10 µg of each toxin were well tolerated without prolonged behavioral effects, so this was the maximal dose used.

After determination of baseline thresholds of withdrawal, saline (n = 6) or one of either antagonist (n = 6–7) in doses of 1 or 10 µg was administered intrathecally and the withdrawal threshold was determined 30 min later to determine the effect of antagonists themselves. Then 15 µg of clonidine was injected intrathecally. Withdrawal threshold was determined at 30, 60, 90, and 120 min after clonidine injection.

Drugs for intrathecal injection were dissolved in normal saline, except MT-7, which was dissolved in sterile distilled water according to the manufacturer’s instructions. Intrathecal injections were performed with a volume of 10 µL, and followed by a 15-µL flush with normal saline.

Western Analysis
Male Harlan Sprague-Dawley rats weighing 200–225 g were prepared under halothane anesthesia. In 8 animals, the left L5 and L6 spinal nerves were isolated and ligated tightly with 6-0 silk suture as described above. Another group of eight animals was prepared with the same surgical procedure, but with only exposure, and not ligation of spinal nerves. These animals served as sham controls. Mechanical withdrawal threshold was determined using von Frey filaments before and after the surgery in all animals. Animals were killed between 4–5 wk after surgery by decapitation under deep halothane anesthesia and spinal cords were rapidly dissected on ice. Dorsal parts of the lumbar spinal cords were collected and divided into left (ipsilateral) and right (contralateral) halves. Samples were mechanically disrupted by sonication in 10 mM Tris-HCl, and 1% sodium dodecyl sulfate (SDS) buffer solution (pH 7.4) on ice, and boiled at 100°C for 5 min and then cooled on ice for 5 min. Cells were lysed with a solution containing 4% SDS, 20% glycerol, 0.02% bromphenol blue, and 200 mM dithiothreitol in 0.5 M Tris-HCl (pH 6.8). To visualize m1 and m4 receptor subtype proteins, proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, electroblotted onto pure nitrocellulose membranes (Bio-Rad, Hercules, CA), and blocked overnight with 2% normal goat serum in Tris-buffered saline/Tween 20 buffer. All primary and secondary antibodies were applied in the same buffer. Primary rabbit polyclonal antibodies specific for m1 and m4 receptors were purchased from Research & Diagnostic Antibodies (Benicia, CA). These antibodies were generated using synthetic peptide analogs of carboxyl termini of these proteins (17). The m1 receptor antibody was applied in a dilution of 1:800 and the m4 was applied in a dilution of 1:400. Then, purified horseradish peroxidase coupled-goat anti-rabbit immunoglobulin G antibody (Sigma) diluted 1:2500 was applied. The blots were developed using the enhanced diaminobenzidine reaction, digitally scanned, and quantified using SigmaGel (SPSS Inc., Chicago, IL). Density of bands at the appropriate molecular weights for these receptors on each gel (17) were normalized across gels to bands from a lane on each gel containing 50 µg of pooled protein from normal rat spinal cord.

Data were presented as mean ± SEM and were analyzed by two-way repeated-measures ANOVA followed by Dunnett’s test. A P value < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mechanical withdrawal threshold before spinal nerve ligation was 19 ± 2.7 g. Withdrawal threshold decreased to 2.1 ± 0.64 g within 2 wk after the surgery and was maintained thereafter. Intrathecal catheters remained in the intrathecal space until the experiment had finished in all animals, as determined by response to lidocaine testing at the end of experiments.

Antagonist Studies
Intrathecal injection of MT-7 did not change the baseline withdrawal threshold 30 min later (Fig. 1). Additionally, animals showed no remarkable change in spontaneous activity with MT-7 treatment. Intrathecal injection of 15 µg of clonidine increased the withdrawal threshold within 30 min and this increase remained stable until about 120 min in intrathecal saline pretreated rats (Fig. 1). Clonidine injection was accompanied by decreased spontaneous locomotion and by urination. The antihypersensitivity effect of intrathecal clonidine was not changed significantly by pretreatment with 1 µg or 10 µg of MT-7. Similarly, behavioral sedation and urination produced by clonidine were not affected by MT-7 pretreatment.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Withdrawal threshold to von Frey filament testing in rats after spinal nerve ligation receiving, at time 0, either intrathecal saline (filled circles), or the m1 selective antagonist, muscarinic toxin (MT)-7, 1 µg (open circles) or 10 µg (filled triangles). Clonidine, 15 µg, was then injected at time 30 min. MT-7 did not affect withdrawal threshold 30 min after its injection, and did not affect the increase in withdrawal threshold produced by clonidine. Each value represents the mean ± SE of 6–7 animals.

 
Treatment with 1 µg of MT-3 intrathecally did not change the withdrawal threshold itself, but it significantly inhibited the antihypersensitivity effect produced by intrathecal clonidine (Fig. 2). Unlike our preliminary experiments in normal animals, injection of 10 µg of MT-3 in animals with spinal nerve ligation resulted in hyperactivity and increased withdrawal threshold from 2.0 ± 0.5 g before MT-3 to 7.6 ± 2.6 g after MT-3, precluding assessment of the effects of clonidine at this dose.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. Withdrawal threshold to von Frey filament testing in rats after spinal nerve ligation receiving, at time 0, either intrathecal saline (filled circles), or the m4 selective antagonist, muscarinic toxin (MT)-3, 1 µg (open circles). Clonidine, 15 µg, was then injected at time 30 min. MT-3 did not affect withdrawal threshold 30 min after its injection, and significantly reduced the increase in withdrawal threshold produced by clonidine. Each value represents the mean ± SE of six animals. *P < 0.05 compared with saline/clonidine group.

 
Western Analyses
m1 and m4 receptors were visualized by immunostained band of 60 and 65 kDa (17), respectively. There was no difference between left and right lumbar dorsal spinal cord in m1 receptor protein quantity after spinal nerve ligation, and these values did not differ from sham-operated controls (Fig. 3).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Western analysis of m1 (solid bars) and m4 (open bars) receptor protein in the lumbar dorsal horn on the right (contra = contralateral) and left (ipsi = ipsilateral) side in sham-operated animals and those with left L5 and L6 spinal nerve ligation. Each value represents the mean ± SE of six animals, and is expressed as the ratio of band density to a pooled spinal cord protein sample from normal animals run on each gel. There was no significant differences among or between groups for either m1 or m4 protein.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intrathecal clonidine reduces hypersensitivity after peripheral nerve injury by a direct interaction with spinal {alpha}2-adrenergic receptors (2). Unlike intrathecally administered opioids, which lose potency and efficacy after nerve injury, {alpha}2-adrenergic agonists maintain or even increase potency and efficacy (18), and epidural clonidine effectively treats neuropathic pain in patients in whom morphine has failed (3). The reasons for this pharmacologic plasticity of opioid and {alpha}2-adrenergic analgesia after nerve injury are unknown, but may reflect loss of opioid receptors by degenerating small-diameter afferents after nerve injury (19) and a new interaction between {alpha}2-adrenergic receptors to result in muscarinic receptor activation (6) and spinal nitric oxide synthesis (20) after nerve injury. Because clonidine is effective clinically, is totally reversed by spinal atropine in animals (6), and spinal injection of the cholinesterase inhibitor, neostigmine, is effective in patients with neuropathic pain (21), understanding the muscarinic receptor subtype involved in this interaction may lead to better approaches to the treatment of neuropathic pain.

Five muscarinic receptor subtypes have been identified by molecular cloning (22,23). Most tissues express multiple muscarinic receptors (24) involved in regulation of important physiologic functions (25). Muscarinic receptors have been demonstrated in the spinal cord of humans and animals using autoradiographic techniques (26,27), and intrathecal administration of cholinergic receptor agonists (5) or a cholinesterase inhibitor produces analgesia (28). However, it remains unclear which specific muscarinic receptor subtypes are involved in antinociception. Some radioligand binding studies indicated the presence of all muscarinic receptor subtypes in spinal cord (7,8,29), but others failed to observe one or more subtypes by immunohistochemistry or pharmacology (9–11). Presynaptic m3 receptors have been suggested in one study to enhance acetylcholine release and antinociception stimulated by peripheral formalin injection (30). In mice, central administration of antisense oligodeoxynucleotides to m1 receptors reduces antinociception by noxious heat (31). Knockout of m2 receptors in mice (32), but not m4 receptors (33), reduces antinociception from nonspecific cholinergic agonists. However, none of these studies examined the interaction of {alpha}2-adrenergic drugs and spinal cholinergic systems, and none of them addressed the complex plasticity that occurs after peripheral nerve injury and is thought to underlie neuropathic pain.

Because there are few ligands or agonists and antagonists that interact with muscarinic receptor subtypes with high selectivity, it is difficult to assign individual receptor subtypes to specific muscarinic functions. Several peptides have been isolated from snake venoms and used as pharmacologic tools. Muscarinic antagonists used in this study are isolated from the green mamba snake, Dendroaspis angusticeps, and two of them are now considered as the most selective ligands for m1 (MT-7) and m4 (MT-3) receptors available (13). Using this MT-3 toxin, in the same dose as the current study, Ellis et al. (12) suggested that m4 receptors located in the spinal cord might have a role as a mediator of antinociception in normal animals.

Although the current study, using these highly selective antagonists, supports a role for m4 receptors in the {alpha}2-adrenergic/muscarinic interaction to relieve mechanical hypersensitivity after peripheral nerve injury, we recognize that there are limitations to this interpretation. The distribution of these peptides in the intrathecal space and spinal cord and their nonspecific effects are unknown, but previous work with intrathecal injection (12) and the current study suggest that specific antagonism is possible with these drugs. We were limited in our ability to test whether MT-3 could fully antagonize the effect of clonidine because of behavioral disruption at the larger dose studied. This hyperactivity is consistent with a previous report that m4 receptor-deficient mice exhibit a significant increase in basal locomotor activity, likely because of uninhibited dopamine receptors (33). Finally, we did not test the relevance of other muscarinic subtypes than m1 and m4 in the current study.

Given the behavioral observation that m4 receptors may be important in the {alpha}2-adrenergic/muscarinic interaction, we quantified protein content in spinal cords of sham and nerve-injured animals for this subtype as well as the m1 receptor. Although no differences were observed, it is conceivable that alterations in locations of muscarinic receptors or in G protein coupling efficacy could underlie the increased reliance of {alpha}2-adrenergic agonists on spinal muscarinic mechanisms after peripheral nerve injury. These possibilities are now under investigation.

In summary, using intrathecal injection of highly selective muscarinic antagonists, the current study demonstrates a role for spinal m4, but not m1 receptors, in the antihypersensitivity effect of clonidine in peripheral nerve-injured rats. This is not reflected by a gross increase in the amount of m4 receptor protein in the lumbar dorsal spinal cord. These results add to the evidence that {alpha}2-adrenergic-mediated analgesia in neuropathic pain states is reliant on activation of cholinergic neurons in the spinal cord and on spinal muscarinic receptor stimulation.


   Acknowledgments
 
Supported in part by National Institutes of Health Grants GM35523 and NS41386.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Arner S, Meyerson BA. Lack of analgesic effect of opioids on neuropathic and idiopathic forms of pain. Pain 1988; 33: 11–23.[Web of Science][Medline]
  2. Yaksh TL, Pogrel JW, Lee YW, Chaplan SR. Reversal of nerve ligation-induced allodynia by spinal alpha-2 adrenoceptor agonists. J Pharmacol Exp Ther 1995; 272: 207–14.[Abstract/Free Full Text]
  3. Eisenach JC, DuPen S, Dubois M, et al. Epidural clonidine analgesia for intractable cancer pain. Pain 1995; 61: 391–9.[Web of Science][Medline]
  4. Detweiler DJ, Eisenach JC, Tong C, Jackson C. A cholinergic interaction in alpha2 adrenoceptor-mediated antinociception in sheep. J Pharmacol Exp Ther 1993; 265: 536–42.[Abstract/Free Full Text]
  5. Yaksh TL, Dirksen R, Harty GJ. Antinociceptive effects of intrathecally injected cholinomimetic drugs in the rat and cat. Eur J Pharmacol 1985; 117: 81–8.[Web of Science][Medline]
  6. Pan HL, Chen SR, Eisenach JC. Intrathecal clonidine alleviates allodynia in neuropathic rats: interaction with spinal muscarinic and nicotinic receptors. Anesthesiology 1999; 90: 509–14.[Web of Science][Medline]
  7. Iwamoto ET, Marion L. Characterization of the antinociception produced by intrathecally administered muscarinic agonists in rats. J Pharmacol Exp Ther 1993; 266: 329–38.[Abstract/Free Full Text]
  8. Naguib M, Yaksh TL. Characterization of muscarinic receptor subtypes that mediate antinociception in the rat spinal cord. Anesth Analg 1997; 85: 847–53.[Abstract]
  9. Sheardown MJ, Shannon HE, Swedberg MD, et al. M1 receptor agonist activity is not a requirement for muscarinic antinociception. J Pharmacol Exp Ther 1997; 281: 868–75.[Abstract/Free Full Text]
  10. Sauerberg P, Olesen PH, Sheardown MJ, et al. Muscarinic agonists as analgesics: antinociceptive activity versus M1 activity—SAR of alkylthio-TZTP’s and related 1,2,5-thiadiazole analogs. Life Sci 1995; 56: 807–14.[Web of Science][Medline]
  11. Höglund AU, Baghdoyan HA. M2, M3 and M4, but not M1, muscarinic receptor subtypes are present in rat spinal cord. J Pharmacol Exp Ther 1997; 281: 470–7.[Abstract/Free Full Text]
  12. Ellis JL, Harman D, Gonzalez J, et al. Development of muscarinic analgesics derived from epibatidine: role of the M4 receptor subtype. J Pharmacol Exp Ther 1999; 288: 1143–50.[Abstract/Free Full Text]
  13. Adem A, Karlsson E. Muscarinic receptor subtype selective toxins. Life Sci 1997; 60: 1069–76.[Web of Science][Medline]
  14. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992; 50: 355–63.[Web of Science][Medline]
  15. Storkson RV, Kjorsvik A, Tjolsen A, Hole K. Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods 1996; 65: 167–172.[Web of Science][Medline]
  16. Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53: 55–63.[Web of Science][Medline]
  17. Ndoye A, Buchli R, Greenberg B, et al. Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis. J Invest Dermatol 1998; 111: 410–6.[Web of Science][Medline]
  18. Puke MJ, Wiesenfeld-Hallin Z. The differential effects of morphine and the {alpha}2-adrenoceptor agonists clonidine and dexmedetomidine on the prevention and treatment of experimental neuropathic pain. Anesth Analg 1993; 77: 104–9.[Abstract/Free Full Text]
  19. Stevens CW, Kajander KC, Bennett GJ, Seybold VS. Bilateral and differential changes in spinal mu, delta and kappa opioid binding in rats with a painful, unilateral neuropathy. Pain 1991; 46: 315–26.[Web of Science][Medline]
  20. Xu Z, Chen SR, Eisenach JC, Pan HL. Role of spinal muscarinic and nicotinic receptors in clonidine-induced nitric oxide release in a rat model of neuropathic pain. Brain Res 2000; 861: 390–8.[Web of Science][Medline]
  21. Klamt JG, Dos Reis MP, Neto JB, Prado WA. Analgesic effect of subarachnoid neostigmine in two patients with cancer pain. Pain 1996; 66: 389–91.[Web of Science][Medline]
  22. Bonner TI, Buckley NJ, Young AC, Brann MR. Identification of a family of muscarinic acetylcholine receptor genes. Science 1987; 237: 527–32.[Abstract/Free Full Text]
  23. Kubo T, Fukuda K, Mikami A, et al. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 1986; 323: 411–6.[Medline]
  24. Levey AI. Immunological localization of m1–m5 muscarinic acetylcholine receptors in peripheral tissues and brain. Life Sci 1993; 52: 441–8.[Web of Science][Medline]
  25. Caulfield MP. Muscarinic receptors: characterization, coupling and function. Pharmacol Ther 1993; 58: 319–79.[Web of Science][Medline]
  26. Gillberg PG, Nordberg A, Aquilonius SM. Muscarinic binding sites in small homogenates and in autoradiographic sections from rat and human spinal cord. Brain Res 1984; 300: 327–33.[Web of Science][Medline]
  27. Kayaalp SO, Neff NH. Regional distribution of cholinergic muscarinic receptors in spinal cord. Brain Res 1980; 196: 429–36.[Web of Science][Medline]
  28. Hood DD, Mallak KA, Eisenach JC, Tong CY. Interaction between intrathecal neostigmine and epidural clonidine in human volunteers. Anesthesiology 1996; 85: 315–25.[Web of Science][Medline]
  29. Bartolini A, Ghelardini C, Fantetti L, et al. Role of muscarinic receptor subtypes in central antinociception. Br J Pharmacol 1992; 105: 77–82.[Web of Science][Medline]
  30. Honda K, Harada A, Takano Y, Kamiya H. Involvement of M3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice. Brain Res 2000; 859: 38–44.[Web of Science][Medline]
  31. Ghelardini C, Galeotti N, Bartolini A. Loss of muscarinic antinociception by antisense inhibition of M1 receptors. Br J Pharmacol 2000; 129: 1633–40.[Web of Science][Medline]
  32. Gomeza J, Shannon H, Kostenis E, et al. Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 1999; 96: 1692–7.[Abstract/Free Full Text]
  33. Gomeza J, Zhang L, Kostenis E, et al. Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 1999; 96: 10483–8.[Abstract/Free Full Text]
Accepted for publication January 22, 2003.




This article has been cited by other articles:


Home page
 Anesth. Analg.Home page
D.-H. Roh, H.-W. Kim, S.-Y. Yoon, H.-S. Seo, Y.-B. Kwon, H.-J. Han, A. J. Beitz, and J.-H. Lee
Intrathecal Clonidine Suppresses Phosphorylation of the N-Methyl-D-Aspartate Receptor NR1 Subunit in Spinal Dorsal Horn Neurons of Rats with Neuropathic Pain
Anesth. Analg., August 1, 2008; 107(2): 693 - 700.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, Y.-J.
Right arrow Articles by Eisenach, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, Y.-J.
Right arrow Articles by Eisenach, J. C.
Related Collections
Right arrow Trauma
Right arrow Regional Anesthesia
Right arrow Pain

Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2003 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press