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Research Report

Icilin-evoked behavioral stimulation is attenuated byalpha2-adrenoceptor activation

Jae Kima, Alan Cowanb,c, Renata Liseka, Natalie Raymondia, Aaron Rosenthala,Daniel D. Hirscha, Scott M. Rawlsa,b,⁎aDepartment of Pharmaceutical Sciences, Temple University Health Sciences Center, Philadelphia, PA 19140, USAbCenter for Substance Abuse Research, Temple University Health Sciences Center, Philadelphia, PA 19140, USAcDepartment of Pharmacology, Temple University Health Sciences Center, Philadelphia, PA 19140, USA

A R T I C L E I N F O

⁎ Corresponding author at: Department of PhaPhiladelphia, PA, 19140, USA. Fax: +1 215 707

E-mail address: scott.rawls@temple.edu (S.

0006-8993/$ – see front matter © 2011 Elsevidoi:10.1016/j.brainres.2011.02.002

A B S T R A C T

Article history:Accepted 2 February 2011

Icilin is a transient receptor potential cation channel subfamily M (TRPM8) agonist thatproduces behavioral activation in rats and mice. Its hallmark overt pharmacological effect iswet-dog shakes (WDS) in rats. The vigorous shaking associated with icilin is dependent onNMDA receptor activation and nitric oxide production, but little else is known about thebiological systems thatmodulate the behavioral phenomenon. The present study investigatedthe hypothesis that alpha2-adrenoceptor activation inhibits icilin-inducedWDS. Rats injectedwith icilin (0.5, 1, 2.5, 5 mg/kg, i.p.) displayed dose-related WDS that were inhibited bypretreatment with a fixed dose of clonidine (0.15mg/kg, s.c.). Shaking behavior caused by afixed dose (2.5 mg/kg) of icilin was also inhibited in a dose-related manner by clonidinepretreatment (0.03–0.15 mg/kg, s.c.) and reduced by clonidine posttreatment (0.15mg/kg, s.c.).Pretreatment with a peripherally restricted alpha2-adrenoceptor agonist, ST91 (0.075, 0.15mg/kg), also decreased the incidence of shaking elicited by 2.5 mg/kg of icilin. Pretreatment withyohimbine (2 mg/kg, i.p.) enhanced the shaking induced by a lowdose of icilin (0.5 mg/kg). Theimidazoline site agonists, agmatine (150 mg/kg, i.p.) and 2-BFI (7 mg/kg, i.p.), did not affecticilin-evoked shaking. These results suggest that alpha2-adrenoceptor activation inhibitsshaking induced by icilin and that increases in peripheral, as well as central, alpha2-adrenoceptor signaling oppose the behavioral stimulant effect of icilin.

© 2011 Elsevier B.V. All rights reserved.

JEL classification:Neurotransmitters, Modulators,Transporters, andReceptorsInteractions betweenneurotransmittersBehavioral pharmacology

Keywords:ClonidineIcilinTRPM8Wet-dog shakeAlpha2-adrenoceptorAgmatineST91Yohimbine

1. Introduction

Icilin (AG-3-5) activates transient receptor potential cationchannel subfamily M member 8 (TRPM8) (McKemy et al., 2002;

rmaceutical Sciences, Te3678.M. Rawls).

er B.V. All rights reserved

Peier et al., 2002; Reid et al., 2002; Nealen et al., 2003; Story et al.,2003; Bandell et al., 2004; Biró et al., 2005; Liu et al., 2006). TRPM8channels are densely expressed in sensory neurons within thedorsal root ganglion, and their activation by icilin and other

mple University Health Sciences Center, 3307 North Broad Street,

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Fig. 1 – Clonidine pretreatment reduces WDS induced by afixed dose of icilin. Rats pretreated with clonidine (0.03, 0.06,0.15 mg/kg) or saline were injected 15 min later with icilin(2.5 mg/kg). Data from 6 to 8 rats per group are expressed asmean WDS+S.E.M. *P<0.5 or **P<0.01 compared to ratspretreated with saline.

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cooling compounds (e.g., menthol, WS-12, and CPS-369) pro-duces sodium and calcium ion influx that results in cellulardepolarization and the feeling of cold (Nealen et al., 2003; Babeset al., 2004). Despite the potential of TRPM8 ligands as analgesic,antipruritic and antiarthritic agents, the endogenous andexogenous substances that modulate their pharmacologicaleffects in vivo are largely unknown. The trademark overtpharmacological effect of icilin in rats is vigorous wet-dogshakes (WDS) (Wei, 1976, 1981; Werkheiser et al., 2006, 2007,2009), and this shaking behavior provides a sensitive, reproduc-ible, and quantifiable endpoint to investigate TRPM8 pharma-cology in vivoand to identify, and exclude, receptor systems thatshape the pharmacological response to TRPM8 channelactivation.

Our studies have traditionally focused on the identificationof downstream signaling events that underlie icilin-inducedbehavioral activation. For example, we have demonstrated thaticilin-inducedWDSaredependent onNMDAreceptor activationand nitric oxide production but not on AMPA receptor orglutamate transporter subtype 1 (GLT-1) activation (Werkheiseret al., 2009). We have also shown that mu and kappa opioidreceptor agonists reduce icilin-induced shaking (Werkheiseret al., 2006). Thepresent study tested the overall hypothesis thatalpha2-adrenoceptor activation opposes icilin-induced behav-ioral activation. The hypothesis was based in part on the tenetthat increased sympathetic activity contributes to shakingmovements in rats and that suppression of sympatheticdischarge by an alpha2-adrenoceptor agonist (clonidine)would reduce shaking caused specifically by icilin (Wei andSeid, 1983). The hypothesis was also based on a recent findingthat alpha2-adrenoceptors and TRMP8 channels are co-expressed on peripherally located sensory neurons and thatalpha2-adrenoceptor activation inhibits TRPM8 channel signal-ing (Bavencoffe et al., 2010).We specifically investigated theeffect of: 1) clonidine on the development and maintenance oficilin-induced shaking; 2) N-(2,6-diethylphenyl)-4,5-dihydro-1H-imidazol-2-amine hydrochloride (ST-91), a peripherallyrestricted alpha2-adrenoceptor agonist, on the development oficilin-induced shaking; and 3) yohimbine, an alpha2-adreno-ceptor antagonist, on icilin-induced shaking. We also studiedtheeffects of imidazolinesiteagonistson icilin-inducedshakingbehavior.

2. Results

2.1. Clonidine pretreatment reduces icilin-induced shaking

Effects of graded doses of clonidine (0.03, 0.6, 0.15 mg/kg) onWDS produced by a fixed dose of icilin (2.5 mg/kg) arepresented in Fig. 1. One-way ANOVA revealed a significantdrug effect [F (3, 31)=5.351, P=0.0049] for the experiment.Injection of icilin (2.5 mg/kg) by itself produced 148±32.5 WDSover the 30-min observation interval. Dunnett's post-hocanalysis indicated that the total number of WDS producedby icilin was significantly inhibited by pretreatment witheither 0.06 mg/kg (P<0.05) or 0.15 mg/kg (P<0.01) clonidine.Specifically, 0.06 mg/kg and 0.15 mg/kg of clonidine inhibitedthe incidence of shaking by approximately 41% and 77%,respectively. The onset of shaking, and excessive grooming,

began within 2 min of icilin injection. The behaviors persistedfor the duration of the 30-min observation period, and werepreceded, in all cases, by abdominal writhing. Althoughclonidine pretreatment (0.06, 0.15 mg/kg) did significantlyinhibit the total extent of shaking induced by icilin, it did notsignificantly delay the onset of shaking. The administration ofclonidine (0.03, 0.06, 0.15 mg/kg) by itself did not elicitabdominal writhing, wet-dog shaking, excessive grooming,or sedation.

Separate experiments examined the effect of a fixed dose ofclonidine (0.06mg/kg) onWDS elicited by progressively increas-ing doses of icilin (0.5, 1, 2.5, 5 mg/kg) (Fig. 2). Two-way ANOVA(pretreatment, icilin dose) indicated significant effects ofpretreatment [F (1, 48)=61.37, P<0.0001] and dose [F (3, 48)=16.92, P<0.0001]. Icilin by itself produced dose-related shaking(0.5 mg/kg, 58±16.1; 1 mg/kg, 144±19.8; 2.5 mg/kg, 189±27.0;and 5 mg/kg, 190±18.2). Post-hoc analysis indicated that WDSassociated with each icilin dose (0.5, 1, 2.5, 5 mg/kg) wassignificantly inhibited by pretreatment with clonidine(0.15mg/kg). Specifically, clonidine pretreatment (0.15 mg/kg)produced the following effects on icilin-induced shaking (icilindose, % inhibition, significance): (0.5 mg/kg, 75%, P<0.01); (1 mg/kg, 78%, P<0.01); (1 mg/kg, 60%, P<0.01); and (2.5 mg/kg, 52%,P<0.05).

2.2. Clonidine posttreatment attenuates icilin-inducedshaking

Results from the experiment investigating the effect ofclonidine posttreatment (0.6 mg/kg) on WDS produced byicilin (2.5 mg/kg) are presented as time-course data in Fig. 3.

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Fig. 2 – Pretreatmentwitha fixeddoseof clonidine (0.15 mg/kg)reducesWDS evoked by graded doses of icilin. Rats pretreatedwith clonidine (0.15mg/kg) or salinewere injected 15min laterwith icilin (0.5, 1, 2.5, 5 mg/kg). Data from 6 to 8 rats per groupare expressed as mean WDS+S.E.M. *P<0.05 or **P<0.01compared to respective icilin group pretreated with saline.

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Two-way ANOVA (treatment, time) revealed significanteffects of treatment [F (1, 10)=77.98, P<0.0001] and time [F (6,60)=13.97, P<0.0001]. Significantly less shaking was observed

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Fig. 3 – Clonidine posttreatment reduces WDS produced by afixed dose of icilin. Rats injected with icilin (2.5 mg/kg) wereinjected 5 min later with clonidine (0.15 mg/kg) or saline.Time-course data from 6 rats per group are expressed asmean WDS+S.E.M post-clonidine or post-saline treatment.*P<0.05 or **P<0.01 compared to icilin+vehicle group.

in icilin (2.5 mg/kg)-exposed rats that were posttreated 5 minlater with clonidine (0.06 mg/kg) than in icilin (2.5 mg/kg)-exposed rats that received saline posttreatment. The inhibi-tory effect of clonidine was both rapid in onset, as a significantattenuation (P<0.05) of icilin-induced shaking was observed5 min post-clonidine injection, and persistent in duration, as itendured for the entire 35-min observation interval (Fig. 3).

2.3. ST-91 pretreatment reduces icilin-induced shaking

Effects of graded doses of the peripherally restricted alpha2-adrenoceptor agonist, ST-91 (0.0375, 0.075, 0.15 mg/kg), onWDS produced by a fixed dose of icilin (2.5 mg/kg) arepresented in Fig. 4. One-way ANOVA revealed a significantdrug effect [F (3, 28)=3.298, P=0.0348]. Dunnett's post-hocanalysis indicated that WDS elicited by icilin were significant-ly inhibited by pretreatment with either 0.075 or 0.15 mg/kg ofST-91 (P<0.05). Specifically, 0.0375 and 0.15 mg/kg of ST-91inhibited the incidence of shaking by approximately 42% and51%, respectively. The lowest dose of ST-91, 0.0375 mg/kg,reduced the number of WDS induced by icilin by approxi-mately 20% but the effect did not reach statistical significance(P>0.05). The injection of ST-91 (0.15 mg/kg) by itself did notelicit abdominal writhing, wet-dog shaking, excessive groom-ing, or sedation.

2.4. Yohimbine pretreatment enhances WDS elicited by alow dose of icilin

Effects of graded doses of the alpha2-adrenoceptor antagonistyohimbine (0.5, 1, 2 mg/kg) on WDS elicited by two doses oficilin, 0.5 and 2.5 mg/kg, are presented in Fig. 5. For

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Fig. 4 – ST-91 pretreatment reduces WDS induced by a fixeddose of icilin. Rats pretreated with ST-91 (0.0375, 0.075,0.15 mg/kg) or saline were injected 15 min later with icilin(2.5 mg/kg). Data from 6 to 8 rats per group are expressed asmeanWDS+S.E.M. *P<0.05 compared to rats pretreated withsaline.

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Fig. 5 – Yohimbine enhances WDS induced by a low dose oficilin. Rats pretreated with yohimbine (YOH) (0.5, 1, 2 mg/kg)or saline were injected 15 min later with icilin (0.5, 2.5 mg/kg). Data from 6 to 8 rats per group are expressed as meanWDS+S.E.M. *P<0.05 compared to rats pretreated withsaline.

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experiments with the low dose of icilin, one-way ANOVArevealed a significant drug effect [F (3, 27)=1.48, P<0.0001](Fig. 5, Panel A). Dunnett's post-hoc analysis indicated that0.5 mg/kg of icilin produced a significantly greater number ofWDS in rats pretreated with 2 mg/kg of yohimbine ascompared to rats pretreated with saline (P<0.01). For experi-

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Fig. 6 – Imidazoline site agonists, agmatine and 2-BFI, do not reduagmatine (150 mg/kg) or saline were injected 15 min later with icexpressed as mean WDS+S.E.M. Box) Rats pretreated with 2-BFI(2.5 mg/kg). Data from 6 to 8 rats per group are expressed as me

ments with the higher dose of icilin (2.5 mg/kg), one-wayANOVAdid not reveal a significant drug effect [F (3, 28)=0.9647,P>0.05] (Fig. 5, Panel B).

2.5. Pretreatment with imidiazoline site agonists do notaffect icilin-induced shaking

The effects of a fixed dose of agmatine (150 mg/kg) on WDSelicited by icilin (0.5, 1, 2.5, 5 mg/kg) are presented in Fig. 6.Two-way ANOVA (pretreatment, icilin dose) revealed asignificant effect of dose [F (3, 46)=12.58, P<0.0001] but not asignificant effect of pretreatment [F (1, 46)=0.07886, P=0.7802] ,thus indicating that icilin-induced shaking was not signifi-cantly affected by agmatine exposure. Lower doses of agma-tine (25, 75 mg/kg) were equally ineffective as they failed tosignificantly alter WDS induced by 2.5 mg/kg of icilin (P>0.05)(data not shown). In separate experiments, rats injected with2.5 mg/kg of icilin following pretreatment with 2-BFI (7 mg/kg)did not display shaking that was significantly different fromicilin (2.5 mg/kg)-exposed rats pretreated with saline (P>0.05,Student's t-test) (Fig. 6, box).

3. Discussion

Icilin is a cold-inducing compound that produces a behavioralstimulant effect comprised of WDS, hyperthermia, writhing,and excessive grooming in rats following intraperitonealinjection (Collier et al., 1974; Wei, 1976, 1981; Collier et al.,1981; Cowan and Watson, 1978; Cowan, 1981; Werkheiseret al., 2006, 2007, 2009; Ding et al., 2008). Consistent with priorwork, icilin elicited a dose-dependent increase in shakingbehavior in rats in the present experiments. Although the

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ce WDS elicited by icilin. Rats pretreated with a fixed dose ofilin (0.5, 1, 2.5, 5 mg/kg). Data from 6 to 8 rats per group are(7 mg/kg) or saline were injected 15 min later with icilinan WDS+S.E.M.

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dramatic stimulant effect of icilin was identified 38 years ago(Burford and Chappel, 1972), its cellular sites of action of icilinhave only recently been identified. The TRPM8 channel is onesite of action (McKemy et al., 2002; Peier et al., 2002; Story et al.,2003; Liu et al., 2006). A second possible site of action is theTRPA1 channel (Bandell et al., 2004), although this finding wascontradicted by another study which demonstrated thatTRPA1 channels are insensitive to icilin and cold (Jordt et al.,2004). TRPM8 and TRPA1 channels are located in the peripheryon dorsal root ganglia and trigeminal neurons and are part ofthe TRP superfamily of ion channels that share significantsequence homology (Moran et al., 2004). Mechanisms under-lying the behavioral stimulant effect of icilin, includingpotential roles for TRPM8 and TRPA1 channels, remain poorlyunderstood. Wei and Seid (1983) originally proposed that icilinexerts its stimulant actions through a peripheral mechanism,an interpretation now supported by evidence that directadministration of icilin into the lateral ventricle or dorsalstriatum fails to cause WDS or hyperthermia (Werkheiseret al., 2006; Ding et al., 2008). These observations are alsosupported by molecular evidence that TRPM8 and TRPA1channels are located primarily, if not exclusively, in theperiphery (Fonfria et al., 2006; Voets et al., 2007). Furthermore,pretreatment of rats with a small molecule TRPM8 channelantagonist (JNJ-39267631) inhibits shaking produced by intra-peritoneal icilin injection (Colburn et al., 2008). Taken togeth-er, these data suggest that activation of TRPM8 channelslocated outside of the CNS is responsible for the developmentof icilin-induced shaking.

The first significant finding in the present study was thatclonidine pretreatment and posttreatment reduced icilin-evokedWDS. These outcomes suggest that systemic clonidineadministration inhibits both the development and mainte-nance of icilin-evoked shaking.

One clue to the mechanism by which clonidine inhibitedicilin-evoked shaking in our experiments may reside in itswide-ranging efficacy against WDS induced by a panoply ofpharmacological manipulations, including kainic acid, nalox-one-precipitated morphine withdrawal, carbachol, benzylide-neaminooxycarbonic acid derivatives, and thyrotropin (TRH)-releasing hormone analogues (Jahn and Mixich, 1976; Byrskaet al., 1980; Turski et al., 1981a,b; Wei, 1983; Ohno et al., 1987;Velísek et al., 1994). The fact that dissimilar pharmacologicalmanipulations evoke a similar behavioral stimulant effect,WDS, suggests that a common downstream signaling path-way underlies the phenomenon and that alpha2-adrenoceptoractivation by clonidine inhibits the normal activation orrecruitment of the said pathway.

One possibility is that clonidine disrupts the centralglutamate component of icilin-induced shaking. The fullstimulant effect of icilin is dependent on increased glutamatetransmission in the brain, as the shaking response is accom-panied by an elevation in extracellular striatal glutamate andinhibited by NMDA receptor antagonism (Werkheiser et al.,2007, 2009). Neurochemical data obtained from conscious ratsreveal that clonidine reduces extracellular glutamate andinhibits, through alpha2-adrenoceptor activation, presynapticglutamate release in the brain and spinal cord (Tingley andArnerić, 1990; Kamisaki et al., 1992). Thus, when administeredwith clonidine, icilin still activates TRPM8 channels, but the

ensuing downstream increase in glutamate transmission thatunderlies a significant proportion of the behavioral stimulanteffect is suppressed because of alpha2-adrenoceptor activationby clonidine, leading to an overall reduction in icilin efficacy.The behavioral stimulant effect of icilin is also dependent on anon-glutamate component, and it is possible that clonidinetargets substrates within this component to inhibit theshaking response, perhaps through indirect opioid receptoractivation or inhibition of nitric oxide production (Werkheiseret al., 2006, 2009). A role for 5-HT2A receptors in thepharmacological effects of icilin has not been identified, butit is documented that 5-HT2A receptor activation elicits WDS(Kitamura et al., 2002; Umeda et al., 2007). In vivomicrodialysisstudies demonstrating that clonidine reduces extracellularserotonin in brain regions that trigger stimulant activitysuggest clonidine efficacy may have been partly dependenton its ability to reduce serotonergic transmission at the 5-HT2A

receptor (Pudovkina et al., 2003).The positive results with clonidine suggest that alpha2-

adrenoceptor activation suppresses the behavioral stimulanteffect of icilin, but they do not discriminate between periph-eral and central alpha2-adrenoceptor mechanisms. Becauseclonidine is a lipophilic compound that readily penetrates theCNS, its systemic administration activates alpha2-adrenocep-tors located in the peripheral and central nervous systems.Thus, we conducted experiments to determine the effect of aperipherally restricted alpha2-adrenoceptor agonist (ST91) onicilin-induced shaking. What we found was that pretreatmentof rats with ST91 dose-dependently inhibited shaking inducedby icilin. The degree of inhibition by ST91 was significant butstill less than that of clonidine, an observation which suggeststhat both peripheral and central alpha2-adrenoceptor activationinhibits icilin-induced shaking. Support for this interpretation isprovided by in vivo studies in rats which demonstrated that thepotency of ST91 is about ten times less than that of clonidinefollowing systemic injection, whereas ST91 displays the samedegree of potency as clonidine after injections into the cerebralventricles (Chariot et al., 1988; Taylor et al., 1988; Eshel et al.,1990). Furthermore, ST91, when administered by the same routeand within the same dose range (0.075–0.15mg/kg) as in ourstudy, did not alter the extent of shaking precipitated bynaloxone in morphine-dependent rats (Taylor et al., 1988). It isequally important to note that administration of ST91 into themidbrain almost completely inhibits WDS produced by nalox-one-precipitated morphine withdrawal (Taylor et al., 1988). Thecombined findings with ST91 suggest that its effects againsticilin aremediatedbyperipherally located alpha2-adrenoceptorsand that activation of peripheral alpha2-adrenoceptors inhibitsicilin-evoked behavioral activation. In fact, alpha2-adrenocep-tors and TRPM8 channels are co-expressed by sensory neuronsin the dorsal root ganglia and activation of these alpha2-adrenoceptors is postulated to reduce signaling events initiatedby TRPM8 channel activation (Bavencoffe et al., 2010). Thus,enhanced activity at the subpopulation of peripheral alpha2-adrenoceptors, through a reduction in TRPM8 channel signaling,may be one mechanism that counters the normal behavioralactivation produced by icilin.

Another interesting finding was that rats pretreated withthe alpha2-adrenoceptor antagonist, yohimbine, displayed anincreased incidence of WDS following icilin administration.

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Yohimbine did not produce shaking per se. These resultssuggest that endogenous adrenergic tone exerted at alpha2-adrenoceptors inhibits icilin-induced shaking. Since yohim-binewas administered systemically and penetrates the CNS, itis unclear whether peripheral or central alpha2-adrenoceptorsare involved in the effect. The ability of yohimbine to enhanceicilin-induced shaking was, however, dependent on the doseof icilin. Yohimbine enhanced the shaking produced by a lowdose (0.5 mg/kg) of icilin but not by a higher dose (2.5 mg/kg).These data suggest that tonic alpha2-adrenoceptor activationcounters the shaking elicited by a low level of TRPM8, orperhaps TRPA1, channel activity. The ineffectiveness of analpha2-adrenoceptor block on WDS induced by a higher doseof icilin may be due to a “ceiling effect.” In this case, a greaterlevel of TRPM8 channel activity resulting from the adminis-tration of higher icilin doses may cause a maximum degree ofshaking that cannot be further enhanced. Alternatively, theendogenous tone exerted at alpha2-adrenoceptors may besimply incapable of countering the greater degree of shaking(i.e., the shaking overwhelms the compensatory adrenergicresponse).

A final series of experiments tested the effect of agmatineon icilin-evoked shaking. Agmatine is a biogenic amine that isproduced in the brain from arginine by the catalytic activity ofarginine dexarboxylase (Reis and Regunathan, 2000). Perhapsthe most notable pharmacological effect of agmatine is itsbiphasic modulation of mu opioid receptor function, namelyenhancement of acute morphine analgesia and inhibition ofmorphine-induced analgesic tolerance, physical dependence,and behavioral sensitization (Su et al., 2003; Regunathan, 2006;Wei et al., 2007). Agmatine is a pharmacologically diversesubstance. It acts through multiple mechanisms, includingimidazoline site activation, NMDA receptor antagonism, nitricoxide synthesis inhibition, and peptide hormone release(Loring, 1990; Li et al., 1994; Piletz et al., 1995; Gilad et al.,1996; Fairbanks et al., 2000; Reis and Regunathan, 2000).Agmatine also interacts with the target of interest in ourexperiments, alpha2-adrenoceptors, but its actual effects atthese receptors remain unclear. Some studies suggest that anumber of the pharmacological effects of agmatine aredependent on active alpha2-adrenoceptors whereas otherreports suggest that agmatine binds to alpha2-adrenoceptorsbut neither inhibits nor activates these receptors (Li et al.,1994; Pinthong et al., 1995; Kotagale et al., 2010). In the presentstudy, we found that agmatine, even when administered atdoses as great as 150 mg/kg, failed to affect icilin-inducedshaking. Moreover, 2-BFI, a selective imidazoline site agonist,also lacked efficacy against icilin-induced shaking (Macinnesand Duty, 2004). The ineffectiveness of agmatine and 2-BFI donot support a major role for imidazoline sites in themechanism of icilin-induced shaking, especially since othermechanisms of action of agmatine (i.e., NMDA receptorantagonism and inhibition of nitric oxide production), dosignificantly reduce behavioral activation produced by icilin(Ding et al., 2008; Werkheiser et al., 2009). Although it isunclear why the NMDA receptor antagonist LY 235959attenuates icilin-induced shaking and agmatine is ineffective,it may be related to the mechanisms of the compounds. LY235959 is a selective NMDA antagonist that competitivelyblocks the glutamate recognition site at the NMDA receptor

complex whereas agmatine displays properties that aresimilar to the pharmacological profiles of NMDA receptorchannel blockers such as phencyclidine and ketamine (Yangand Reis, 1999). Evidence also suggests that compoundstargeting non-NMDA receptors, including AMPA receptorantagonists and GLT-1 activators, do not affect icilin-inducesshaking (Werkheiser et al., 2009).

In conclusion, our results confirm and expand the originalobservation byWei (1976, 1983) that shakingmovements in ratsare dependent on the level of adrenergic activity. Specifically,we demonstrate that the alpha2-adrenoceptor agonist clonidinereduces both the development and maintenance of icilin-induced shaking. We also demonstrate that a peripherallyrestricted alpha2-adrenoceptor agonist reduces icilin-inducedshaking. Taken with the recent finding by Bavencoffe et al.(2010) that sensory neurons co-express alpha2-adrenoceptorsand TRPM8 channels, our results suggest that activation ofalpha2-adrenoceptors located in both theperipheral and centralnervous systems reduce the behavioral stimulation elicited byicilin.

4. Experimental procedures

4.1. Animals and compounds

Male Sprague–Dawley rats (Ace Laboratories, Boyertown, PA),weighing 100–125 g,were housed in groups of three for five daysprior to use, with food and water available ad libitum. Thetemperature in the roomwas 23±1 °C and a standard light–darkcycle was maintained with a timer-regulated light period from0700 to 1900 h. The studies were conducted in accordance withthe NIH Guide for the Care and Use of Laboratory Animals andapproved by the Institutional Animal Care and Use Committeeat Temple University. Icilin, a gift from Delmar Chemicals Ltd.(Montreal, Canada), was suspended in 1% Tween 80/distilledwater and injected interapertioneally (i.p.). Clonidine hydro-chloride, ST91, yohimbine hydrochloride, agmatine sulfate, and2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) werepurchased from Tocris Laboratories (Ellisville, MO, USA),dissolved in physiological saline, and injected i.p. All com-pounds were injected in a volume of 1 ml/kg.

4.2. Experimental design

Experiments were performed during the light phase betweennoon and 5 p.m. Each rat was weighed and acclimated in aPlexiglas observation box (22 cm long; 18 cmwide; 25 cm high)90 min before administration of test compounds as describedpreviously (Werkheiser et al., 2006, 2009; Ding et al., 2008). Forthe clonidine studies, three separate experiments wereperformed in which rats were injected with: (1) clonidine(0.03, 0.06, 0.15 mg/kg) or saline 15 min prior to a fixed dose oficilin (2.5 mg/kg); (2) clonidine (0.15 mg/kg) or saline 15 minprior to one progressively increasing icilin dose (0.5, 1, 2.5,5 mg/kg); or (3) clonidine (0.15 mg/kg) or saline 5 min followinginjection of icilin (2.5 mg/kg). For the ST91/icilin experiments,rats were pretreated with ST91 (0.0375, 0.075, 0.15 mg/kg) orsaline 15 min prior to the injection of a fixed dose of icilin(2.5 mg/kg). For the yohimbine/icilin experiments, rats were

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pretreated with yohimbine (0.5, 1, 2 mg/kg) or saline 15 minprior to the injection of icilin (0.5, 2.5 mg/kg). For the agmatine/icilin and 2-BFI/icilin experiments, rats were injected withagmatine (150 mg/kg) 15 min prior to icilin (0.5, 1, 2.5, 5 mg/kg),and with 2-BFI (7 mg/kg) 15 min prior to icilin (2.5 mg/kg). Theincidence of WDS was counted for 30 min after icilinadministration in all experiments except the clonidineposttreatment experiment, in which the number of WDSwere counted for 35 min. Following experimentation, each ratwas placed in the prone position on the Perspex lid of theobservation chamber, and tested for sedation. If the rat movedfreely off the lid and on to the bench top, it was considered“not behaviorally depressed.” None of the rats in ourexperiment were observed to be behaviorally depressedfollowing experimentation. Rats were used only once foreach experiment and euthanized immediately followingexperimentation. Doses of icilin and the test compounds, aswell as pretreatment intervals, were based on previous in vivostudies in our, and other, laboratories (Taylor et al., 1988;Werkheiser et al., 2006, 2009; Aggarwal et al., 2009).

4.3. Data analysis

Data are expressed as mean wet-dog shakes±S.E.M. (Graph-Pad Prism). For experiments testing a fixed dose of clonidine oragmatine against multiple doses of icilin, two-way ANOVA(pretreatment, treatment) was used to evaluate overalldifferences between groups followed by Bonferroni's post-hocanalysis to compare individual groups. For experimentstesting multiple doses of clonidine, ST91, or yohimbineagainst a fixed dose of icilin, one-way ANOVA was used todetermine a significant main effect followed by a Dunnett'spost-hoc analysis to compare individual groups to the iclin-treated group. For the 2-BFI experiments, two groups werecompared using a Student's t-test. Values of P<0.05 wereconsidered statistically significant.

Acknowledgment

This studywas supported by NIDA grants P30DA01342 and T32DA07237-17.

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