peripheral nerve injury alters spinal nicotinic acetylcholine receptor pharmacology
TRANSCRIPT
European Journal of Pharmacology 590 (2008) 163–169
Contents lists available at ScienceDirect
European Journal of Pharmacology
j ourna l homepage: www.e lsev ie r.com/ locate /e jphar
Peripheral nerve injury alters spinal nicotinic acetylcholine receptor pharmacology
Tracey Young, Shannon Wittenauer, Renee Parker, Michelle Vincler ⁎Department of Anesthesiology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA
⁎ Corresponding author. Department of AnesthesioHealth Sciences, Medical Center Blvd., Winston-Salem, N3452; fax: +1 336 716 6744.
E-mail address: [email protected] (M. Vincler).
0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.06.020
A B S T R A C T
A R T I C L E I N F OArticle history:
Nicotinic acetylcholine rece Received 1 April 2008Received in revised form 22 May 2008Accepted 5 June 2008Available online 11 June 2008Keywords:Neuropathic painAntinociceptionNicotineNociception
ptors are widely expressed in the rat spinal cord and modulate innocuous andnociceptive transmission. The present studies were designed to investigate the plasticity of spinal nicotinicacetylcholine receptors modulating mechanosensitive information following spinal nerve ligation. A tonicinhibitory cholinergic tone mediated by dihydro-β-erythroidine- (DHβE) and methyllycaconitine- (MLA)sensitive nicotinic acetylcholine receptors was identified in the normal rat spinal cord and cholinergic tone atboth populations of nicotinic acetylcholine receptors was lost ipsilateral to spinal nerve ligation. Theadministration of intrathecal nicotinic acetylcholine receptor agonists reduced mechanical paw pressurethresholds with a potency of epibatidine=A-85380NNnicotineNcholine in the normal rat. Following spinalnerve ligation, intrathecal epibatidine and nicotine produced an ipsilateral antinociception, but intrathecal A-85380 and choline did not. The antinociceptive response to intrathecal nicotine was blocked with the α7⁎and α9α10⁎-selective nicotinic acetylcholine receptor antagonist, MLA, and the αβ heteromeric nicotinicacetylcholine receptor antagonist, DHβE. The antinociceptive effects of both intrathecal nicotine andepibatidine were mediated by GABAA receptors. Spinal [3H]epibatidine saturation binding was unchanged inspinal nerve-ligated rats, but spinal nerve ligation did increase the ability of nicotine to displace [3H]epibatidine from spinal cord membranes. Spinal nerve ligation altered the expression of nicotinicacetylcholine receptor subunits ipsilaterally, with a large increase in the modulatory α5 subunit. Takentogether these results suggest that pro- and antinociceptive populations of spinal nicotinic acetylcholinereceptors modulate the transmission of mechanosensitive information and that spinal nerve ligation-inducedchanges in spinal nicotinic acetylcholine receptors likely result from a change in subunit composition ratherthan overt loss of nicotinic acetylcholine receptor subtypes.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
A preponderance of behavioral evidence supports the importance ofspinal nicotinic acetylcholine receptors in the transmission of nocicep-tive stimuli. Intrathecal administration of nicotinic acetylcholinereceptor agonists increases blood pressure and heart rate, and producesagitation, nociceptivebehaviors (e.g., vocalizations), and antinociceptionto thermal stimuli (Khan et al., 1994a,b, 1997). Careful pharmacologicalstudies conducted over the past several years have shown that differentnicotinic acetylcholine receptor subtypes, located on distinct spinalstructures, are responsible for each of these responses (Khan et al., 2001,2004; Rashid and Ueda, 2002). Importantly, the nociceptive and
logy, Wake Forest UniversityC 27157, USA. Tel.: +1 336 716
l rights reserved.
antinociceptive responses can be attributed to different nicotinicacetylcholine receptor subtypes (Rueter et al., 2000).
In the rat central nervous system, eight α (α2–α7, α9–α10) andthree β (β2–β4) subunits have been identified (Le Novere et al., 2002;Léna et al., 1999; Lips et al., 2002). Heterologous expression andknockout experiments have identified numerous heteropentamericcombinations as well as some homopentameric combinations (α7,α9) of these subunits. The most prevalent nicotinic acetylcholinereceptors in rat brain are the α4β2⁎ and α7⁎ receptors (the asteriskindicates the native subunit composition is unknown (Lukas et al.,1999; Marks et al., 1986). In contrast, spinal cord nicotinic acetylcho-line receptors have been far less characterized. Radioligand bindingstudies support the presence of several distinct populations ofnicotinic acetylcholine receptor subtypes in the spinal cord (Khanet al., 1997, 1994b).
In the spinal cord, nicotinic acetylcholine receptors are expressed onprimary afferents (Genzen and McGehee, 2003; Miao et al., 2004;Roberts et al., 1995; Khan et al., 2004; Li et al., 1998), descendingnoradrenergic (Li et al., 2000) and serotoninergic (Cordero-Erausquin
Fig. 1. Dose–response relationships of intrathecal nicotinic acetylcholine receptorantagonists (A) and agonists (B) on paw withdrawal threshold to mechanical pressure inthe normal rat. A) The mean percent of baseline paw withdrawal thresholds± S.E.M.following the intrathecal administration of nicotinic acetylcholine receptor antagonistsdihydro-β-erythroidine (DHβE) and methyllycaconitine (MLA) are shown. B) The meanpercent of baseline paw withdrawal thresholds±S.E.M. following the intrathecaladministration of nicotinic acetylcholine receptor agonists epibatidine, A-85380, nicotine,and choline are shown. Each data point represents the mean±S.E.M of 8 animals.
164 T. Young et al. / European Journal of Pharmacology 590 (2008) 163–169
and Changeux, 2001) fibers presynaptically, as well as postsynapticallyon spinal inhibitory and excitatory neurons (Cordero-Erausquin et al.,2004; Genzen and McGehee, 2005; Bradaia and Trouslard, 2002a,b).Previous studies suggest that theα4β2⁎ andα7⁎ nicotinic acetylcholinereceptors on primary afferent C-fibers are likely responsible for thenociceptive responses while an α3β4⁎ or a previously undescribednicotinic acetylcholine receptor may be responsible for the antinoci-ceptive properties (Khan et al., 2001; Rueter et al., 2000).
Peripheral nerve injury produces a variety of changes within thespinal cord both ipsilaterally and contralaterally, including changes inthe expression of nicotinic acetylcholine receptors (Yang et al., 2004).Transection of the sciatic nerve greatly upregulates transcripts for theα5 and β2 nicotinic acetylcholine receptor subunits within the spinalcord dorsal horn (Yang et al., 2004). Spinal nerve ligation also increasedthe numbers of cells expressing the α3 subunit and the number offibers expressing the α5 subunit (Vincler and Eisenach, 2004). Thebehavioral implications of injury-induced changes in spinal nicotinicacetylcholine receptors have not been investigated thoroughly. Partialsciatic nerve injury in the mouse results in an increased spinalantinociceptive potency of nicotinic agonists and a loss of cholinergic-stimulated GABAergic inhibitory tone atα4β2⁎ nicotinic acetylcholinereceptors (Rashid and Ueda, 2002; Rashid et al., 2006). In the rat, tibialnerve transection results in a novel, antinociceptive effect of spinalnicotinic acetylcholine receptor agonists by increasing spinal glyciner-gic transmission (Abdin et al., 2006).
The current series of studieswere undertaken to further examine therole of spinal nicotinic acetylcholine receptorsmodulating the transmis-sion of nociceptive mechanical stimuli and to define injury-inducedchanges in nicotinic acetylcholine receptor function that may underliechanges in spinal nicotinic acetylcholine receptor pharmacology.
2. Materials and methods
2.1. Animals
All animals used in this study were male Sprague–Dawley rats(200–250 g; Harlan, IN), housed in pairs prior to surgery andindividually post-catheter implantation with free access to food andwater. Protocols and procedures were approved by the Animal Careand Use Committee (Wake Forest University Health Sciences,Winston-Salem, NC).
2.1.1. Surgical preparations
2.1.1.1. Intrathecal catheter implantation. Lumbosacral intrathecalcatheters were implanted as described previously (Storkson et al.,1996), with slight modifications (Milligan et al., 1999). Cathetersconsisted of PE-10 tubing stretched to reduce the overall diameter.Briefly, under halothane anesthesia, an incision was made in the skinof the lower back and a sterile 20 G needlewas used as a guide cannulaand was inserted between the L5 and L6 vertebrae. A tail flickconfirmed entry into the intrathecal space. The stretched PE10catheter containing a guide wire was gently fed through the needleuntil the catheter extended 3 cm beyond the tip of the needle to reachthe lumbar enlargement. The needle and guide wire were gentlyremoved. A loosely tied knot was made in the catheter and threesutures were used to hold the catheter in place. A small fistula (amodified 1 cc syringe hub (Milligan et al., 1999)) was sutured to themuscle surface and the catheter was fed through the fistula. Theremaining externalized catheter was coiled into the fistula and therubber plug sealed with a small amount of silicon sealant. The deadspace of the catheter ranged from 7–10 µl and, therefore, all drugadministrations were followed by a 10 µl saline flush.
2.1.1.2. Spinal nerve ligation. Rats underwent spinal nerve ligation asdescribed previously (Kim and Chung, 1992). Under halothane
anesthesia (2–3% halothane in 100% oxygen), the left L5 and L6 spinalnerves were isolated adjacent to the vertebral column and tightlyligated with 6.0 silk suture. The incision was closed and the animalsreturned to their home cages for 12–14 days post-ligation to allow forthe development of mechanical allodynia.
2.2. Behavioral testing
All behavioral testing was conducted 12–14 days post-surgerybetween the hours of 9:00 AM and 4:00 PM. Paw withdrawalthresholds were determined for left and right hind paws using theRandall–Selitto paw pressure technique (Randall and Selitto, 1957).The Analgesy-meter (Ugo Basile, Italy) uses a conical Teflon applicatorto apply a constant rate of increasing pressure (16 g per second) to thehind paws. The cut-off pressure was set at 250 g. Prior to experimentaltesting, animals were first subjected to 4 training sessions prior tospinal nerve ligation to stabilize baseline responses (Taiwo et al.,1989). Each hind paw was tested 2 times with a 5 min intertrialinterval. In spinal nerve-ligated rats, the mean paw withdrawalthresholds for the ipsilateral and contralateral hind paws werecompared to determine the presence of mechanical hypersensitivity.Mechanical hypersensitivity was defined as the presence of at least a40% decrease in paw withdrawal thresholds for the ipsilateral hindpaw.
For pharmacological testing, all drugswere dissolved in sterile 0.9%saline and administered intrathecally in a volume of 10 µl. Pawwithdrawal thresholds were measured at 5, 10, 15, 30, and 60 minfollowing the intrathecal administration of agonists and antagonists.When administered in combination, the antagonists were adminis-tered 10 min prior to agonists. Data are expressed as the mean pawwithdrawal thresholds±S.E.M. in grams or as a percentage of pre-drugbaseline responses (% Baseline=Post-drug paw withdrawal threshold/Pre-drug paw withdrawal threshold×100).
Fig. 2. Spinal nerve ligation alters the pharmacology of intrathecal nicotinic acetylcholine receptor agonists and antagonists. A) Intrathecal epibatidine (Epibat) and nicotine (Nic)showed a novel, unilateral antinociceptive response on the hind paw ipsilateral (Ipsi) to ligation at concentrations that produced no effect or pronociceptive responses contralaterally(Contra). B) Intrathecal administration of A-85380 and choline failed to produce antinociception in spinal nerve-ligated rats. C) Antinociceptive cholinergic tone is lost at spinalDHβE-sensitive nicotinic acetylcholine receptors ipsilateral (Ipsi) to spinal nerve-ligated while this tone remains apparent on the contralateral (Contra) hind paw. Changes in pawwithdrawal thresholds (PWTs) in grams of spinal nerve-ligated and uninjured (Normal) rats to 10 nmol intrathecal DHβE over time are shown. D) Antinociceptive cholinergic tone islost at spinal MLA-sensitive nicotinic acetylcholine receptors ipsilateral (Ipsi) to spinal nerve ligation but not contralaterally (Contra). Changes in pawwithdrawal thresholds (PWT) ingrams (g) of spinal nerve-ligated and uninjured (Normal) rats to 11 nmol MLA over time are shown. Data are depicted as the mean percent of baseline pawwithdrawal thresholds±S.E.M. (A and B) or mean paw withdrawal thresholds±S.E.M. (C and D) for 8–10 animals per experimental group. In A and B, significant antinociceptive (# Pb0.05) and pronociceptive(⁎ Pb0.05, ⁎⁎ Pb0.01, ⁎⁎⁎ Pb0.005) effects are indicated. In C and D, the pronociceptive of DHβE and MLA on the contralateral hind paw in spinal nerve-ligated rats (^ Pb0.05,^^ Pb0.01) and in normal rats (⁎⁎ Pb0.01, ⁎⁎⁎ Pb0.005).
165T. Young et al. / European Journal of Pharmacology 590 (2008) 163–169
2.3. Radioligand binding
2.3.1. Spinal cord tissue preparationThe dorsal half (normal rats) or ipsilateral dorsal quadrant (spinal
nerve-ligated rats) of the L4–L6 spinal cord tissue was placed in 10volumes (w/v) of ice-cold hypotonic buffer (14.4 mMNaCl, 0.2 mM KCl,0.2mMCaCl2, 0.1mMMgSO4, 2.0mMHEPES, pH 7.5) and homogenizedusing a Kinematica polytron. Homogenized sampleswere centrifuged at25,000 g for 15min. The pelletwas resuspended in hypotonic buffer andagain centrifuged. The resuspension/centrifugation cycle was repeatedtwo more times. The resulting pellet was stored frozen at −80 °C underfresh hypotonic buffer until ready for use.
2.3.2. Radioligand bindingAt the time of assay, the pellet was thawed and resuspended with
Tris–HCl buffer (50mMTris–HCl,120mMNaCl, 5mMKCl,1mMMgCl2,2 mM CaCl2, pH 7.5) supplemented with 0.1 mM PMSF and 5 mMiodoacetamide. The assay mixture consisted of 200 µg of membraneprotein in a final incubation volume of 60 µl. Incubations were carriedout in a cold room on a gentle shaker for 60 min. Assays were initiatedwith the addition of the membrane suspension with rapid mixing topolypropylene tubes containing tritiated ligands. Incubations wereterminated by the addition of 3 ml of ice-cold assay buffer followed byrapid filtration through Whatman GF/B filter papers previouslyequilibrated with 0.5% polyethyleneimine at 4 °C using a Brandel cellharvester. Samples were then washed four times with 4 ml of ice-coldassay buffer (Tris–HCl buffer supplemented with 10 µM atropinesulfate). The filters were then placed in counting vials, mixedvigorously with scintillation fluid and counted the next day in aBeckman Coulter LS 6500 liquid scintillation counter. All assays were
done in triplicate and protein was assayed by the Bradford proteinassay.
For saturation binding, final concentrations of (±)-[3H]epibatidine(Perkin-Elmer, USA) varied between 0.02 nM and 4.3 nM; stocksolutions are prepared in assay buffer. Nonspecific binding isdetermined by including 40 µM nicotine. For competition binding, aconcentration of 0.8 nM (±)-[3H]epibatidine was used with variousconcentrations of nicotine or cytisine.
2.4. Western blotting
The lower lumbar (L4–L6) spinal cord was removed and the dorsalhalf (normal rats) or ipsilateral dorsal quadrants (spinal nerve-ligatedrats) were homogenized in lysis buffer (Mammalian Cell Lysis Kit,Sigma). Protein concentrations were quantified using a Bio-RadProtein Assay Kit, aliquoted (50 µg), and stored at −80 °C until use.Proteins were separated using SDS-PAGE electrophoresis in a 10%acrylamide gel (Bio-Rad) and transferred to a nitrocellulose mem-brane. Membranes were blocked with 5% dehydrated milk andincubated with primary antibody generated against the α4 (rabbitpolyclonal, Santa Cruz Biotechnology; 1:500), α5 (goat polyclonal,Santa Cruz Biotechnology, Inc.; 1:100),α7 (goat polyclonal, Santa CruzBiotechnology; 1:500), and β2 (rabbit polyclonal, Santa Cruz Biotech-nology; 1:100) nicotinic acetylcholine receptor subunits overnight at4 °C. Membranes werewashedwith 0.01M PBS and incubated with anHRP-conjugated secondary antibody for 1 h at room temperature.Protein bands were visualized with a chemiluminescence detectionsystem (Amersham) and exposed to autoradiography film.Membraneswere then stripped using ReblotWestern Blot Recycling Kit (Millipore)and re-probed with an antibody to β-actin (Cell Signaling; 1:1000). β-
Fig. 3. The antinociceptive effects of intrathecal nicotine and epibatidine in spinal nerve-ligated rats. A)Theantinociceptive effects of intrathecal nicotine (2.2nmol) and epibatidine(0.36 nmol) are blocked by pretreatment with the GABAA receptor antagonist bicuculline(0.3 µg). Significant pronociceptive (⁎ Pb0.05) and antinociceptive (# Pb0.05) effects areshown. ^ Pb0.05 compared to nicotine or epibatidine treatment alone. B) Theantinociceptive effects of intrathecal nicotine (2.2 nmol) are blocked by pretreatmentwith DHβE (10 nmol) or MLA (11 nmol) in spinal nerve-ligated rats. ⁎ Pb0.05 compared tonicotine administration alone. Data are shown as the mean percent of baseline pawwithdrawal thresholds±S.E.M. for 8–10 animals per treatment group.
Fig. 4. Radioligand binding of [3H]epibatidine in spinal cord membranes from normaland spinal nerve-ligated rats. A) Saturation radioligand binding of [3H]epibatidine inmembranes prepared from the dorsal half (Normal) or ipsilateral dorsal quadrant(spinal nerve ligation, SNL) of the lower lumbar spinal cord. Inset is the Scatchard plotshowing the presence of high and low affinity sites. B) Competitive radioligand bindingof nicotine displacement of [3H]epibatidine in membranes prepared from the dorsalhalf (Normal) or ipsilateral dorsal quadrant (SNL) of the lower lumbar spinal cord.
166 T. Young et al. / European Journal of Pharmacology 590 (2008) 163–169
Actin was visualized as described above. Protein band density wasmeasured using SigmaScan Pro 5.
2.5. Materials
Nicotine hydrate tartrate salt, (±)-epibatidine dihydrochloride, 3-(2(S)-Azetidinylmethoxy) pyridine HCl (A-85380), choline chloride, (−)-bicuculline methiodide, dihydro-β-erythroidine, methyllycaconitine,and all chemical components of buffers were purchased from Sigma.[3H]-Epibatidine was purchased from Perkin-Elmer.
2.6. Statistics
Behavioral pharmacology was analyzed using one-way or two-wayrepeated measures ANOVA where indicated. Radioligand bindingcurves were calculated by nonlinear regression and were comparedbetween groups using the four parameter Hill equation in Prism 4(GraphPad).
3. Results
3.1. Pro- and antinociceptive populations of spinal nicotinic acetylcholinereceptors modulate the transmission of nociceptive mechanical stimuli
To assess the functionality of spinal nicotinic receptors in themodulation of mechanosensitive stimuli, normal rats were adminis-tered 2 commonly used nicotinic acetylcholine receptor antagonistsintrathecally and paw withdrawal thresholds to mechanical pressure
were measured. Baseline pawwithdrawal thresholds ranged from 135to 149 g and data were normalized to baseline paw withdrawalthresholds for each rat prior to drug administration. Both nicotinicacetylcholine receptor antagonists tested dose-dependently reducedpaw withdrawal thresholds (Fig. 1A). Dihydro-β-erythroidine (DHβE),which functionally blocks αβ heteromeric nicotinic acetylcholinereceptors with some preference for the α4β2 subtype, dose-dependently reduced paw withdrawal thresholds following theadministration of 2, 10, 28, or 140 nmol [F(4,28)=8.4, Pb0.001].Intrathecal methyllycaconitine (MLA), which preferentially blocks α7and α9α10 nicotinic acetylcholine receptors, dose-dependentlyreduced paw withdrawal thresholds following the administration of1, 11, or 57 nmol [F(3,20)=14, Pb0.0001]. The greatest reductions inpawwithdrawal thresholds following DHβE and MLAwas observed at15 min with a complete return to baseline levels at 60 min (datashown in Fig. 2C and D).
Intrathecal administration of nicotinic acetylcholine receptoragonists also reduced paw withdrawal thresholds to mechanicalpressure in normal rats with a rank order potency of A-85380=epi-batidineNNnicotineNcholine (Fig. 1B). The reduction of paw with-drawal thresholds was dose-dependent for each agonist. A-85380,which is selective for β2-containing nicotinic receptors, significantlyreduced paw withdrawal thresholds [F(3,32) =17.5, Pb0.001].Intrathecal administration of epibatidine [F(6,43)=4.1, Pb0.005] andnicotine [F(4,25)=6.2, Pb0.005], which are potent agonists atheteromeric nicotinic acetylcholine receptors, also produced dose-dependent reductions in nociceptive thresholds. Choline, an α7⁎selective nicotinic receptor agonist, also produced significant mechan-ical hypersensitivity [F(3,17)=6.6, Pb0.01]. The peak effect of nicotinicacetylcholine receptor agonists ranged from 5–15 minwith significanthypersensitivity remaining at 45 min at the highest doses adminis-tered (data not shown).
Table 1[3H]Epibatidine binding to spinal cord membranes
KD1 (nM) Bmax1 (fmol/mg) KD2 (nM) Bmax2 % High affinity
Normal 0.079±0.05 1.7±0.6 1.74±0.1 27.5±4 5.8±2SNL 0.076±0.01 2.6±0.2 2.432±0.7 33.6±2 7.2±1
Mean affinities (KD) and number of binding sites (Bmax)±S.E.M. for high and low [3H]epibatidine-sensitive nAChRs in normal and spinal nerve-ligated (SNL) rats. The meanpercentage of high affinity sites of total [3H]epibatidine binding sites±S.E.M. is also shown.
Table 2Displacement of [3H]epibatidine binding to spinal cord membranes by nicotine
IC50 (nM) % Total binding
High affinity Low affinity High affinity Low affinity
Normal 16.2±1.5 531.6±1.4 41.59±7% 58.41±7%SNL 8.406±1.7 401.8±1.7 43.5±8% 56.5±8%
Mean IC50 IC50±S.E.M. of nicotine displacement of 0.8 nM [3H]epibatidine to spinal cordmembranes fromnormal and spinal nerve-ligated (SNL) rats. Themean percentage±S.E.M. of high and low affinity nicotine-sensitive sites in spinal cord membranes fromnormal and SNL rats. Italicized text indicates statistical significance of Pb0.05.
Fig. 5. Spinal nerve ligation differentially alters nicotinic acetylcholine receptor subunitexpression in the dorsal horn of the lower lumbar rat spinal cord. A) The expression ofthe α4 subunit is significantly decreased (^ Pb0.05) ipsilateral (Ipsi) to spinal nerveligation (SNL) compared to the levels of expression in the normal rat spinal cord. Theexpression of the α5 and β2 subunits is significantly increased (⁎ Pb0.05) compared tothe expression of these subunits in the normal rat spinal cord. Data are expressed as themean percentage of the levels of expression in normal rats (% Normal)±S.E.M. of 4separate experiments per nicotinic acetylcholine receptor subunit. B) RepresentativeWestern blots for nicotinic acetylcholine receptor subunit expression in the spinal cordsof normal (Nor) rats and SNL rats ipsilateral (Ipsi) and contralateral (Contra) to ligation.
167T. Young et al. / European Journal of Pharmacology 590 (2008) 163–169
3.2. Spinal nerve ligation alters nicotinic acetylcholine receptorpharmacology
Rats underwent spinal nerve ligation and mechanical hypersensi-tivity developed 14 days post-ligation. Paw withdrawal thresholdsdecreased from143±5 g at baseline to 73±4 g in the ipsilateral hindpaw14 days post-spinal nerve ligation. No change in baseline pawwithdrawal thresholdswas observed on the contralateral side. Intrathe-cal epibatidine significantly increased paw withdrawal thresholds onthe ipsilateral hind paw at 0.036 pmol and 0.36 pmol while these samedoses produced no or pronociceptive effects, respectively, on thecontralateral side (Fig. 2A). At a higher dose of epibatidine (360 pmol),however, a pronociceptive effect was observed bilaterally. Similar toepibatidine, intrathecal nicotine produced antinociceptive effectsipsilateral to spinal nerve ligation at lower doses (2.2 and 6.5 nmol)while these same doses produced no significant effect on thecontralateral side. This antinociceptive effect could be overcome athigherdoses,with 22nmol reducingpawwithdrawal thresholds equallyon the ipsilateral and contralateral sides. Conversely, theα7 andα9α10nicotinic acetylcholine receptor agonist, choline, did not produceantinociception. Intrathecal choline produced either no effect orreduced paw withdrawal thresholds equally in the ipsilateral andcontralateral hindpaws. Similarly, intrathecal A-85380 failed to produceantinociception in spinal nerve-ligated rats (Fig. 2B), although thepharmacology was modified compared to normal rats.
Spinal nerve ligation produced significant mechanical hypersensi-tivity ipsilateral to ligation with mean paw withdrawal thresholdsranging from 84–95 g 14 days post-ligation (Fig. 2C and D). Theintrathecal administration of DHβE (10 nmol) to spinal nerve-ligatedrats significantly reduced paw withdrawal thresholds on the hindpawcontralateral to ligation [F(5,47)=3.7, Pb0.01] but had no effect on theipsilateral hind paw (Fig. 2C). MLA (11 nmol) produced a similar effectwhen administered intrathecally to spinal nerve-ligated rats (Fig. 2D),significantly reducing paw withdrawal thresholds contralateral tospinal nerve ligation [F(5,22)=2.9, Pb0.05], but not ipsilaterally.
3.3. Spinal nicotinic acetylcholine receptor agonist antinociception ismediated by GABA
The antinociceptive effects of intrathecal epibatidine (0.36 pmol) andnicotine (2.2 nmol) ipsilateral to spinal nerve ligationwere antagonizedby intrathecal administration of the GABAA receptor antagonist bicucul-line (Fig. 3A). Although bicuculline has been reported to producebehavioral hypersensitivity following intrathecal administration pre-viously, this dose of bicuculline (0.3 µg/10 µl) did not alter pawwithdrawal thresholds in normal or spinal nerve-ligated rats.
To determine which populations of nicotinic acetylcholine recep-tors contribute to the antinociceptive effects of intrathecal nicotine onthe ipsilateral hind paw, nicotinic acetylcholine receptor antagonistswere administered intrathecally 10 min prior to intrathecal nicotine.Fig. 3B shows the antinociceptive effect of 2.2 nmol intrathecal nicotineipsilateral to spinal nerve ligation. Similar to the rats in Fig. 2,intrathecal MLA (11 nmol.) and DHβE (10 nmol) do not alter pawwithdrawal thresholds ipsilateral to SNL 15 min post-administration(Fig. 3B, white bars). Pretreatment with MLA and DHβE, however,completely blocked the antinociceptive effect of intrathecal nicotine(Fig. 3B, gray bars).
3.4. Spinal nerve ligation-induced changes in epibatidine- and nicotine-sensitive nicotinic acetylcholine receptors in the rat spinal cord
Because spinal nerve ligation reduced the pronociceptiveresponses of intrathecal epibatidine and nicotine, we investigatedwhether changes in epibatidine and nicotine binding sites in theipsilateral dorsal horn could underlie these effects. Saturationradioligand binding of [3H]epibatidine was performed in spinal cordmembranes from the dorsal half (normal rats) or ipsilateral dorsalquadrant (spinal nerve-ligated rats) of the lower lumbar (L4–L6)spinal cord. Epibatidine binding was saturable in normal and spinalnerve ligation spinal cord membranes with 2 binding sites beingobserved (Fig. 4A and Table 1). Apparent KD values of 0.079±0.05 nMand 1.74±0.1 nM for the higher and lower affinity sites in the normalrat, respectively, were calculated by nonlinear regression. Spinal nerveligation did not alter [3H]epibatidine binding [F(4,52)=1.17, PN0.05] orthe percentage of [3H]epibatidine binding sites in the higher affinitypopulation (Table 1).
Competition radioligand binding of [3H]epibatidine (0.8 nM) bynicotine was performed to identify spinal nerve ligation-inducedchanges in nicotine-sensitive nicotinic acetylcholine receptor popula-tions. Nicotine bound 2 sites rat spinal cord membranes of normal ratswith IC50s of 16.2±1.5 and 531.6±1.4 for the higher and lower affinitysites, respectively (Fig. 4B and Table 2). Spinal nerve ligationsignificantly altered nicotine binding [F(1,68) =7.2, Pb0.0001],
168 T. Young et al. / European Journal of Pharmacology 590 (2008) 163–169
significantly reducing the IC50s of both the higher and lower affinitysites (Table 2).
3.5. Spinal nerve ligation-induced changes in spinal nicotinicacetylcholine receptor subunit expression
Changes in the expression of individual nicotinic acetylcholinereceptor subunit proteins by Western blotting following spinal nerveligation has not been reported previously. Spinal nerve ligationsignificantly altered the expression of α4, α5, and β2 nicotinicacetylcholine receptor subunits, but had no effect on the expression ofthe α7 subunit when compared to levels of expression in normal ratspinal cord (Fig. 5). The expression of the α4 subunit was significantlydecreased ipsilateral to spinal nerve ligation, while the expression ofthe α5 and β2 subunits was significantly increased.
4. Discussion
The results of these studies show that multiple populations ofspinal nicotinic acetylcholine receptors function to facilitate andinhibit the transmission of nociceptive mechanical stimuli in thenormal rat spinal cord. A variety of nicotinic acetylcholine receptoragonists reduced paw withdrawal thresholds in a dose-dependentmanner while the nicotinic acetylcholine receptor antagonists DHβEand MLA blocked a tonic cholinergic antinociceptive tone within thenormal rat spinal cord. Spinal nerve ligation differentially alterednicotinic acetylcholine receptor pharmacology resulting in theappearance of a novel unilateral antinociceptive effect of intrathecalepibatidine and nicotine, but not A-85380 or choline, and the loss ofapparent cholinergic tone at DHβE- and MLA-sensitive nicotinicacetylcholine receptors. The antinociceptive effects of intrathecalepibatidine and nicotine could be blocked with the GABAA receptorantagonist, bicuculline, supporting a role for nicotinic acetylcholinereceptor-evoked GABA release in SNL rats. The antinociceptive effectsof intrathecal nicotine in spinal nerve-ligated rats were antagonizedby both DHβE and MLA, nicotinic acetylcholine receptor subtypes thatwere identified as antinociceptive in normal rats. This suggests thatalthough the inhibitory cholinergic tone is lost ipsilateral to SNL, thereceptors themselves are still present. These spinal nerve ligation-induced changes in behavioral pharmacology could not be explainedby changes in the number of [3H]epibatidine binding sites or changesin affinity. However, nicotine-sensitive sites were altered by spinalnerve ligation. The underlying mechanism of this spinal nerveligation-induced change in nicotine affinity may be the largeupregulation of the modulatory α5 nicotinic acetylcholine receptorsubunit.
Our studies show the presence of an inhibitory cholinergic tone inthe spinal cord of the normal rat, similar to the cholinergic regulationof thermal stimuli reported in the mouse cord (Rashid and Ueda,2002; Rashid et al., 2006). In the mouse spinal cord, this tone ismediated by α4β2⁎ nicotinic acetylcholine receptors, but not α7⁎nicotinic acetylcholine receptors (Rashid et al., 2006). Similar to theseobservations, the intrathecal administration of DHβE in the currentstudies dose-dependently produced mechanical hypersensitivitysuggesting the presence of a population of α4β2⁎ nicotinic acetylcho-line receptors that function to inhibit the transmission of noxiousmechanical stimuli. However, DHβE is, at best, only moderatelyselective for α4β2⁎ nicotinic acetylcholine receptors and theconcentration of this antagonist at spinal α4β2⁎ nicotinic acetylcho-line receptors following intrathecal administration is unknown. Theselective nature of DHβE for α4β2⁎ nicotinic acetylcholine receptorsin this model is unclear, and, therefore, we limit our interpretation ofthe effects of DHβE to that of an αβ heteromeric nicotinic acetylcho-line receptor. In addition to the effects of DHβE, we observedpronociceptive effects of MLA at doses that produced no effect inthe mouse spinal cord (Rashid et al., 2006). Differences in spinal
nicotinic acetylcholine receptor pharmacology between rats and micehave been noted previously (Damaj et al., 2000; Khan et al., 2001).
The intrathecal administration of nicotinic acetylcholine receptoragonists in the normal rat spinal cord has been reported previously toproduce a short-lived thermal antinociception and a more prolongedtouch-evoked hypersensitivity (Khan et al., 2001). Our resultsdemonstrate that the intrathecal administration of nicotine, epibati-dine, A-85380, and choline only reduced paw withdrawal thresholdsto mechanical pressure with the expected potencies. Previous studieshave shown that the nocifensive behaviors (e.g., vocalization) ofintrathecal nicotinic acetylcholine receptor agonists are mediated viaglutamate release following the direct activation of nicotinic acet-ylcholine receptors on primary afferent C-fibers (Khan et al., 1998,1996). However, mechanical stimulation is transmitted to the spinalcord by Aδ and Aβ fibers which express nicotinic acetylcholinereceptor subtypes distinct to those localized on C-fibers (Rau et al.,2005). The dependence of the pronociceptive effect of intrathecalnicotinic acetylcholine receptor agonists on mechanical paw with-drawal thresholds on the release of glutamate was not examined inour studies.
Peripheral nerve injury altered spinal endogenous inhibitory tonewith an apparent loss of cholinergic tone at αβ heteromeric (DHβE-sensitive) and α7⁎ or α9α10⁎ (MLA-sensitive) nicotinic acetylcholinereceptors. The antinociceptive populations of nicotinic acetylcholinereceptors stimulated by intrathecal nicotinic agonists following spinalnerve ligation appear to be comprised of the same antinociceptivenicotinic acetylcholine receptors under tonic cholinergic regulation inthe normal rat spinal cord. Nicotine-induced antinociception ipsilat-eral to spinal nerve ligation was mediated by a population of αβheteromers (DHβE-sensitive) and α7⁎ or α9α10⁎ (MLA-sensitive)nicotinic acetylcholine receptors, andwas dependent upon the releaseof GABA. The contribution of spinal GABA correlates with previousfindings in the mouse spinal cord (Rashid et al., 2006), but contrastswith the findings of Abdin et al. (2006) in the rat who reported thatthe neuropathy-specific effects of intrathecal nicotinic agonists weredependent on glycine, but not GABA release (Abdin et al., 2006). Thedifference between the studies of Abdin et al. (2006) and the currentstudy is likely due to the intensity of the mechanical stimuli used.Spinal GABAA and glycine receptors inhibit tactile allodynia (Sivilottiand Woolf, 1994; Sorkin et al., 1998), but only GABAA receptors inhibithypersensitivity to high intensity stimuli (Sorkin et al., 1998).
Similar to our observations with nicotinic antagonists, spinal nerveligation altered the behavioral pharmacology of nicotinic agonists.Nicotine and epibatidine produced antinociception on the hind pawipsilateral to spinal nerve ligation but produced pronociceptive effectscontralaterally. These results are similar to those of Rashid and Ueda(2002) and Abdin et al. (2006) who observed a neuropathy-specificantinociceptive effect of intrathecal nicotine and epibatidine followingperipheral nerve injury. In contrast, no antinociceptive effect ofintrathecal choline was observed, despite using a lower dose thanAbdin et al. (2006). The previous studies by Abdin et al. (2006)measured antinociception using von Frey filaments while the currentstudies utilized a more intense mechanical stimulus, paw pressure.Choline is a fairly weak antinociceptive agonist, exhibiting ananalgesic ED50 to thermal stimuli of approximately 0.47 µM and thisanalgesic effect is likely overcome at higher stimulus intensities suchas paw pressure (Damaj et al., 2000). The failure A-85380 to elicitantinociception in spinal nerve-ligated rats is likely due to thestimulation of a distinct population of spinal nicotinic acetylcholinereceptors. Epibatidine and A-85380 produce antinociception tothermal stimuli that is mediated via separable populations of spinalnicotinic acetylcholine receptors localized at different preterminalsites (Khan et al., 2001). The antinociceptive effects of intrathecal A-85380 rely on the release of norepinephrine whereas the antinoci-ceptive effects of epibatidine are not dependent on norepinephrinerelease (Khan et al., 2001).
169T. Young et al. / European Journal of Pharmacology 590 (2008) 163–169
Although the loss of epibatidine binding in the rat spinal cord hasbeen correlated previously with a reduced nociceptive response tointrathecal nicotinic acetylcholine receptor agonists, no changes inepibatidine binding were observed in spinal nerve ligation exhibitinginjury-induced antinociception. This suggests that an overt loss ofpronociceptive spinal nicotinic acetylcholine receptors does notaccount for spinal nerve ligation-induced antinociception. This ideais further supported by our behavioral data showing that thepronociceptive effects of intrathecal nicotine and epibatidine areelicited at higher doses.
Competitive nicotine binding showed a small but significantchange in affinity in spinal nerve-ligated rats; a result that is moreconsistent with a change in nicotinic acetylcholine receptor subunitcomposition rather than the up- or down-regulation of wholenicotinic acetylcholine receptor subtypes. The increased expressionof the modulatory α5 subunit presents a possible mechanismunderlying the subtle change in nicotine affinity. Inclusion of the α5subunit in heteromeric nicotinic acetylcholine receptors alters theaffinity ofmany nicotinic acetylcholine receptor agonists, although theeffect logically depends upon the identities of the α and β subunits.Consistent with the current study, the presence of theα5 subunit doesnot alter epibatidine saturation binding or the affinity of epibatidinefor α3β2 or α3β4 nicotinic acetylcholine receptors (Wang et al., 2002,1996). However, the rate of epibatidine-induced desensitization isincreased for both receptors when the α5 is included (Wang et al.,1996). The impact of α5 inclusion on nicotine affinity has not beenreported, but inclusion of the α5 subunit in α3β4 nicotinic acetylcho-line receptors reduces the EC50 for nicotine from 6.8 to 1.9 µM.Behaviorally, the increased expression of α5 in the spinal cordcontributes to the presence of mechanical hypersensitivity in SNL rats(Vincler and Eisenach, 2005). Therefore, the upregulation of the α5subunit in the spinal cord of SNL rats may alter the pharmacology ofendogenous ACh and exogenous nicotinic acetylcholine receptoragonists. The identification of which spinal nicotinic acetylcholinereceptors include the α5 subunit in SNL rats will be critical to a morethorough understanding of the observed changes in behavioralpharmacology.
Acknowledgement
This work was supported by NIH grant R01 NS048158 (M.V.).
References
Abdin, M.J., Morioka, N., Morita, K., Kitayama, T., Kitayama, S., Nakashima, T., Dohi, T.,2006. Analgesic action of nicotine on tibial nerve transection (TNT)-inducedmechanical allodynia through enhancement of the glycinergic inhibitory system inspinal cord. Life Sci. 80, 9–16.
Bradaia, A., Trouslard, J., 2002a. Fast synaptic transmission mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in lamina X neurones ofneonatal rat spinal cord. J. Physiol. 544, 727–739.
Bradaia, A., Trouslard, J., 2002b. Nicotinic receptors regulate the release of glycine ontolamina X neurones of the rat spinal cord. Neuropharmacology 43, 1044–1054.
Cordero-Erausquin, M., Changeux, J.P., 2001. Tonic nicotinic modulation of serotoni-nergic transmission in the spinal cord. Proc. Natl. Acad. Sci. U. S. A. 98, 2803–2807.
Cordero-Erausquin, M., Pons, S., Faure, P., Changeux, J.P., 2004. Nicotine differentiallyactivates inhibitory and excitatory neurons in the dorsal spinal cord. Pain 109,308–318.
Damaj, M.I., Meyer, E.M., Martin, B.R., 2000. The antinociceptive effects of alpha7nicotinic agonists in an acute pain model. Neuropharmacology 39, 2785–2791.
Genzen, J.R., McGehee, D.S., 2003. Short- and long-term enhancement of excitatorytransmission in the spinal cord dorsal horn by nicotinic acetylcholine receptors.Proc. Natl. Acad. Sci. U. S. A. 100, 6807–6812.
Genzen, J.R., McGehee, D.S., 2005. Nicotinic modulation of GABAergic synaptictransmission in the spinal cord dorsal horn. Brain Res. 1031, 229–237.
Khan, I.M., Taylor, P., Yaksh, T.L., 1994a. Cardiovascular and behavioral responses tonicotinic agents administered intrathecally. J. Pharmacol. Exp. Ther. 270, 150–158.
Khan, I.M., Taylor, P., Yaksh, T.L., 1994b. Stimulatory pathways and sites of action ofintrathecally administered nicotinic agents. J. Pharmacol. Exp. Ther. 271,1550–1557.
Khan, I.M., Marsala, M., Printz, M.P., Taylor, P., Yaksh, T.L., 1996. Intrathecal nicotinicagonist-elicited release of excitatory amino acids as measured by in vivo spinalmicrodialysis in rats. J. Pharmacol. Exp. Ther. 278, 97–106.
Khan, I.M., Yaksh, T.L., Taylor, P., 1997. Epibatidine binding sites and activity in the spinalcord. Brain Res. 753, 269–282.
Khan, I.M., Buerkle, H., Taylor, P., Yaksh, T.L., 1998. Nociceptive and antinociceptiveresponses to intrathecally administered nicotinic agonists. Neuropharmacology 37,1515–1525.
Khan, I.M., Stanislaus, S., Zhang, L., Taylor, P., Yaksh, T.L., 2001. A-85380 and epibatidineeach interact with disparate spinal nicotinic receptor subtypes to achieve analgesiaand nociception. J. Pharmacol. Exp. Ther. 297, 230–239.
Khan, I.M., Wennerholm, M., Singletary, E., Polston, K., Zhang, L., Deerinck, T., Yaksh, T.L.,Taylor, P., 2004. Ablation of primary afferent terminals reduces nicotinic receptorexpression and the nociceptive responses to nicotinic agonists in the spinal cord.J. Neurocytol. 33, 543–556.
Kim, S.H., Chung, J.M., 1992. An experimental model for peripheral neuropathyproduced by segmental spinal nerve ligation in the rat. Pain 50, 355–363.
Le Novere, N., Corringer, P.J., Changeux, J.P., 2002. The diversity of subunit compositionin nicotinic acetylcholine receptors: evolutionary origins, physiologic and pharma-cologic consequences. J. Neurobiol. 53, 447–456.
Léna, C., de Kerchove, D.'E., Cordero-Erausquin, M., Le Novere, N., Mar Arroyo-Jimenez,M., Changeux, J.P., 1999. Diversity and distribution of nicotinic acetylcholinereceptors in the locus ceruleus neurons. Proc. Natl. Acad. Sci. U. S. A. 96,12126–12131.
Li, X., Rainnie, D.G., McCarley, R.W., Greene, R.W., 1998. Presynaptic nicotinic receptorsfacilitate monoaminergic transmission. J. Neurosci. 18, 1904–1912.
Li, X., Pan, H.L., Xu, Z., Tobin, J.R., Eisenach, J.C., 2000. Nicotinic cholinergic receptormediates norepinephrine release in rat spinal cord: an in vivo microdialysis study.Soc. Neurosci. Symp. 26, 950.
Lips, K.S., Pfeil, U., Kummer, W., 2002. Coexpression of alpha9 and alpha10 nicotinicacetylcholine receptors in rat dorsal root ganglion neurons. Neuroscience 115, 1–5.
Lukas, R.J., Changeux, J.P., Le Novere, N., Albuquerque, E.X., Balfour, D.J., Berg, D.K.,Bertrand, D., Chiappinelli, V.A., Clarke, P.B., Collins, A.C., Dani, J.A., Grady, S.R., Kellar,K.J., Lindstrom, J.M., Marks, M.J., Quik, M., Taylor, P.W., Wonnacott, S., 1999.International Union of Pharmacology. XX. Current status of the nomenclature fornicotinic acetylcholine receptors and their subunits. Pharmacol. Rev. 51, 397–401.
Marks, M.J., Stitzel, J.A., Romm, E., Wehner, J.M., Collins, A.C., 1986. Nicotinic bindingsites in rat and mouse brain: comparison of acetylcholine, nicotine, and alpha-bungarotoxin. Mol. Pharmacol. 30, 427–436.
Miao, F.J., Green, P.G., Benowitz, N., Levine, J.D., 2004. Central terminals of nociceptorsare targets for nicotine suppression of inflammation. Neuroscience 123, 777–784.
Milligan, E.D., Hinde, J.L., Mehmert, K.K., Maier, S.F., Watkins, L.R., 1999. A method forincreasing the viability of the external portion of lumbar catheters placed in thespinal subarachnoid space of rats. J. Neurosci. Methods 90, 81–86.
Randall, L.O., Selitto, J.J., 1957. A method for measurement of analgesic activity oninflamed tissue. Arch. Int. Pharmacodyn. CXI, 409–419.
Rashid, M.H., Ueda, H., 2002. Neuropathy-specific analgesic action of intrathecalnicotinic agonists and its spinal GABA-mediated mechanism. Brain Res. 953, 53–62.
Rashid, M.H., Furue, H., Yoshimura, M., Ueda, H., 2006. Tonic inhibitory role ofalpha4beta2 subtype of nicotinic acetylcholine receptors on nociceptive transmis-sion in the spinal cord in mice. Pain 125, 125–135.
Rau, K.K., Johnson, R.D., Cooper, B.Y., 2005. Nicotinic AChR in subclassified capsaicinsensitive and insensitive nociceptors of the rat DRG. J. Neurophysiol. 93 (3),1358–1371.
Roberts, R.G., Stevenson, J.E., Westerman, R.A., Pennefather, J., 1995. Nicotinicacetylcholine receptors on capsaicin-sensitive nerves. NeuroReport 6, 1578–1582.
Rueter, L.E., Meyer, M.D., Decker, M.W., 2000. Spinal mechanisms underlying A-85380-induced effects on acute thermal pain. Brain Res. 872, 93–101.
Sivilotti, L., Woolf, C.J., 1994. The contribution of GABAA and glycine receptors to centralsensitization: disinhibition and touch-evoked allodynia in the spinal cord.J. Neurophysiol. 72, 169–179.
Sorkin, L.S., Puig, S., Jones, D.L., 1998. Spinal bicuculline produces hypersensitivity ofdorsal horn neurons: effects of excitatory amino acid antagonists. Pain 77, 181–190.
Storkson, R.V., Kjorsvik, A., Tjolsen, A., Hole, K., 1996. Lumbar catheterization of thespinal subarachnoid space in the rat. J. Neurosci. Methods 65, 167–172.
Taiwo, Y.O., Coderre, T.J., Levine, J.D., 1989. The contribution of training to sensitivity inthe nociceptive paw-withdrawal test. Brain Res. 487, 148–151.
Vincler, M., Eisenach, J.C., 2004. Plasticity of spinal nicotinic acetylcholine receptorsfollowing spinal nerve ligation. Neurosci. Res. 48, 139–145.
Vincler, M.A., Eisenach, J.C., 2005. Knock down of the alpha5 nicotinic acetylcholinereceptor in spinal nerve-ligated rats alleviates mechanical allodynia. Pharmacol.Biochem. Behav. 80, 135–143.
Wang, F., Gerzanich, V., Wells, G.B., Anand, R., Peng, X., Keyser, K., Lindstrom, J., 1996.Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2,and beta4 subunits. J. Biol. Chem. 271, 17656–17665.
Wang, N., Orr-Urtreger, A., Chapman, J., Rabinowitz, R., Nachman, R., Korczyn, A.D.,2002. Autonomic function in mice lacking alpha5 neuronal nicotinic acetylcholinereceptor subunit. J. Physiol. 542, 347–354.
Yang, L., Zhang, F.X., Huang, F., Lu, Y.J., Li, G.D., Bao, L., Xiao, H.S., Zhang, X., 2004.Peripheral nerve injury induces trans-synaptic modification of channels, receptorsand signal pathways in rat dorsal spinal cord. Eur. J. Neurosci. 19, 871–883.