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Research Report

Mouse cerebellar nicotinic–cholinergic receptor modulationof Δ9-THC ataxia: Role of the α4β2 subtype

Aaron D. Smith, M. Saeed Dar⁎

Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 252 744 3203.E-mail address: darm@ecu.edu (M.S. Dar).

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.07.075

A B S T R A C T

Article history:Accepted 24 July 2006Available online 24 August 2006

In spite of widespread association of nicotine and cannabinoids in humans, very few studiesin which nicotine and cannabinoids are co-administered have been reported. Previously, wehave reported that intracerebellar (ICB) Δ9-tetrahydrocannabinol (Δ9-THC) produces dose-dependent cerebellar ataxia. The present study investigated the functional consequences ofICB microinfusion of nicotine on ICB Δ9-THC ataxia in CD-1 male mice. Nicotine (0.625, 1.25,2.5, 5 ng; ICB)markedly attenuated Δ9-THC ataxia dose dependently, whichwas abolished byICB hexamethonium (5 μg), thus suggesting that the attenuation by nicotine occurred via thenicotinic acetylcholine receptor (nAChR). To further investigate which specific nAChRsubtype was involved, ICB microinfusion of RJR-2403 (250, 375, 500, 750 ng), a α4β2 selectivenAChR agonist, markedly attenuated Δ9-THC ataxia. DHβE (500 ng), a α4β2 selective nAChRantagonist, virtually abolished RJR-2403-induced attenuation of Δ9-THC ataxia. ICBmicroinfusion of MLA, a α7 selective nAChR antagonist (1, 5 μg) failed to antagonizenicotine or RJR-2403-induced attenuation of Δ9-THC ataxia. This suggested a lack of a role ofthe α7 subtype and further reinforced the significance of α4β2. Additionally, ICB treatmentwith DHβE virtually abolished nicotine-induced attenuation of Δ9-THC ataxia that suggestedα4β2 as the primary cerebellar nAChR subtype. Lack of effect of ICB DHβE or MLA alone on Δ9-THC ataxia ruled out a tonic effect of the α4β2 subtype. The results of the presentinvestigation, therefore, strongly support involvement of the cerebellar α4β2, but not α7,nicotinic receptor subtype in the mediation via nicotine and RJR-2403 on attenuation of Δ9-THC ataxia.

© 2006 Elsevier B.V. All rights reserved.

Keywords:Δ9-THCAtaxiaNicotineRJR-2403α4β2 nAChR subtypeCerebellum

Abbreviations:AUC, area under curveaCSF, artificial cerebrospinal fluidΔ9-THC, Δ9-tetrahydrocannabinolnAChR, nicotinic acetylcholinereceptorDHβE, dihydro-β-erythroidineDMSO, dimethyl sulfoxideRJR-2403, metanicotineMLA, methyllycaconitinecpt-cAMP, 8-(4-chlorophenylthio)cAMPGABA, gamma-aminobutyric acidIP, intraperitonealICB, intracerebellar

1. Introduction

The cannabis sativa plant,morewidely knownasmarijuana, isknown to contain Δ9-tetrahydrocannabinol (Δ9-THC) as the

er B.V. All rights reserved

major psychoactive component, and is the most widelyconsumed illicit drug in humans (Adams and Martin, 1996).Additionally, there is a high frequency of association in theabuse of marijuana and nicotine (via the cigarette) together

.

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(Watson et al., 2000). It has been well established that Δ9-THChas a profound impact on motor effects, including that ofhypokinesia, catalepsy, and ataxia (Dewey, 1986). These effectsthat Δ9-THC and other cannabinoids have on motor functionhave led to widespread investigation of cannabinoids forpossible use in the treatment and pharmacotherapy of motordisorders such as tremor and dystonias (Clifford, 1983). A hugeleap in the field of cannabinoids occurredwith the discovery ofthe brain cannabinoid (CB1) receptor population (Devane et al.,1988). Localization studies revealed that there is a dense CB1

receptor population within the cerebellum (Matsuda et al.,1993), which may have a role in the ataxia, immobility, andcatalepsy that is observed after acute administration of Δ9-THCand other cannabinoids (Fonseca et al., 1998).

The active constituent found in the dried leaves of tobaccoplants (more specifically N. tabaum and N. rustica) is nicotine.Nicotine is most frequently used via smoking of a cigarettecontaining 1.5% nicotine by weight and nicotine being 95% oftotal alkaloid content (Benowitz and Jacob, 1999). Nicotine andits related agonists are known to activatenAChRswhichhaveasignificant impact upon cognitive performance, locomotoractivity, body temperature and pain perception (Lloyd andWilliams, 2000). More specifically, in dealing with the cerebel-lum, there is a clear role for nicotine and related agonists, asboth curare and hexamethonium sensitive sites are presenthere which act to mediate excitatory or inhibitory effects ofnicotine, respectively (De la Garza et al., 1987). Electrophysio-logical studies have revealed that two nicotinic receptorsubtypes which predominate in the cerebellum include theα4β2, which is found in the soma and dendrites of granuleneurons (De Filippi et al., 2001) as well as the α7, primarilylocated in the Purkinje neurons (Caruncho et al., 1997). Inaddition to α4β2 and α7 nAChR subtypes, immunohistochem-ical localization studies have revealed that α6, α3 and β4

subtypes are also locatedwithin the cerebellum (Grahamet al.,2002).

Research has been limited addressing the relationshipbetween the co-administration of nicotine and Δ9-THC, andtheir impact on motor impairment. One study has shown thatnicotine strongly facilitates the hypothermic, antinociceptiveand hypolocomotive effects induced by acute Δ9-THC admin-istration (Valjent et al., 2002). Interestingly, it has been shownthat a dose of nicotine may produce a decrease in locomotiveperformance, with these decreases in activity seen via activitymonitoring boxes (Morrison, 1969). However, this same dose ofnicotine can also result in improvement in locomotorperformance, as evidenced via increases in performance ona Rotorod treadmill (Morrison, 1969). This interesting relation-ship between nicotine and locomotor function shows thecomplexity of the behavioral effects of nicotine. Thus it is clearthat further investigation needs to be conducted in the arearegarding nicotine, Δ9-THC and their relationship to motorcoordination. It was the goal of this study to elucidate thefunctional interaction between nicotine and Δ9-THC withregard to motor impairment, and to further delineate thespecific nAChR subtype(s) responsible for the attenuation ofΔ9-THC ataxia.

The present investigation, therefore, was intended toevaluate the consequences of direct intracerebellar micro-infusion of nicotine on Δ9-THC ataxia. Once the involvement

of nicotine in Δ9-THC ataxia had been observed, furtherinvestigation regarding the specific subtype(s) of the nAChRmodulating Δ9-THC ataxia was carried out. Two subtypes ofthe nAChR, α4β2 and α7, were chosen to be the focus of theinvestigation. These subtypes were selected because of theirsignificant distribution within the cerebellar cortex (DeFilippi et al., 2001; Nakayama et al., 1997) and literaturereports that implicate the nicotinic α4β2 subtype in nicotineaddiction (Tapper et al., 2004). We hypothesize that the α4β2

nAChR subtype, and not α7, mediates the functional interac-tion between nicotine and Δ9-THC within the cerebellarcortex.

2. Results

Based on our previous dose response studies (Dar, 2000), amedian dose of Δ9-THC at 20 μg was selected because of itsability to produce significant ataxia. Therefore, for all experi-ments conducted in this investigation, an intracerebellar doseof 20 μg/1 μl of Δ9-THC was administered. However, thevolume for all other drugs microinfused in the present studywas 100 nl. Fig. 1A shows a dose–response profile for nicotinein the presence of Δ9-THC. Intracerebellar nicotine microinfu-sion was made at four dosages, beginning from 0.625 ng andincreasing up to 5 ng. As shown in Fig. 1A, there is a clear dose-dependent attenuation of acute intracerebellar Δ9-THC-induced ataxia. Nicotine at 2.5 and 5 ng resulted in an almostcomplete abolishment of Δ9-THC ataxia. The “aCSF+Δ9-THC”treatment was used as a positive functional control to ensureproper drug action within the superficial layers of thecerebellar cortex. Fig. 1B shows the Area under the Curve(AUC) of the same treatment groups as in Fig. 1A. Area underthe curve is another quantitative measure to look at the totallevel of ataxia present between the different treatmentgroups. The larger a treatment groups AUC is, the greaterthe amount of ataxia present. The relationship between AUCand the dose of nicotine is inversely proportional. In otherwords, as the dose of nicotine increases, the total AUCdecreases. Thus from Fig. 1B, it is clear that the AUC decreasesin a dose-dependent manner as the dose of nicotine increasesfrom the 0.625 ng to 5 ng. In addition, the AUC clearlydemonstrates that the “aCSF+Δ9-THC” treatment has thelargest AUC, which similarly correlates with this treatmentgroup having the largest degree of ataxia present. Informationregarding drug treatments in Fig. 1A was shown over thecorresponding bars in Fig. 1B.

Once we observed that nicotine pretreatment markedlyattenuated Δ9-THCataxia,we subsequently demonstrated thatnicotine-induced attenuation of Δ9-THC ataxia was mediatedvia the nAChR(s). Intracerebellarmicroinfusion of hexametho-nium, a non-selective nAChR antagonist, at either a 1 μg or 5 μgdose prior to nicotine administration, significantly attenuatedor virtually abolished nicotine's ability to decrease Δ9-THCataxia (Fig. 2A). Conversely, to ascertain if the initial antagon-ismof the cerebellarnAChRbyhexamethoniumwasnecessaryto block nicotine's action on Δ9-THC ataxia, administration ofnicotine was given at 5 ng first followed by hexamethonium at5 ng and lastly Δ9-THC i.e., the order of microinfusion ofnicotine and hexamethonium was reversed. When nicotine

Fig. 2 – Effect of acute intracerebellar microinfusion of nAChRantagonist hexamethonium (1 μg or 5 μg), onnicotine-induced attenuation of Δ9-THC (20 μg) cerebellarataxia inmice. Each point represents themean±SE of 10mice.(D) nicotine 5 ng+Δ9-THC; (▲) nicotine 5 ng+hexamethonium 5 μg+Δ9-THC; (■) hexamethonium 1 μg+nicotine 2.5 ng+Δ9-THC; (□) hexamethonium 5 μg+nicotine5 ng+Δ9-THC; (•) hexamethonium 5 μg+Δ9-THC;(○) aCSF+Δ9-THC. A significant drug treatment and timeinteraction was observed (F15,187=24.45; P<0.0001).Pretreatment with nicotine (5 ng) prior to hexamethonium(5 μg) revealed significant (p<0.01) attenuation ofΔ9-THC-induced ataxia at 10, 20 and 30 min post Δ9-THCinfusion. In contrast, the treatments of both“hexamethonium 1 μg+nicotine 2.5 ng+Δ9-THC” and“hexamethonium 5 μg+nicotine 5 ng+Δ9-THC”, as well as“hexamethonium 5 μg+Δ9-THC” did not produce significantattenuation of Δ9-THC-induced ataxia as compared to“aCSF+Δ9-THC” control. Panel B shows AUC data for the sametreatment groups as in panel A with drug treatments shownabove the corresponding bar. Although not indicated, Δ9-THCwas administered to all groups.

Fig. 1 – Effect of intracerebellar microinfusion of nicotine(0.625, 1.25, 2.5, 5 ng) on intracerebellar Δ9-THC (20μg) ataxiain mice. Each point represents the mean±SE of 10 mice. (•)nicotine 5 ng+Δ9-THC; (▲) nicotine 2.5 ng+Δ9-THC; (□)nicotine 1.25 ng+Δ9-THC; (▼) nicotine 0.625 ng+Δ9-THC;(○) aCSF+Δ9-THC. A significant drug treatment and timeinteraction was observed (F12,127=30.02; P<0.0001).Significant attenuation of Δ9-THC ataxia was presentfollowing 5 ng (p<0.01), 2.5 ng (p<0.01) and 1.25 ng (p<0.01)doses of nicotine at 10, 20 and 30 post Δ9-THC intervals. At10 min post Δ9-THC infusion, the 0.625 ng nicotine treatmentcaused significant (p<0.05) attenuation of ataxia ascompared to “aCSF+Δ9-THC” treated control. The ED50 valueand 95% confidence limits for nicotine was 1.28 ng(0.95–1.72). Panel B shows AUC data for the same treatmentgroups as in panel A with drug treatments shown above thecorresponding bar. Although not indicated, Δ9-THC wasadministered to all groups.

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was administered prior to hexamethonium, significantattenuation of Δ9-THC ataxia was evidenced. Thus, the orderof drug administration here plays a crucial role in the beha-vioral outcome, and clearly demonstrates the critical impor-tance of initial activation of the cerebellar nAChR by nicotine.Fig. 2Bdemonstrates theAUCof thehexamethonium/nicotine/Δ9-THC data. As is evidenced in Fig. 2B, the AUC is significantlydiminished when pretreatment with nicotine occurs prior tohexamethonium administration. However, when hexametho-nium (either 1 μg or 5 μg) is given prior to nicotine, AUC issignificantly increased closer to “aCSF+Δ9-THC” AUC levels.

Upon establishing that intracerebellar nicotine attenuatesacute intracerebellar Δ9-THC ataxia through the cerebellarnAChR, it was important to delineate the specific cerebellar

nAChR subtypes responsible for the attenuation of Δ9-THCataxia. In the present investigation, we focused on twofunctionally important cerebellar nAChR subtypes, α4β2 andα7. RJR-2403 (metanicotine), a α4β2 selective nicotinic agonist,was microinfused in a dose–response paradigm followed byΔ9-THC administration. Fig. 3A illustrates a clear dose-dependent decrease in Δ9-THC ataxia as the dose of RJR-2403 increases. Thus RJR-2403, at 750 ng was able tocompletely abolish Δ9-THC ataxia in the acute paradigm. Inaddition, significant attenuation of Δ9-THC ataxia by RJR-2403was present at 500 ng and 375 ng. Thus, a clear role for theα4β2 nAChR in mediating attenuation of Δ9-THC ataxia was

Fig. 4 – Effect of intracerebellar microinfusion of DHβE (α4β2

selective nAChR antagonist; 250/500 ng) on RJR-2403(750 ng)-induced attenuation of acute intracerebellar Δ9-THC(20 μg) ataxia in mice. Each point represents the mean±SE of10 mice. (▲) DHβE 250 ng+RJR-2403+Δ9-THC;(□) DHβE 500 ng+RJR-2403+Δ9-THC; (▼) aCSF+Δ9-THC. Asignificant drug treatment and time interactionwas observed(F6,60=9.85; P<0.0001). RJR-2403 in the presence of DHβE at250 ng was able to significantly attenuate (p<0.01)Δ9-THC-induced ataxia at 10, 20 and 30 min post Δ9-THCinfusion. However, at a 500 ng dose of DHβE, inhibition ofRJR-2403 attenuation of Δ9-THC ataxia was present, makingthis treatment statistically insignificant from “aCSF+Δ9-THC”control. Panel B shows AUC data for the same treatmentgroups as in panel A with drug treatments shown above thecorresponding bar. Although not indicated, Δ9-THC wasadministered to all groups.

Fig. 3 – Effect of intracerebellar microinfusion of RJR-2403(α4β2 selective agonist; 250, 375, 500, 750 ng) onintracerebellar Δ9-THC (20 μg) ataxia in mice. Each pointrepresents the mean±SE of 10 mice. (○) RJR-2403 250 ng+Δ9-THC; (▼) RJR-2403 375 ng+Δ9-THC; (□) RJR-2403 500 ng+Δ9-THC; (▲) RJR-2403 750 ng+Δ9-THC; (■) aCSF+Δ9-THC. Asignificant drug treatment and time interactionwas observed(F9,95=11.48; P<0.0001). Significant attenuation (p<0.01) ofΔ9-THC ataxia was present at the 10, 20 and 30 min postΔ9-THC infusion times for RJR-2403 following 750 ng and500 ng doses. RJR-2403 at 375 ng significantly attenuated(p<0.05) Δ9-THC ataxia at the 20 min interval. RJR-2403 at250 ng was not significant in the attenuation ofΔ9-THC-induced ataxia. The ED50 value and 95% confidencelimits for RJR-2403 was 471.4 ng (417.1–561.2). Panel B showsAUC data for the same treatment groups as in panel A withdrug treatments shown above the corresponding bar.Although not indicated, Δ9-THC was administered to allgroups.

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observed. Fig. 3B demonstrates a similar relationship withRJR-2403 as is evidenced with nicotine in Fig. 1B. That is, asthe dose of RJR-2403 increases from 250 ng up to 750 ng, thetotal AUC decreases in a dose-dependent manner. Onceagain as in Fig. 1B, the “aCSF+Δ9-THC” group proved to havethe largest AUC in Fig. 3B.

To obtain further evidence that the α4β2 nAChR subtype isresponsible for attenuation of Δ9-THC ataxia, DHβE, a α4β2

selective antagonist, wasmicroinfused at 250 or 500 ng prior toRJR-2403 (750 ng) and finally Δ9-THC. DHβEwas able to producea virtual blockade of RJR-2403's ability to attenuate Δ9-THCataxia at 500 ng, thus further indicating the importance of theα4β2 nAChR subtype in attenuation of Δ9-THC ataxia (Fig. 4A).

Fig. 4B demonstrates that the group receiving a 250 ng dose ofDHβE has a smaller AUC as compared to the AUC for the 500 ngDHβE treatment. These observations correlate to Fig. 4A, sincethe larger AUC (500 ng DHβE) indicates a stronger antagonismof RJR-2403-induced attenuation of Δ9-THC ataxia as comparedto the 250 ng dose of DHβE.

We also investigated the possible role of the cerebellar α7

nAChR subtype in Δ9-THC ataxia. Administration of MLA(methyllycaconitine) has been proven from past literature tobe active and eliciting a full blockade of α7 receptors in themicromolar range (Grottick et al., 2000), and thuswhywe chosea 1 μg dose of MLA. Therefore, we administered MLA at thisdose in the presence of either nicotine (5 ng) or RJR-2403(750 ng). Fig. 5A shows that MLA displays no significant role inthe attenuation of Δ9-THC ataxia in mouse cerebellum. Boththe nicotine and RJR-2403 groups, in the presence of MLA at1 μg, displayed an almost complete abolishment of Δ9-THCataxia as compared to “aCSF+Δ9-THC” control. To ensure that

Fig. 5 – Effect of intracerebellar microinfusion of MLA(α7 antagonist) on intracerebellar RJR-2403 (750 ng) ornicotine's (5 ng) ability to attenuate Δ9-THC (20 μg) ataxia.Each point represents the mean±SE of 10 mice.(▲) MLA 1 μg+nicotine+Δ9-THC; (□) MLA 1 μg+RJR-2403+Δ9-THC; (D) MLA 5 μg+nicotine+Δ9-THC; (▼) aCSF+Δ9-THC.A significant drug treatment and time interaction wasobserved (F9,92=17.77; P<0.0001). MLA at 1 μg was unable toantagonize RJR-2403 (p<0.01) nor nicotine's (p<0.01) abilityto attenuate Δ9-THC-induced ataxia. In addition, when thedose of MLA was increased to 5 μg, MLA was still not able toantagonize nicotine (p<0.01) attenuation of Δ9-THC ataxia.Panel B shows AUC data for the same treatment groups as inpanel A with drug treatments shown above thecorresponding bar. Although not indicated, Δ9-THC wasadministered to all groups.

Fig. 6 – Effect of intracerebellar microinfusion of DHβE onnicotine (5 ng)-induced attenuation of intracerebellar Δ9-THC(20 μg) ataxia in mice. Each point represents the mean±SE of10 mice. (▲) DHβE 500 ng+nicotine+Δ9-THC; (□) DHβE 1 μg+nicotine+Δ9-THC; (▼) aCSF+Δ9-THC. A significant treatmentand time interaction was not observed (F6,58=0.260; P=0.42).Significant antagonism of nicotine-induced attenuation ofΔ9-THC-induced ataxia occurred when DHβE wasadministered in the presence of nicotine at either 500 ng or1 μg. Panel B shows AUC data for the same treatment groupsas in panel A with drug treatments shown above thecorresponding bar. Although not indicated, Δ9-THC wasadministered to all groups.

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MLAat the dose selected in the study significantly antagonizedα7, a 5-fold higher dose of MLA (5 μg) was given in the presenceof nicotine (5 ng), followed by ICB microinfusion of Δ9-THC.Even at the higher dose of MLA, nicotine was able to almostcompletely abolish Δ9-THC ataxia indicating virtually noinvolvement of the α7 subtype. These findings helped tostrengthen the postulation that the α4β2 is the importantsubtype of the cerebellar nAChR involved in the mediation ofnicotine-induced attenuation of Δ9-THC cerebellar ataxia. Fig.5B reinforces the data presented in Fig. 5A, in that both groups,whethernicotine or RJR-2403 treated, showedvirtuallynoAUC,indicating no ataxia upon antagonism of the α7 receptor ateither dose of MLA.

Following the demonstration that the cerebellar α4β2

nAChR is the main subtype responsible for attenuation ofmotor impairment due to Δ9-THC, we evaluated the effect ofits selective antagonist DHβE in the presence of nicotine. This

experiment could confirmwhether or not any other cerebellarnAChR subtype(s) is/are possibly involved in the attenuationof Δ9-THC ataxia. We microinfused DHβE at either a 500 ng or1 μg dose, followed by microinfusion of nicotine at 5 ng andthen Δ9-THC. As shown in Fig. 6A, pretreatment with bothdoses of DHβE completely abolished nicotine-inducedattenuation of Δ9-THC ataxia. Thus, antagonism of thecerebellar α4β2 nAChR subtype alone was sufficient incompletely eliminating nicotine's ability to attenuate Δ9-THCataxia. Fig. 6B shows the AUC for the same treatments as inFig. 6A. It is clear that the total AUC is not different betweenthe groups that received either the 500 ng or 1 μg dose of DHβE,and neither group has a significant AUC difference from thatof “aCSF+Δ9-THC” control, meaning that full antagonism ofnicotine's attenuation of Δ9-THC ataxia was present.

We also wanted to investigate if the nAChR antagonists,DHβE or MLA, exerted any tonic activity on the nAChR in thepresence of Δ9-THC. Fig. 7A shows that both DHβE and MLAhave no effect on Δ9-THC ataxia, and thus, exhibited no tonicinfluence on cerebellar nAChRs. Demonstrating the lack oftonic activity on the nAChRs helped to rule out any possible

Fig. 7 – Evaluation of tonic activity of MLA or DHβE followingintracerebellar microinfusion on Δ9-THC (20 μg) ataxia. Eachpoint represents the mean±SE of 10 mice. (▲) MLA (1 μg)+Δ9-THC; (○) DHβE (500 ng)+Δ9-THC; (▼) aCSF+Δ9-THC. Asignificant treatment and time interaction was not observed(F6,60=0.854; P=0.53). No significant difference from “aCSF+Δ9-THC” control was evidenced when either DHβE or MLAwas administered alone with Δ9-THC. Panel B shows AUCdata for the same treatment groups as in panel A with drugtreatments shown above the corresponding bar. Althoughnot indicated, Δ9-THC was administered to all groups.

Fig. 8 – Effect of intracerebellar (ICB) microinfusion of nicotine(5 ng) on intraperitoneal (ip) Δ9-THC (4 mg/kg) ataxia in mice.Each point represents the mean±SE of 10 mice. (■) nicotine+ICB Δ9-THC (20 μg); (□) nicotine+ip Δ9-THC;(○) aCSF+ICB Δ9-THC (20 μg); (▲) aCSF+ip Δ9-THC. Asignificant drug treatment and time interaction was observed(F9,91=15.8; p<0.0001). Intracerebellar nicotine pretreatmentsignificantly attenuated (p<0.01) ip Δ9-THC ataxia at alltime intervals as compared to “aCSF+ip Δ9-THC” controls.Panel B shows AUC data for the same treatment groups as inpanel A with drug treatments shown above thecorresponding bar.

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confounding factor due to these antagonists. The slopes ofresponse curves following “MLA+Δ9-THC,” “DHβE+Δ9-THC”and “aCSF+Δ9-THC” treatments were similar (Fig. 7A). Theresults of the tonic activity experiments shown in Fig. 7A havebeen expressed as AUC data in Fig. 7B. The AUC bars for bothDHβE and MLA treatment groups were nearly identical withthat of the “aCSF+Δ9-THC” control group (Fig. 7B).

After demonstrating that ICB nicotine/RJR-2403 markedlyattenuated ICB Δ9-THC ataxia, we decided to confirm if ICBnicotine would also attenuate Δ9-THC ataxia following sys-temic administration of Δ9-THC via intraperitoneal (ip) injec-tion. We first administered Δ9-THC at a dose of 2 mg/kg, butobserved no significant Rotorod impairment. However, ahigher dose (4 mg/kg) of Δ9-THC produced a significant ataxia(Fig. 8A; ICB aCSF+ip Δ9-THC). Once the ataxic effect ofsystemic Δ9-THC was observed, the effect of pretreatmentwith ICB nicotine (5 ng) on Δ9-THC (4 mg/kg) ataxia wasevaluated. Fig. 8A shows that ICB nicotine significantlyattenuated systemic Δ9-THC ataxia as compared to “aCSF+ipΔ9-THC” control providing further support to the behavioralnicotine/Δ9-THC interaction. Fig. 8B shows the AUC for theRotorod data presented in Fig. 8A. It is evident that a muchlarger AUC is present in “ICB aCSF+ip Δ9-THC” treated mice ascompared to the “ICB nicotine+ip Δ9-THC” treated group.

We also evaluated if ip nicotine (similar to ICB nicotine)attenuates ICB Δ9-THC ataxia. Two doses (0.25 and 0.5 mg/kg,ip) of nicotine were selected for the study. Fig. 9A shows that0.25mg/kgof ipnicotinehasno significant effect on ICBΔ9-THCataxia. However, the 0.5 mg/kg dose of nicotine significantlyattenuated ICB Δ9-THC ataxia, providing further support to thenicotine/Δ9-THC behavioral interaction. Fig. 9B shows the AUCof the same treatments used in Fig. 9A. The AUC wassignificantly lower in the case of “ip 0.5 mg/kg nicotine+ICBΔ9-THC” compared to “aCSF+Δ9-THC” control.

3. Discussion

From the results of the present investigation, it is evident thatthere is a strong functional interaction between nicotine andits ability to attenuate Δ9-THC ataxia. Intracerebellar nicotinein a dose-dependent manner attenuated Δ9-THC ataxia, pro-ducing full abolishment of ataxia at higher doses of nicotine.Additionally, hexamethonium microinfusion prior to theadministration of nicotine and Δ9-THC antagonized nicotine's

Fig. 9 – Effect of intraperitoneal (ip) injection of nicotine (0.25,0.5 mg/kg) on intracerebellar Δ9-THC (20 μg) ataxia in mice.Each point represents the mean±SE of 10 mice. (□) ICBnicotine 5 ng+Δ9-THC; (▼) ip nicotine (0.5 mg/kg)+Δ9-THC;(D) ip nicotine (0.25 mg/kg)+Δ9-THC; (▲) aCSF+Δ9-THC. Asignificant drug treatment and time interactionwas observed(F9,83=20.9; p<0.0001). Nicotine (0.5 mg/kg, ip) significantlyattenuated (P<0.01) intracerebellar Δ9-THC ataxia at 10, 20and 30 min post Δ9-THC infusion. Nicotine (0.25 mg/kg, ip)displayed no significant attenuation of Δ9-THC ataxia. Panel Bshows AUC data for the same treatment groups as in panel Awith drug treatments shown above the corresponding bar.Although not indicated, Δ9-THC was administered to allgroups.

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ability to attenuate Δ9-THC ataxia, thus demonstrating thatnicotine-induced attenuation of Δ9-THC ataxia occurredthrough participation of nAChRs. Upon establishing thatnicotine mediates the attenuation of Δ9-THC ataxia throughparticipation of the cerebellar nAChR, the next logical pro-gression was to investigate the cerebellar nAChR subtype(s)involved in the functional interaction between nicotine andΔ9-THC. The results of the study demonstrated that RJR-2403, aα4β2 nAChR selective agonist, attenuated Δ9-THC ataxia dose-dependently, thus indicating that nicotine-induced attenua-tion most likely occurred through mediation of its cerebellarα4β2 subtype.

To further confirm that nicotine attenuated Δ9-THC ataxiaspecifically through the cerebellar α4β2 nAChR subtype, weevaluated the effect of intracerebellar microinfusion of DHβE,a selective α4β2 nAChR antagonist, on nicotine-induced atte-nuation of Δ9-THC ataxia. In the presence of DHβE, nicotinewas unable to attenuate ataxia due to Δ9-THC administration.This provided further evidence to the suggestion that nico-

tine-induced attenuation of Δ9-THC ataxia occurs through thecerebellar α4β2 nAChR subtype. The results also indicated thatthe failure by RJR-2403 to attenuate Δ9-THC ataxia in thepresence of DHβE was primarily due to blockade of the α4β2

receptor. Although the data from RJR-2403 and DHβE experi-ments strongly suggested an involvement of the nicotinic α4β2

subtype in nicotine-induced attenuation of Δ9-THC ataxia, thedata did not rule out additional participation of other nAChRsubtype(s). Therefore, to rule out the involvement of any othersubtype of nAChR in the functional interaction of nicotine andΔ9-THC, we evaluated the role of the cerebellar α7 subtype inthe nicotine-induced attenuation of Δ9-THC ataxia. Thedecision to evaluate the role of nAChR α7 subtype was madebecause the α7 subtype is known to be present in high densitywithin the cerebellum (Caruncho et al., 1997). In the absence ofcommercial availability of a selective α7 agonist, only methyl-lycaconitine (MLA), a selective α7 nAChR subtype antagonist,was used in the study. The dose range of MLA used in ourstudy (either 1 or 5 μg) has been shown to produce significantattenuation of nicotine-induced seizures (Damaj et al., 1999).Thus, MLA is active and able to fully antagonize α7 when givenin the lowmicrogram range.WhenMLAwas given at 5 μg, withno effect on nicotine-induced attenuation of Δ9-THC ataxia, itmost likely suggested that the α7 subtype does not appear tomodulate nicotine-induced attenuation of Δ9-THC ataxia. Thisalso helped strengthen the notion that the α4β2 nAChRsubtype may be a primary nicotinic receptor subtype involvedin the nicotine-induced attenuation of Δ9-THC ataxia. Theintraceberebellar administration of the various nicotinic drugsused in the present study had relative potencies thatcorresponded with their binding affinities based on valuesreported in the literature (Sharples and Wonnacott, 2001).

We also considered the possibility that intracerebellar nico-tine treatment may desensitize nAChRs and the latter mayhave played a role in our results. Nicotine is well known toexert a dual action: desensitization of nAChRs at lowdoses andactivation at higher doses. Our results in the present studyindicated that intracerebellar nicotine administration pro-duced no desensitization but rather activation of nAChRsbecause: (i) nicotine-induced attenuation of Δ9-THC ataxiawasvirtually abolishedby theantagonist hexamethonium (Fig. 2A);and (ii) nicotine markedly attenuated Δ9-THC ataxia dose-dependently (Fig. 1A). Theseobservations supportedactivationand not desensitization of nicotinic receptors. The functionalconsequence of receptor blockade by its antagonist andreceptor desensitization by its agonist would be the same. Inpreviously published reports from our laboratory (Dar et al.,1993, 1994) as well as in the present investigation, stimulationof the nAChR by nicotine or RJR-2403 did not alter normalmotor coordination when infused alone. However, when infu-sion of nicotine or RJR-2403 each was followed by microinfu-sionofΔ9-THC, attenuationof Δ9-THCataxiawas alwaysnoted.Thus, the doses of nicotine and RJR-2403 used in the presentinvestigation had no effect on normal motor coordination butexerted a marked attenuating effect on motor incoordinationproduced by Δ9-THC.

We also wanted to explain how nicotine or RJR-2403following intracerebellar microinfusion may act to abolish/attenuate Δ9-THC ataxia. Intracerebellar nicotine stimulatesnAChRs located at the Mossy fiber–granule cell synapse

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resulting in glutamate activation (Fedele et al., 1998) withsubsequent enhanced glutamate release (De Filippi et al., 2001;Reno et al., 2004). This released glutamate results in an en-hanced glutamatergic neurotransmission in the glutamatergicgranule cell–parallel fiber tract. Because parallel fibers synapseonto Purkinje cell dendrites, the enhanced glutamatergic neuro-transmission may likely oppose Purkinje cell firing therebyinhibiting GABA release. Inhibition of GABA release ultimatelydisinhibits deep cerebellar nuclei with consequent attenuation/abolition of Δ9-THC ataxia. Therefore, nicotine pretreatmentmay functionally oppose the Δ9-THC-produced decrease inglutamatergic neurotransmission. This may also explain whyit is critical that nicotine must be administered first to blockΔ9-THC ataxia. In the case of post Δ9-THC nicotine administra-tion, the possible GABAergic stimulation by Δ9-THC (Takahashiand Linden, 2000) may be difficult to be overcome by nicotine-induced increases in glutamatergic neurotransmission.

The parallel fiber terminals, basket cells, stellate cells andpossibly the climbing fiber terminals are the sites of localiza-tion of CB1 receptors in the cerebellum (Herkenhamet al., 1991;Mailleux and Vanderhaeghen, 1992). Activation of the CB1

receptor by Δ9-THC may impair neurotransmission from eachaxon terminal of these cerebellar interneurons (Takahashi andLinden, 2000). The combined overall effect on these neuronalsubtypes may explain the observed Δ9-THC ataxia. We havepreviously observed that forskolin and cpt-cAMP attenuatedcannabinoid-induced ataxia (unpublished data). This observa-tion provides further support to interference with basket cellneurotransmission because the GABAergic transmission frombasket cells onto Purkinje cells is thought to be highly depen-dent on cAMP signaling (Mitoma and Konishi, 1996). Increasesin cAMPsignaling can enhanceGABA release and thus possiblyreverse the inhibitory influence of CB1 receptor activation(Mitoma and Konishi, 1996). Electrophysiological data haveprovided evidence that increases in intracellular cAMP byforskolin or noradrenergic drugs in GABAergic interneurons(basket and stellate) lead to a long-lasting robust potentiationof inhibitory neurotransmission onto Purkinje cells and theresulting inhibition of Purkinje cell firing results in enhancedmotor function and motor learning (Mitoma and Konishi,1996). This basket-Purkinje synapse has been postulated to beresponsible for cerebellar motor impairment by cannabinoids,because disinhibition of the Purkinje cell would decrease firingof the deep cerebellar nuclei, a consequence that theoreticallycould impair motor function (Patel and Hillard, 2001).

The study, therefore, providednew informationwith regardto functional interactions between nicotine, nAChR subtypesand Δ9-THC ataxia. Direct evidence of the ability of nicotine toattenuate Δ9-THC ataxia primarily through the α4β2 nAChRsubtype was presented for the first time. Thus, our studydelineated the importance of the cerebellar α4β2 nAChRsubtype and seems to suggest little or no involvement of α7

or other cerebellar nAChR subtypes in the attenuation of Δ9-THC ataxia. However, further studies to elucidate the signalingmechanisms in the functional interaction between nicotineandΔ9-THCwith regard tomotor impairmentmaybe essential.

In summary, the results of this investigation demonstratedthat nicotine markedly attenuated Δ9-THC ataxia in a dose-related manner. The results further indicated that nicotine-induced attenuation of Δ9-THC ataxia was via the α4β2 nAChR

subtype and little or no involvement of other nAChR subtypesin the functional interaction between nicotine and Δ9-THCataxia occur.

4. Experimental procedures

4.1. Animals

Male CD-1 mice were purchased from Charles River Labs(Raleigh, NC). The mice were 5 to 6 weeks old and weighedbetween 23 and 28 g at the time of behavioral experiments. Themice were maintained in a housing facility under controlledhumidity (60–80%) and temperature (23 to 25 °C) and kept on a12-h light/dark cycle (lights on 07:00 h). Following theimplantation of a stainless steel guide cannula for directmicroinfusion into the cerebellum, each animalwas housed inits own individual plastic cage. Mice had free access to waterand commercial mouse chow ad libitum. Each animal was usedonly once in the Rotorod experiment. All experiments wereevaluated and approved by the Animal Use and Care Commit-tee of East Carolina University.

4.2. Stereotaxic surgery

Two days after arrival, mice were anesthetized with chloralhydrate (450 mg/kg, ip) prior to placement in a small stereo-taxic frame (Model 900; David Kopf Instruments, Tujunga, CA).The head of each mouse was trimmed of hair, scrubbed withpovidone–iodine (The Clinipad Co., Rocky Hill, CT) via swabstick and then wiped clean with an isopropyl alcohol swab.With the skull flat, a 2 cm longmid-sagittal incision wasmadeby sterile scalpel in order to expose the skull. Cannulation ofthe cerebellum was performed aseptically according to thefollowing coordinates of Slotnick and Leonard (1975): AP−6.4 mm (from bregma); ML ±0.8 mm; DV −1.0 mm from theskull surface. The stainless steel guide cannula (22 gauge,10 mm length) was lowered through a drilled craniotomy holevia aMasterlight® Hand Piece (Henry Schein, PortWashington,NY) into the superficial layers of the anterior lobe region of thecerebellum. Durelon® cement (Premier Dental Products Co.,Norristown, PA) was used to anchor the cannula to the skullsurface. A removable stainless steel wire plug was placedinside the guide cannula to prevent occlusion. Followingsurgery, each animal received 3000 units, subcutaneously,Durapen® (procaine and benzathine penicillin G (VEDCO Inc.,St. Joseph, MO) to prevent infection during post-surgicalrecovery. Each animal also received an injection of ketorolactromethamine (Abbott Labs, N. Chicago, IL), 2 mg/kg, foranalgesia shortly after surgery and again 4 to 6 h later. Animalswere allowed to recover in their own individual cages at theBrody School ofMedicine animal care facility for aminimumof5 days before behavioral testing.

4.3. Drugs

Drug solutions were prepared the day of use in behavioralexperiments. The following drugs were used in experiments:CB1 receptor agonist Δ9-THC (Supplied free by DHHS, NIDAResearch Triangle Institute, Research Triangle Park, NC); (−)-

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nicotine-di-L-tartrate; non-selective nicotinic acetylcholinereceptor antagonist hexamethonium; CNS selective α4β2

antagonist dihydro-beta-erythroidine hydrobromide (DHβE)(Sigma-Aldrich, St. Louis, MO); CNS selective α4β2 nicotinicreceptor agonist RJR-2403 fumarate; α7 neuronal nicotinicreceptor antagonist methyllycaconitine citrate (MLA) (Tocris,Ellisville, MO). Δ9-THC was dissolved in 100% dimethylsulf-oxide (DMSO) and all nicotinic drugswere dissolved in artificialcerebrospinal fluid (aCSF). The composition of aCSF was thefollowing (in mM): NaCl, 127.65; KCl, 2.55; CaCl2, 0.05; MgCl2,0.94; Na2S2O5, 0.05 (at pH 7.4). Δ9-THC for ip injections wasdissolved in a dispersion of Tween 80 (Fisher Scientific,Pittsburgh, PA) and 0.9% saline (1:19 final volume). (−)-nicotine-di-L-tartrate for ip injections was dissolved in 0.9%saline.

4.4. Intracerebellar microinjections

A Harvard Model 22 (Harvard Apparatus, Holliston, MA)microinfusion syringe pump was used for drug infusionsduring which a drug solution was injected at a constant rateof 0.1 μl/min for 1min, for a total volume of 100 nl. Δ9-THCwasinfused at 1 μl/min yielding a total volume of 1 μl. Drugs weremicroinfused through PE-10 (Clay Adams; Parsippany, NJ)polyethylene tubing by a microinjection pump fitted with a25 μl Hamilton Syringe. The sterile stainless steel injectioncannula (30 gauge; 0.31 mm diameter) was fitted to the PE-10tubing so that the total length of exposed cannula was 11mm.This allowed for protrusion of the injector cannula 1 mmbeyond the lower tip of the guide cannula. Injection cannulaewere left in guide cannulae for one additional minute to allowfor adequate diffusion of the solution. An air bubble separatingdrug solution and water in the tubing was monitored forcontinuous movement to indicate that blockage was notoccurring and that the desired drug dose was administered.Otherdetailswere the sameas explained inour previous report(Dar, 2000).

4.5. Motor incoordination evaluation

Mice were evaluated for motor coordination using a standardRotorod treadmill (Ugo Basil, Verese, Italy) set at a fixed speedof 24 rpm. As previously described, normalmotor coordinationwas arbitrarily defined as the ability of a mouse to walkcontinuously on the Rotorod, without falling off, for 180 s (Daret al., 1993). All mice were screened prior to intracerebellarmicroinjection in order to establish normal motor coordina-tion, and thus, the mice served as their own controls.Screening was performed the morning of the experimenttypically 20 min prior to microinjection and subsequentRotorod evaluations. Any mouse unable to walk 180 s in 3attempts during screening was considered to have abnormalcoordination and was excluded from the experiment. In thepresent investigation, no animal failed the Rotorod screeningtest. Motor coordination experiments were performedbetween 8 and 11AM.

The Rotorod experiments were conducted 5 days aftersurgery to allow the animals to recover from the effects of theanesthetic and surgical trauma. The Rotorod evaluation timesused were 10, 20, 30 and 40 min starting from the moment of

Δ9-THC microinjection. After evaluation on the Rotorod ateach time point, the mouse was placed back into its originalcage until the next evaluation time. Each treatment groupconsisted of 10 mice. Results are expressed in seconds and180 s represented the normalmotor coordination based on ourestablished criterion. The longer the animals walked on theRotorod, the lesser the motor incoordination and vice versa.Accentuation or attenuation of intracerebellar Δ9-THC ataxiaby other drugs is thus indicated by either a decrease or anincrease, respectively, in the time period the animals walkedon the Rotorod.

4.6. Histology

In order to confirm the accuracy of drug microinjections, micewere microinjected with Fast Green dye at the end of eachRotorod experiment. The mice were killed by cervical disloca-tion and decapitation after light anesthesia via isofluorane.The guide cannula and brain were carefully removed and thesite of microinjection was then verified by examining thelocation of the dye in the cerebellar anterior lobe region. Overthe past 15 years in our laboratory, cannulation success rateshave been in excess of 95%. In the present study, all cannu-lations were confirmed to be correct and thus all drug micro-infusions were at the desired sight within the cerebellum.

4.7. Statistical analysis

Motor incoordination data were analyzed by a two-way re-peated measure analysis of variance (ANOVA) in order toevaluate the effect of various drug doses and time on Rotorodmotor coordination using the multivariate criterion of Wilk'slambda (Λ). Significance in drug versus time interaction wasevaluated with a one-way ANOVA. A Dunnett's C post hoc testwas performedwhenever significancewas foundon treatmentand/or time. A P value <0.05 was taken as the level ofsignificance in all statistical tests. Statistical analyses wereperformed using SPSS for Windows, version 13.0. Area underthe curve (AUC) analysiswas performedusing GraphPad Prism4.0©. ED50 calculations were performed using the EPA ProbitAnalysis Program version 1.5.

R E F E R E N C E S

Adams, I.B., Martin, B.R., 1996. Cannabis: pharmacology andtoxicology in animals and humans. Addiction 91, 1585–1614.

Benowitz, N., Jacob, P., 1999. Pharmacokinetics and metabolism ofnicotine and related alkaloid. In: Aneric, S., Brioni, J. (Eds.),Neuronal Nicotinic Receptors: Pharmacology and TherapeuticOpportunities. Wiley-Liss, New York, pp. 213–234.

Caruncho, H.J., Guidotti, A., Lindstrom, J., Costa, E., Pesold, C.,1997. Subcellular localization of the alpha 7 nicotinicreceptor in rat cerebellar granule cell layer. NeuroReport 8,1431–1433.

Clifford, D.B., 1983. Tetrahydrocannabinol for tremor in multiplesclerosis. Ann. Neurol. 13, 669–671.

Damaj, M.I., Glassco, W., Dukat, M., Martin, B.R., 1999.Pharmacological characterization of nicotine-induced seizuresin mice. J. Pharmacol. Exp. Ther. 291, 1284–1291.

Dar, M. Saeed, 2000. Cerebellar CB1 receptor mediation ofΔ9-THC-induced motor incoordination and its potentiation by

25B R A I N R E S E A R C H 1 1 1 5 ( 2 0 0 6 ) 1 6 – 2 5

ethanol and modulation by the cerebellar adenosinergic A1

receptor in the mouse. Brain Res. 864, 186–194.Dar, M.S., Li, C., Bowman, E.R., 1993. Central behavioral

interactions between ethanol, (−)-nicotine, and (−)-cotinine inmice. Brain Res. Bull. 32, 23–28.

Dar, M.S., Bowman, E.R., Li, C., 1994. Intracerebellarnicotinic–cholinergic participation in the cerebellaradenosinergic modulation of ethanol-induced motorincoordination in mice. Brain Res. 644, 117–127.

De Filippi, G., Baldwinson, T., Sher, E., 2001. Evidence for nicotinicacetylcholine receptor activation in rat cerebellar slices.Pharmacol. Biochem. Behav. 70, 447–455.

De la Garza, R., Bickford-Wimer, P.C., Hoffer, B.J., Freedman, R.,1987. Heterogeneity of nicotine actions in the rat cerebellum:an in vivo electrophysiologic study. J. Pharmacol. Exp. Ther.240, 689–695.

Devane, W.A., Dysarz, F.A.I., Johnson, M.R., Melvin, L.S., Howlett,A.C., 1988. Determination and characterization of acannabinoid receptor in rat brain. Mol. Pharmacol. 34, 561–564.

Dewey, W.L., 1986. Cannabinoid pharmacology. Pharmacol. Rev.38, 151–178.

Fedele, E., Varnier, G., Ansaldo, M.A., Raiteri, M., 1998. Nicotineadministration stimulates the in vivo N-methyl-D-aspartatereceptor/nitric oxide/cyclic GMP pathway in rathippocampus through glutamate release. Br. J. Pharmacol.125, 1042–1048.

Fonseca, F.R., Arco, I.D., Martin-Calderon, J.L., Gorriti, M.A.,Navarro, M., 1998. Role of endogenous cannabinoid system inthe regulation of motor activity. Neurobiol. Dis. 5, 483–501.

Graham, A., Court, J.A., Martin-Ruiz, C.M., Jaros, E., Perry, R.,Volsen, S.G., Bose, S., Evans, N., Ince, P., Kuryatov, A.,Lindstrom, J., Gotti, C., Perry, E.K., 2002.Immunohistochemical localization of nicotinic acetylcholinereceptor subunits in human cerebellum. Neuroscience 113,493–507.

Grottick, J. Andrew, Trube, G., Corrigall, A. William, Huwyler, J.,Malherbe, P., Wyler, R., Higgins, A. Guy, 2000. Evidence thatnicotinic α7 receptors are not involved in the hyperlocomotorand rewarding effects of nicotine. J. Pharmacol. Exp. Ther. 294,1112–1119.

Herkenham, M., Groen, B.G.S., Lynn, A.B., DeCosta, B.R., Richfield,E.K., 1991. Neuronal localization of cannabinoid receptors andsecond messengers in mutant mouse cerebellum. Brain Res.552, 301–310.

Lloyd, G.K., Williams, M., 2000. Neuronal nicotinic acetylcholine

receptors as novel drug targets. J. Pharmacol. Exp. Ther. 292,461–467.

Mailleux, P., Vanderhaeghen, J.J., 1992. Distribution of neuronalcannabinoid receptor in the adult rat brain: a comparativereceptor binding radioautography and in situ hybridizationhistochemistry. Neuroscience 48, 665–668.

Matsuda, L.A., Bonner, T.I., Lolait, S.J., 1993. Localization ofcannabinoid receptor mRNA in rat brain. J. Comp. Neurol. 327,535–550.

Mitoma, H., Konishi, S., 1996. Long-lasting facilitation of inhibitorytransmission by monoamingergic and cAMP-dependentmechanism in rat cerebellar GABAergic synapses. Neurosci.Lett. 217, 141–144.

Morrison, F. Cathleen, 1969. Effects of nicotine on motorco-ordination and spontaneous activity in mice. J. Pharm.Pharmacol. 21, 35–37.

Nakayama, H., Shioda, S., Nakajo, S., Ueno, S., Nakashima, T.,Nakai, Y., 1997. Immunocytochemical localization of nicotinicacetylcholine receptor in the rat cerebellar cortex. Neurosci.Res. 29, 233–239.

Patel, S., Hillard, C.J., 2001. Cannabinoid CB1 receptor agonistsproduce cerebellar dysfunction in mice. J. Pharmacol. Exp.Ther. 297, 629–637.

Reno, L.A., Zago, W., Markus, M.P., 2004. Release of[3H]-L-glutamate by stimulation of nicotinic acetylcholinereceptors in rat cerebellar slices. Neuroscience 124, 647–653.

Sharples, G.V., Wonnacott, S., 2001. Neuronal nicotinic receptors.Tocris Rev. 9, 1–12.

Slotnick, B.M., Leonard, C.M., 1975. A Stereotaxic Atlas of AlbinoMouse Forebrain. US Government Printing Office, Washington,DC.

Takahashi, K.A., Linden, D.J., 2000. Cannabinoid receptormodulation of synapses received by cerebellar Purkinje cells.J. Neurophysiol. 83, 1167–1180.

Tapper, A.R., McKinney, S.L., Nashmi, R., Schwarz, J., Deshpande,P., Labarca, C., Whiteaker, P., Marks, M.J., Collins, A.C., Lester,H.A., 2004. Nicotine activation of α4 receptors: sufficient forreward, tolerance, and sensitization. Science 306, 1029–1032.

Valjent, E., Mitchell, J.M., Besson, M.J., Caboche, J., Maldonado, R.,2002. Behavioural and biochemical evidence for interactionsbetween Δ9-tetrahydrocannabinol and nicotine. Br. J.Pharmacol. 135, 564–578.

Watson, S.J., Benson Jr., J.A., Joy, J.E., 2000. Marijuana andmedicine: assessing the science base: a summary of the 1999Institute of Medicine Report. Arch. Gen. Psychiatry 57, 547–552.