tonic inhibitory role of a4b2 subtype of nicotinic acetylcholine

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Tonic inhibitory role of a4b2 subtype of nicotinic acetylcholinereceptors on nociceptive transmission in the spinal cord in mice

Md Harunor Rashid a,b,*, Hidemasa Furue a, Megumu Yoshimura a, Hiroshi Ueda b

a Department of Integrative Physiology, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japanb Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan

Received 23 November 2005; received in revised form 6 April 2006; accepted 3 May 2006

Abstract

In the spinal dorsal horn, activation of the nicotinic acetylcholine receptors (nAChR) by exogenously applied agonists is knownto enhance inhibitory synaptic transmission, and to produce analgesia. However, it is still unknown whether endogenously releasedacetylcholine exerts a tonic inhibition on nociceptive transmission through the nAChRs in the spinal dorsal horn. Here, we reportthe presence of such a tonic inhibitory mechanism in the spinal dorsal horn in mice. In behavioral experiments, intrathecal (i.t.)injection of non-selective nAChR antagonist mecamylamine and a4b2 subtype-selective antagonist dihydro-b-erythroidine (DHbE)dose-dependently induced thermal and mechanical hyperalgesia in mice while the a7-selective antagonist methyllycaconitine (MLA)had no effect. Similarly, antisense knock-down of a4 subunit of nAChR, but not a7 subunit, in spinal cord induced thermal andmechanical hyperalgesia. In whole-cell patch-clamp experiments in spinal cord slice preparation from adult mice, the frequencyof miniature inhibitory postsynaptic currents (mIPSCs) observed in substantia gelatinosa (SG) neurons was decreased by mecamyl-amine and DHbE, but not by MLA. The amplitudes of the mIPSCs were not affected. The nicotinic antagonists decreased thefrequency of both GABAergic and glycinergic IPSCs. On the other hand, the nicotinic antagonists had no effect on the excitatorypostsynaptic currents (EPSCs). Finally, acetylcholine-esterase inhibitor neostigmine-induced facilitation of IPSC frequencies in SGneurons was inhibited by mecamylamine and DHbE. Altogether these findings suggest that nicotinic cholinergic system in the spinaldorsal horn can tonically inhibit nociceptive transmission through presynaptic facilitation of inhibitory neurotransmission in SG viathe a4b2 subtype of nAChR. 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Keywords: Spinal dorsal horn; Nociceptive transmission; Nicotinic receptors; Tonic inhibition; Whole cell patch clamp; Substantia gelatinosa;Inhibitory postsynaptic currents

1. Introduction

In the spinal cord dorsal horn, which is a major site formodulation of sensory information, cholinergic interneu-rons are present (Ribeiro-da-Silva and Cuello, 1990; Bar-ber et al., 1984; Borges and Iversen, 1986; Olave et al.,2002). Their cell bodies are found in lamina III–V and

formaplexusofaxonterminalsinthelaminaIIIandinnerpart of lamina II (Olave et al., 2002). Endogenous acetyl-

choline released by these interneurons might act as amajor neuromodulatory transmitter in the spinal dorsalhorn since receptors for acetycholine are present in laminaII and III, where they are likely located on terminals of primary afferents, spinal interneurons as well as ondescending monoaminergic terminals (Coggeshall andCarlton, 1997; Baba et al., 1998; Cordero-Erausquinand Changeux, 2001; Khan et al., 2003; Zhang et al.,2005). Although the effects of exogenous cholinomimeticdrugs on nociceptive transmission in the spinal cord have

0304-3959/$32.00 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.pain.2006.05.011

* Corresponding author. Present address: Anesthesia Research Unit,McGill University, McIntyre Medical Building Room 1207, 3655Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada.Tel.: +1 514 398 4565; fax: +1 514 398 8241.

E-mail address: [email protected] (M.H. Rashid).

www.elsevier.com/locate/pain

Pain 125 (2006) 125–135

mailto:[email protected]:[email protected]


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been extensively studied (Iwamoto and Marion, 1993;Damaj et al., 1998; Khan et al., 1998), the role of endog-enous acetylcholine on nociceptive transmission remainslargely unexplored. A tonic inhibitory role of the spinalmuscarinic cholinergic system on mechanical nociceptivetransmission had been reported earlier (Zhuo and Geb-

hart, 1991; Zhuo et al., 1993). However, such a tonicinhibitory role of the spinal nicotinic cholinergic systemon pain transmission is still unclear. In a previous study,we demonstrated that intrathecal injections of nicotinicantagonist produce thermal hyperalgesia in normal mice,suggesting the presence of a tonic inhibitory mechanismthrough nicotinic receptors in the spinal cord (Rashidand Ueda, 2002).

Nociceptive information from the periphery is subjected to inhibitory modulation in the spinal dorsal horn,specifically in the substantia gelatinosa (SG; lamina II of Rexed) which is very rich in inhibitory GABAergicand glycinergic interneurons (Cervero and Iggo, 1980;

Narikawa et al., 2000; Furue et al., 2004). Both GAB-Aergic and glycinergic synaptic transmissions in the SGare reported to be enhanced by nicotinic agoniststhrough different subtypes of nAChRs (Kiyosawaet al., 2001; Takeda et al., 2003; Genzen and McGehee,2005). By using single-cell RT-PCR, Changeux and col-leagues (Cordero-Erausquin et al., 2004) reported thatthe majority of inhibitory GABAergic and/or glycinergicinterneurons in the dorsal horn preferentially expressa4a6b2 subunits whereas excitatory or NK1-expressingneurons mainly express a7a3b2 subunits. Nevertheless,it has been proposed that endogenous acetylcholine

may tonically activate the nAChRs located on inhibitoryinterneurons in the dorsal horn (Cordero-Erausquin andChangeux, 2001). This, in conjunction with our previousspeculation for the presence of a tonic nAChR-mediatedinhibitory mechanism in the spinal cord in mice (Rashidand Ueda, 2002), prompted us to further investigate thematter. In the present study, intrathecal injections of non-specific nAChR antagonist mecamylamine anda4b2-selective nAChR antagonist dihydro-b-erythroi-dine drastically reduced nociceptive thermal andmechanical withdrawal thresholds in mice. Moreover,frequencies of miniature inhibitory postsynaptic currents(mIPSCs) observed in SG neurons were decreased bythese antagonists. Our combined behavioral and electro-physiological data demonstrate the presence of a tonicinhibitory mechanism through a4b2 subtype of nAChRon nociceptive transmission in the spinal cord.

Materials and method

1.1. Experimental animals

Male ddY mice weighing 25–30 g (6–8 weeks old) were usedin the present study. All procedures throughout the presentstudy were approved by the Nagasaki University Animal Care

Committee and the Kyushu University Guidelines for AnimalExperimentations, and adhered to the guidelines of theCommittee for Research and Ethical Issues of the Internation-al Association for the Study of Pain. In all circumstances,maximum possible efforts were made to minimize animal suf-ferings and to reduce number of animals used in theexperiments.

1.2. Drugs

The following drugs were obtained from Sigma–Aldrich(St. Louis, MO, USA): mecamylamine hydrochloride, dihy-dro-b-erythroidine hydrobromide (DHbE), methyllycaconitinecitrate (MLA), neostigmine bromide. All drugs were dissolvedin deionized water to make the stock solution. The final dilu-tion was made with saline for behavioral studies and withKrebs solution for electrophysiological studies.

1.3. Intrathecal injection

Intrathecal injections were performed free-hand between L5and L6 lumber space in unanesthetized mice using a 30-gaugeneedle attached to a Hamilton microsyringe according to themethods of Hylden and Wilcox (Hylden and Wilcox, 1980).The accurate placement of the needle tip in the subarachnoidspace was verified by a quick flicking of the mouse’s tailimmediately upon entry of the needle. The injection was givenslowly in a volume of 5 ll.

1.4. Nociceptive tests

Nociceptive tests were performed with either thermal ormechanical stimulus. In thermal paw withdrawal test, thelatency to withdrawal evoked by exposing the right hind paw

to a thermal stimulus was measured. Unanesthetized animalswere placed under Plexiglas cages on top of a glass sheet andadapted in the testing environment for about an hour. Thethermal stimulus (IITC, Woodland Hills, CA, USA) was thenpositioned under the glass sheet to focus the projection bulbexactly on the middle of plantar surface of the mice. Time towithdraw the paw from the thermal stimulus was then auto-matically measured. In mechanical paw pressure test, micewere placed under Plexiglas cages on top of a wire mesh grid,adapted for 1 h, and mechanical stimulus was delivered on theplantar surface of right hand paw using an automated Trans-ducer Indicator (IITC, Woodland Hills, CA, USA), and thewithdrawal thresholds were measured. A cut-off time or pres-sure of 15 s or 15 g was used to minimize tissue damage.

1.5. Antisense oligonucleotides and Western blotting

The antisense oligodeoxynucleotides and its mismatch fora4 (AS-ODN; 5 0 CCC CCG ATC TCC ATG GCT 3 0;MS-ODN 5 0 GCC GCG TTC ACC TTG CCT 3 0) and a7(AS-ODN; 5 0 GCC GCG CAT GTC GCC GGA 3 0; MS-ODN 5 0 CCC CCA GAT CTC CCC CGA 3 0) subunits of the nicotinic acetylcholine receptor were synthesized, freshlydissolved in physiological saline and injected in mice in a vol-ume of 2 ll (10 lg) on 1st, 3rd and 5th day, and nociceptivetests were performed on the 6th day. To confirm the downreg-ulation of the target receptor by the AS-ODN, Western blot-

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ting experiments in freshly isolated spinal cord samples of thetreated animals were performed using standard protocol. Therabbit polyclonal antibodies raised against the a4 and a7 sub-units of nAChR were used in immunoblotting studies (1:500;Santa Cruz Biotechnology, CA, USA).

1.6. Electrophysiological studies

Blind whole-cell patch-clamp recordings were made fromsubstantia gelatinosa (SG) neurons in a transverse slice fromlumbar region of the spinal cord of mice. The method usedfor obtaining the transverse slice preparation was as describedpreviously in the rat (Yoshimura and Jessell, 1989; Yoshimuraand Nishi, 1993; Miyakawa et al., 2005). Briefly, mice wereanesthetized with urethane (1.5 g/kg, i.p.), and a thoraco-lum-bar laminectomy was performed. A portion of the lumbarL4–L6 spinal cord was removed and submerged in ice-cold,pre-oxygenated Krebs solution (in mM: NaCl 117, KCl 3.6,CaCl2 2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 25, and D-glu-cose 11). After removal of dura mater, the dorsal and ventral

roots were cut and then pia-arachnoid membrane wasremoved. A 500–550 lm thick transverse slice was cut on avibratome microslicer. The slice was then placed in the record-ing chamber and perfused continuously at a rate of 10–15 ml/min with Krebs solution which was equilibrated with 95% O 2and 5% CO2 at 36 ± 1 C. Blind whole-cell recordings werethen made in SG with patch pipettes (6–12 MX) filled withinternal solution containing (in mM): Cs2SO4 110, CaCl2 0.5,MgCl2 2, TEA-Cl 5, EGTA 5, Hepes 5, and Mg-ATP 5. Theinhibitory postsynaptic currents were recorded at 0 mV andamplified with an Axopatch 200B amplifier (Axon Instru-ments, CA, USA). Signals were filtered at 5 kHz and digitizedwith an A/D converter. The acquired data were analyzed witha personal computer using pClamp version 8.1 (Axon Instru-

ments) and Mini-analysis program version 6.0.3 (Synaptosoft,Decatur, GA, USA). Drugs were applied by exchanging perfu-sion solution containing a known drug concentration withoutaltering the perfusion rate and temperature. We assumed theeffects of the drugs as ‘decrease’, ‘increase’ or ‘no effect’ if changes compared with control were ‘less than 80%’, ‘morethan 120%’, and ‘between 80 and 120%’, respectively.

1.7. Statistical analysis

The data were analyzed by either Student’s t-test or one-way analysis of variance with suitable post hoc tests. The elec-trophysiological cumulative histogram data were analyzed byKolmogorov–Smirnov test. The criterion of significance wasset at p < 0.05.

2. Results

2.1. Induction of thermal and mechanical hyperalgesia in

naı̈ ve mice by intrathecal injection of non-specific nicotinic

antagonist mecamylamine

In a previous study with peripheral nerve injurymodel (Rashid and Ueda, 2002), we observed thatintrathecal (i.t.) injections of nicotinic antagonist mec-amylamine produced thermal hyperalgesia in control

sham-operated mice, suggesting the presence of a tonicinhibitory mechanism through nicotinic receptors inthe spinal cord. In the present study also, we found thati.t. injection of non-specific nicotinic receptor antagonist,mecamylamine, dose-dependently (0.1 –10 nmol, i.t.)produced thermal and mechanical hyperalgesia in mice

(Fig. 1A and B). When we measured the area under thetime-course curves (AUC) in Fig. 1A and B, it wasobserved that mecamylamine significantly decreasedthermal and mechanical withdrawal thresholds in mice.The induction of hyperalgesia was rapid and long-last-ing. With i.t. 10 nmol of mecamylamine, the paw with-drawal latency or threshold was significantly decreasedat 10 min after injection and the effects continued beforereturning to baseline level at 90 min after the injection(Fig. 1A and B).

2.2. Involvement of a4b 2 subtype of nicotinic receptors for

the induction of hyperalgesia

To further identify the subtype of nAChR thatmediates the endogenous acetylcholine-mediated tonicinhibition on nociceptive transmission in the spinalcord, we used subtype-selective antagonists. We usedantagonists for a4b2 and a7 subtypes of nAChR,the two major nAChR subtypes that had been report-ed to be involved in nicotinic agonists-mediated antin-ociception in the spinal cord in mice (Damaj et al.,1998; Khan et al., 1998; Marubio et al., 1999). Similarto mecamylamine, intrathecal injection of a4b2nAChR antagonist dihydro-b-erythroidine (DHbE)

dose-dependently (1–10 nmol, i.t.) produced thermaland mechanical hyperalgesia (Fig. 2A and B). Howev-er, i.t. injection of 10 nmol of a7 nAChR antagonistmethyllycaconitine (MLA) did not induce any thermalor mechanical hyperalgesia (Fig. 2A and B). Plottingthe data as AUC also indicates that DHbE significant-ly decreased thermal and mechanical withdrawalthresholds while MLA had no significant effects. Sim-ilarly, antisense knockdown of a4 subunit of nAChRinduced thermal and mechanical hyperalgesia(Fig. 2C and D) while the antisense knockdown of a7 subunit had no effect (data not shown). We furtherexamined whether antisense knockdown of a specificnicotinic receptor subunit caused loss of functionthrough that specific subtype of nAChR in the spinalcord in mice. For this purpose, we examined theeffects of i.t. a4b2-selective nAChR antagonist DHbEon the a4 knockdown mice. As shown in Fig. 2E, i.t.injection of 10 nmol of DHbE did not further decreasethe thermal latency in the AS-ODN-treated mice,while DHbE decreased thermal latency in the saline-or MS-ODN-treated mice (Fig. 2E), suggesting lossof functional a4b2 nAChR by the AS-ODN. Similarresults were obtained with the paw pressure test (datanot shown).

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2.3. Reduction in mIPSCs frequency in dorsal horn SG

neurons by nicotinic antagonists

In whole-cell patch-clamp experiments, we recordedthe inhibitory postsynaptic currents (IPSCs) in substan-tia gelatinosa (SG) neurons in adult mice spinal cordslice preparations, and examined the effects of nicotinicantagonists thereon. The cells were held at 0 mV wherethe IPSCs were observed as outward currents sinceinward glutamatergic currents were all minimized at thispotential. As shown in Fig. 3A and B, the frequencies of miniature inhibitory postsynaptic currents (mIPSCs)observed in presence of TTX (1 lM) were reduced bybath application of 10 lM of mecamylamine(61.59 ± 6.41% of control; n = 6). While mecamylaminesignificantly decreased the cumulative probability of mIPSC frequencies, it had no effect on the mIPSCamplitudes (Fig. 3C and D). Fig. 3E shows the timecourse for the effects of mecamylamine on mIPSC fre-quency in a SG neuron. Out of total 62 SG neurons test-ed, application of 10 lM of mecamylamine decreased

the frequency of IPSCs in 20 neurons (control:9.07 ± 0.03 Hz vs mecamylamine: 5.37 ± 0.23 Hz). TheIPSCs amplitudes were unaffected by mecamylamine(control: 29.4 ± 1.1 pA vs mecamylamine: 26.7 ± 0.7A). In three neurons, mecamylamine increased the IPSCfrequency but did not affect the IPSC amplitude (con-trol: 5.74 ± 1.13 Hz vs mecamylamine: 9.24 ± 0.98 Hz;and control: 16.7 ± 0.6 pA vs mecamylamine:18.5 ± 0.8 pA). The rest of the neurons did not respondto mecamylamine (% change in IPSC frequency andamplitudes was between 80% and 120% of control; datanot shown). On the other hand, mecamylamine (10 lM)had no effects on the excitatory postsynaptic currents(EPSCs) recorded at a holding potential of 70 mV(Fig. 4A; n = 7).

Next, we examined the effects of subtype-selectivenAChR antagonists on the IPSCs in SG neurons thatwere responsive to mecamylamine. Out of 15 mecamyl-amine-sensitive neurons, antagonist of the a4b2 subtypeof nAChR dihydro-b-erythroidine (DHbE; 10 lM)decreased the mIPSC frequency in 11 neurons

Fig. 1. Induction of thermal and mechanical hyperalgesia in mice by intrathecal (i.t.) nicotinic antagonist. (A and B) Intrathecal injections of non-specific nicotinic acetylcholine receptor antagonist, mecamylamine (Meca), dose-dependently induced thermal (A) and mechanical (B) hyperalgesia inmice. The paw withdrawal latency (A) or thresholds (B) were drastically reduced by i.t. mecamylamine that persisted for more than an hour. The

right side bar graphs show the area under the curves (AUC) in (A and B), respectively. Each data point represents mean ± SEM from 6 to 8 mice. ‘‘*’’indicates statistically significant difference compared with the vehicle (Veh) saline injection at p < 0.05.



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(Fig. 4E, control: 6.68 ± 0.23 Hz vs DHbE:3.22 ± 0.8 Hz; n = 15). Moreover, DHbE decreased themIPSC frequency in a dose-dependent manner

(Fig. 4F). On the other hand, application of a7-selectivenAChR antagonist methyllycaconitine (MLA; 100 nM)decreased the mIPSC frequency only in 1 cell out of

Fig. 2. Involvement of a4b2 subtype of nicotinic receptor for the induction of hyperalgesia in mice. (A and B) Intrathecal injections of antagonist of

the a4b2 subtype of nicotinic receptor dihydro-b-erythroidine (DHbE), but not a7 subtype antagonist methyllycaconitine (MLA), induced thermal(A) and mechanical (B) hyperalgesia in the mice. The right side bar graphs show the AUC from (A and B), respectively. ‘‘*’’ indicates statisticallysignificant difference compared with the vehicle (Veh) saline injection at p < 0.05. (C and D) Involvement of a4b2 subtype of nAChR for theinduction of such hyperalgesia was further confirmed by antisense blockade of expression of a4 subunit. Pretreatments with antisenseoligodeoxynucleotides for a4 subunit induced thermal and mechanical hyperalgesia in mice. Mice were treated with vehicle saline (Veh), missenseoligodeoxynucleotides (MS) and antisense oligodeoxynucleotides (AS) at day 1, day 3 and day 5, and paw withdrawal latency or thresholds weremeasured at day 6. The upper panel shows Western blots for a4 subunit of nAChR in spinal cord tissues from the different treatment groups. (E) Nofurther decrease in thermal paw withdrawal latency by DHbE in a4 antisense oligodeoxynucleotides (AS)-treated mice. Each data point representsmean ± SEM from 6 to 8 mice. * p < 0.05.



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six mecamylamine-sensitive cells tested (Fig. 4E, control:9.27 ± 1.13 Hz vs MLA: 8.4 ± 1.05 Hz; n = 6). BothDHbE and MLA did not affect the amplitude of themIPSCs. Traces in Fig. 4B–D showed a representativeneuron that was responsive to mecamylamine andDHbE, but not to MLA.

2.4. Nicotinic antagonist decreased frequency of both

GABAergic and glycinergic IPSCs

Two types of IPSCs are usually observed in the SGneurons according to their decay time. One type has ashorter duration and is blocked by strychnine (2 lM),and thus referred to as glycinergic IPSCs; the other typehas a relatively longer duration and is antagonized bybicuculline (20 lM), and thus referred to as GABAergicIPSCs (Yoshimura and Nishi, 1993). Out of 15 neuronsthat were tested for the effects of DHbE, 8 had

GABAergic IPSCs, 5 had glycinergic IPSCs and 2 hadmixed IPSCs as determined by decay time and respon-siveness to specific antagonists. As shown in Fig. 5Aand B, the a4b2-selective antagonist DHbE decreasedthe frequency of both GABAergic (control: 7.08 ±1.18 Hz vs DHbE: 3.41 ± 0.41 Hz; n = 7) and glyciner-gic IPSCs (control: 5.4 ± 0.46 Hz vs DHbE: 2.73 ±0.03 Hz; n = 3). DHbE did not affect the amplitudes of GABAergic (control: 14.4 ± 1.3 pA vs DHbE: 12.9 ±1.2 pA) or glycinergic IPSCs (control: 32.4 ± 3.5 pA vsDHbE: 29.5 ± 3.0 pA).

2.5. Nicotinic antagonists blocked the enhancement of

IPSC frequency induced by acetylcholinesterase inhibitor,

neostigmine

The acetylcholinesterase inhibitor neostigmine isknown to enhance the level of endogenous ACh by

Fig. 3. Reversible decrease in miniature inhibitory postsynaptic currents (mIPSC) frequency in SG neurons by nicotinic antagonist. (A) In whole-cell

patch-clamp experiments in substantia gelatinosa (SG) of spinal dorsal horn of mice, the frequency of mIPSCs observed in presence of 1 lM of TTXwas reversibly decreased by bath application of 10 lM of non-specific nicotinic antagonist, mecamylamine. (B) Traces of mIPSCs on an extendedtime-scale in absence or presence of mecamylamine. (C and D) Analyses of cumulative frequency and amplitude histograms indicate thatmecamylamine significantly decreased the frequency of mIPSCs in SG neurons (C; Kolmogorov–Smirnov test, p < 0.05) while the effect on mIPSCamplitudes was not statistically significant (D; Kolmogorov–Smirnov test, p < 0.05). (E) Time-course for the effects of mecamylamine on mIPSCfrequency in the neuron in (A).



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blocking its metabolism. Previous report demonstratesthat neostigmine can increase the frequency of IPSCsin the spinal dorsal horn in the rat (Baba et al.,1998). In the present report, to strengthen our propo-sition for the presence of an endogenous ACh-medi-ated tonic inhibitory mechanism through nAChR inthe spinal cord, we sought to know whether the neo-stigmine-induced increases in the IPSCs frequenciesin SG neurons are blocked by the nicotinic antago-nists. As shown in Fig. 6A, bath application of 10 lM of neostigmine markedly increased the IPSCfrequency in the SG neurons in mice(303.98 ± 16.25% of control; n = 14). We observedthat pre-application of nAChR antagonists mecamyl-amine and DHbE significantly inhibited the increasein IPSC frequencies that was induced by neostigmine(Fig. 6B–E), further suggesting that the spinal nicotinic

cholinergic system had a tonic inhibitory role on dor-sal horn synaptic transmission.

3. Discussion

Consistent with our previous observation (Rashidand Ueda, 2002), in the present study, intrathecal (i.t.)injection of the nicotinic antagonists induced thermaland mechanical hyperalgesia in naı̈ve mice. In severalprevious studies with the normal rat, i.t. nicotinic antag-onists alone had no effects while they blocked the noci-ceptive or antinociceptive effects of the i.t. nicotinicagonists (Khan et al., 1998; Rueter et al., 2000). Thesedifferences in the effects of i.t. nicotinic antagonists inthe normal mice and rat might be due to species differ-ences and/ or differential expression of nicotinic receptorsubtypes or differential tonic release of acetylcholine in

Fig. 4. Nicotinic antagonists dose-dependently decreased IPSC frequencies and had no effect on EPSC frequencies. (A) The EPSC frequenciesobserved at 70 mV were unaffected by nicotinic antagonist mecamylamine. (B–D) Bath application of non-specific nicotinic antagonistmecamylamine (10 lM) and a4b2 nicotinic antagonist DHbE 10 lM, but not a7 antagonist MLA (100 nM) reversibly decreased the IPSC frequenciesin SG neurons. The traces in (A–D) were taken from the same neuron. (E) Analyses of percent decrease in IPSC frequency in SG neurons by differentnicotinic antagonists. (F) Dose-dependent decrease in IPSC frequency by DHbE. The number of neurons is indicated above each column. * p < 0.05.



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the spinal cord. Nevertheless, in our present experi-ments, i.t. injection of non-specific nicotinic antagonist

mecamylamine and a4b2-selective antagonist dihydro-b-erythroidine (DHbE) caused drastic reduction in thethermal and mechanical nociceptive thresholds in micewhile a7 antagonist methyllycaconitine (MLA) had noeffect. In electrophysiological experiments, the frequencyof miniature inhibitory postsynaptic currents (mIPSCs)observed in SG neurons was decreased by mecamyl-amine and DHbE, but not by MLA. Mecamylamineand DHbE did not affect the amplitude of the mIPSCs,suggesting that the effects were mediated through block-ade of presynaptic nicotinic receptors. Moreover, theincreases in IPSC frequencies induced by the cholines-terase inhibitor neostigmine were blocked by both mec-amylamine and DHbE. All these results suggest thatcholinergic neurons in the spinal dorsal horn tonicallyactivate the inhibitory GABAergic and glycinergic inter-neurons to produce a constant and sustained endoge-nous inhibition on nociceptive transmission.

It is well known that nociceptive transmission in thespinal dorsal horn is subjected to tonic modulation bylocal interneurons and descending fibers. Behavioraland biochemical studies suggest that GABA and gly-cine released by the local GABAergic and glycinergicneurons in the dorsal horn may tonically inhibit noci-ceptive transmission (Ishikawa et al., 2000; Cronin

et al., 2004). Electrophysiological studies also showedthat excitatory inputs in spinal dorsal horn are under

the control of inhibitory interneurons that may mediatepresynaptic inhibition through axo-axonic synapsesand postsynaptic inhibition through axo-dendritic oraxo-somatic synapses (Todd, 1990, 1996; Yoshimuraand Nishi, 1995). Our present results provide evidencethat this inhibitory system is further controlled by cho-linergic inputs through spinal nicotinic receptors. Ourbehavioral results of induction of hyperalgesia in miceby i.t. injection of nicotinic antagonists clearly indicatethe presence of a tonic inhibitory mechanism throughthe spinal nicotinic cholinergic system. A similar tonicinhibitory mechanism through spinal muscarinic cho-linergic system on mechanical nociceptive transmissionhad already been reported in behavioral studies (Zhuoand Gebhart, 1991; Zhuo et al., 1993; Baba et al.,1998). Our electrophysiological data also strongly sup-port our behavioral results of tonic nicotinic inhibitionof nociceptive transmission in the spinal cord. The factthat nicotinic antagonists can decrease the frequency of both GABAergic and glycinergic mIPSCs in SG indi-cates that endogenous acetylcholine spontaneouslyreleased at the presynaptic terminals of inhibitory neu-rons can increase the release probability of GABA andglycine to modulate nociceptive transmission. Adecrease in release probability of inhibitory neurotrans-

Fig. 5. Nicotinic antagonists decreased the frequency of both GABAergic and glycinergic IPSCs. (A) Frequencies of GABAergic IPSCs observed inthe presence of glycine receptor antagonist strychnine were decreased by a4b2 nicotinic antagonist, DHbE. (B) Frequencies of glycinergic IPSCsobserved in the presence of GABA-A receptor antagonist bicuculline were also decreased by DHbE. The lower panels show traces from indicatedparts in Fig. 5 A and B, respectively, on an extended time scale.



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behavioral as well as gene knock-out experiments strong-ly suggest a role for mainly a4b2 subtype of nAChR inspinal nociceptive inhibition (Khan et al., 1998; Marubioet al., 1999; Rashid and Ueda, 2002). The a4b2 subtype of nAChRs is also known to be involved in enhancedGABAergic and glycinergic transmission in the dorsal

horn in newborn rat (Kiyosawa et al., 2001; Cordero-Erausquin et al., 2004; Genzen and McGehee, 2005).Our results for the involvement of a4b2 subtype of nAChR in GABAergic and glycinergic synaptic transmis-sion in the SG in adult mice are consistent with these linesof evidence. On the other hand, Takeda et al. (2003)reported the involvement of a non-a4b2, non-a7 subtypeof nAChR for the enhancement of GABAergic transmis-sion in SG in the adult rat. These differences might be dueto use of different species of animals and different experi-mental conditions.

The reduction in mIPSCs frequency in SG neurons bythe nicotinic antagonist DHbE in our experiments sug-

gests that a4b2 receptors located on the presyanptic ter-minals might be involved in tonic nicotinic inhibition of spinal nociceptive transmission through enhancedrelease of GABA or glycine. In many brain regions,nAChRs expressed on presynaptic terminals are knownto enhance neurotransmitter release (McGehee et al.,1995; Alkondon et al., 1997). In the spinal cord also, nic-otinic agonists are known to increase the mIPSC fre-quency by acting on presynaptic a4b2 nAChRs(Kiyosawa et al., 2001). Recently, Vincler and Eisenach(2004) immunohistochemically labeled various subtypesof nAChRs in the spinal cord in normal and neuropathy

state rat. The immunostaining for a4 subunit wasobserved in lamina II–V of spinal dorsal horn wherethey were located on round cells as well as on punctatefibers, suggesting their presence on soma and axon ter-minals in dorsal horn neurons (Vincler and Eisenach,2004). This is consistent with previous electrophysiolog-ical data where nicotinic agonists mainly increased thefrequency of mIPSCs and in some cases induced post-synaptic inward current in some SG neurons in the rat(Kiyosawa et al., 2001; Takeda et al., 2003). In the pres-ent study with adult mouse spinal cord slice preparation,our data suggests that the endogenous ACh may inducetonic release of GABA or glycine in the SG through thepresynaptic nicotinic receptors. However, a postsynapticcomponent for the effects of acetylcholine in tonic nico-tinic receptor-mediated inhibitory effects cannot beexcluded since previous studies with muscarinic cholin-ergic system suggest involvement of such mechanisms(Baba et al., 1998). Moreover, the possibility of an indi-rect effect for the tonic nicotinic inhibition cannot beexcluded since cholinergic system is also known to acti-vate other inhibitory mechanisms in the spinal cord suchas 5HT and nitric oxide system. It has been reportedthat endogenous acetylcholine may tonically inducerelease of serotonin by directly acting on the nicotinic

receptors located on spinal serotonergic projectionsfrom the raphe magnus (Cordero-Erausquin andChangeux, 2001). Similarly, endogenous acetylcholineis known to induce nitric oxide (NO) synthesis throughboth nicotinic and muscarinic receptors in the spinalcord to produce inhibitory effects (Zhuo et al., 1993;

Xu et al., 2000). However, our results of blockade of mIPSC frequency by the nicotinic antagonists as wellas other electrophysiological studies where nicotinicagonists increased the mIPSC frequencies in presenceof TTX (Kiyosawa et al., 2001; Takeda et al., 2003;Genzen and McGehee, 2005) suggest that nicotinic cho-linergic system may also directly mediate tonic inhibi-tion through presynaptic nAChRs in the spinal cord.

In conclusion, our behavioral and electrophysiologicaldata provide evidence that nociceptive transmission in thespinal cord is under a tonic nicotinic cholinergic inhibi-tion, and the a4b2 subtype of nicotinic receptors locatedon the terminals of GABAergic and glycinergic inhibitory

interneurons in the SG may, at least in part, contribute tothis tonic nicotinic inhibitory mechanism. In continuationof our previous findings (Rashid and Ueda, 2002), wespeculate that a reduction in this tonic nicotinic recep-tor-mediated inhibitory tone might be one of the spinalmechanisms for the induction of neuropathic pain.

Acknowledgements

Research described in this article was supported inparts by funds from Philip Morris U.S.A. Inc. and Phil-

ip Morris International granted to H.U. Parts of thisstudy were also supported by grants from Japan Societyfor the Promotion of Science (JSPS) to M.H.R. andGrants-in-Aid from the Ministry of Education, Science,Sports and Culture of the Govt. of Japan to M.Y. andH.F., and the 21st Century Centre of Excellence(COE) program to M.Y.

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tonic inhibitory role of a4b2 subtype of nicotinic acetylcholine

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