The phytocannabinoid D 9 -tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

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1 Brtsh Journal of Pharmacology (8) 54, 4 5 & 8 Nature Publshng Group All rghts reserved 7 88/8 $3. RESEARCH PAPER The phytocannabnod D 9 -tetrahydrocannabvarn modulates nhbtory neurotransmsson n the cerebellum Y-L Ma, SE Weston, BJ Whalley and GJ Stephens School of Pharmacy, Unversty of Readng, Whteknghts, Readng, Berkshre, UK Background and purpose: The phytocannabnod D 9 -tetrahydrocannabvarn (D 9 -THCV) has been reported to exhbt a dverse pharmacology; here, we nvestgate functonal effects of D 9 -THCV, extracted from Cannabs satva, usng electrophysologcal technques to defne ts mechansm of acton n the CNS. Expermental approach: Effects of D 9 -THCV and synthetc cannabnod agents on nhbtory neurotransmsson at nterneurone-purknje cell (IN-PC) synapses were correlated wth effects on spontaneous PC output usng sngle-cell and mult-electrode array (MEA) electrophysologcal recordngs respectvely, n mouse cerebellar bran slces n vtro. Key results: The cannabnod receptor agonst WIN 55,- () decreased mnature nhbtory postsynaptc current (mipsc) frequency at IN-PC synapses. -nduced nhbton was reversed by D 9 -THCV, and also by the CB receptor antagonst AM5; D 9 -THCV or AM5 acted to ncrease mipsc frequency beyond basal values. When appled alone, D 9 -THCV, AM5 or rmonabant ncreased mipsc frequency. Pre-ncubaton wth D 9 -THCV blocked -nduced nhbton. In MEA recordngs, ncreased PC spke frng rate; D 9 -THCV and AM5 acted n the opposte drecton to decrease spke frng. The effects of D 9 -THCV and were attenuated by the GABA A receptor antagonst bcucullne methodde. Conclusons and mplcatons: We show for the frst tme that D 9 -THCV acts as a functonal CB receptor antagonst n the CNS to modulate nhbtory neurotransmsson at IN-PC synapses and spontaneous PC output. D 9 -THCV- and AM5-nduced ncreases n mipsc frequency beyond basal levels were consstent wth basal CB receptor actvty. -nduced ncreases n PC spke frng rate were consstent wth synaptc dsnhbton; whlst D 9 -THCV- and AM5-nduced decreases n spke frng suggest a mechansm of PC nhbton. Brtsh Journal of Pharmacology (8) 54, 4 5; do:.38/bjp.8.57; publshed onlne 3 March 8 Keywords: cannabnods; CB receptor; GABA A receptor; patch-clamp; mult-electrode array Abbrevatons: acsf, artfcal cerebrospnal flud; AM5, N-(pperdn--yl)--(,4-dchlorophenyl)-5-(4-odophenyl)-4- methyl-h-multpyrazole-3-carboxamde; BMI, bcucullne methodde; CB, cannabnod; DCN, deep cerebellar nucle; IEI, nter-event ntervals; IN-PC, nterneurone-purknje cell; MEA, mult-electrode array; mipsc, mnature nhbtory postsynaptc current; PC, Purknje cell; D 9 -THCV, D 9 -tetrahydrocannabvarn; TTX, tetrodotoxn; WIN 55, -,(R)-( þ )-[,3-dhydro-5-methyl-3-(4-morpholnylmethyl)pyrrolo-[,,3-de]-,4- benzoxazn-6-yl]--naphthalenylmethanone mesylate Introducton Cannabnods represent a dverse group of compounds, ncludng plant-derved phytocannabnods, amnoalkylndoles Correspondence: Dr G Stephens, School of Pharmacy, Unversty of Readng, Whteknghts, PO Box 8, Readng RG6 6AJ, UK. E-mal: g.j.stephens@readng.ac.uk These authors contrbuted equally to ths work. Receved 7 November 7; revsed 7 January 8; accepted 8 January 8; publshed onlne 3 March 8 (such as the prototypc ) and endocannabnods, (D Marzo et al., 999; Howlett et al.,, 4). Cannabnods act on two major subtypes of G proten-coupled receptors, CB and CB (Alexander et al, 7),bothofwhch predomnantly couple to nhbtory G /o subunts. CB s strongly expressed n the CNS, partcularly n the cerebellum (Tsou et al., 998; Egertova and Elphck, ; Dana et al., ). In contrast, CB receptors are prncpally localzed to perpheral, non-neuronal tssue (Howlett et al., ; see also Van Sckle et al., 5; Ashton et al., 6). In addton to

2 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al 5 CB /CB -medated effects, cannabnods may act at further putatve CB receptor subtypes, other non-cb receptors and may have other non-receptor-medated actons (Howlett et al., ; Pertwee, 7). Plant-derved phytocannabnods have recently attracted consderable nterest as novel potental therapeutc agents (Mechoulam, 5, Pertwee, 5a); however, mechansms of acton reman poorly defned. In partcular, D 9 -tetrahydrocannabvarn (D 9 -THCV), the propyl homologue of D 9 -tetrahydrocannabnol, has recently been reported to have a dverse pharmacology and to dsplay tssue-dependent effects (Pertwee, 8). D 9 -THCV was orgnally shown to share D 9 -tetrahydrocannabnol-lke propertes, producng catalepsy n a mouse rng test, although at a lower potency (Gll et al., 97). More recently, [ 35 S]GTPgSbndng assays have shown that D 9 -THCV exhbts an antagonst acton at CB and CB receptors n whole mouse bran membranes and recombnant cells, respectvely (Thomas et al., 5). Our prelmnary data also show that D 9 -THCV has regonal effects n [ 35 S]GTPgS assays, actng as a CB receptor antagonst n cerebellar and prform cortcal membranes (Denns et al, 7). Plant-derved D 9 -THCV also acted as a functonal antagonst n vtro n the solated mouse vas deferens (Thomas et al., 5). Synthetc D 9 -THCV shared these propertes and also acted as an antagonst n ant-nocceptve and hypotherma tests n vvo (Pertwee et al., 7). In contrast, hgher concentratons of D 9 -THCV were shown to nhbt electrcally evoked responses n vas deferens reportedly by a non-cb receptor-medated mechansm (Thomas et al., 5) and to be ant-nocceptve va an agonst effect at CB receptors (Pertwee et al., 7). Despte ths recent nterest, the functonal effects of D 9 -THCV on neuronal exctablty reman unknown. In the cerebellum, actvaton of presynaptc CB receptors at nterneurone-purknje cell (IN-PC) synapses causes nhbton of GABA release (Takahash and Lnden, ; Dana et al., ; Kretzer et al., ; Szabo et al., 4; Yamasak et al., 6), whereas CB receptor actvaton at exctatory synapses nhbts glutamate release (Takahash and Lnden, ; Kretzer and Regehr, ; Yamasak et al., 6). Importantly, PCs represent the sole output of the cerebellar cortex. The major nhbtory drve onto PCs s provded by basket cell nterneurones, whch form numerous synaptc contacts n the pnceau regon surroundng the PC axon ntal segment (Palay and Chan-Palay, 974). The promnent expresson of CB receptors wthn the pnceau (Tsou et al., 998; Egertova and Elphck, ) provdes a unque opportunty for exogenously appled cannabnods, as well as endogenously released endocannabnods, to modulate the overall output of the cerebellum. Actvaton of CB receptors has been shown to play a role n cerebellar dysfuncton, causng severe motor ncoordnaton, ncludng forms of ataxa (DeSanty and Dar, ; Patel and Hllard, ), therefore novel modulators of CB receptors have clear therapeutc potental. Here, we perform sngle-cell and network-level recordngs to show that D 9 -THCV, extracted from Cannnabs satva, acts n a smlar manner to standard CB receptor antagonsts to ncrease nhbtory neurotransmsson at IN-PC synapses and that these actons correlate wth decreases n spontaneous PC output n the cerebellum. Methods Tssue preparaton and solutons All anmal procedures compled wth UK Home Offce regulatons (Anmals (Scentfc Procedures) Act 986). Cerebellar slces were prepared accordng to methods descrbed n detal prevously (Harvey and Stephens, 4). Three- to fve-week-old male TO mce (Harlan, UK) were humanely klled by cervcal dslocaton and decaptated. The bran was removed and transferred to chlled, carboxygenated sucrose-based artfcal cerebrospnal flud (acsf). The cerebellum was dssected out and parasagttal cerebellar slces (3 mm thck) were cut usng a Vbratome (R. & L. Slaughter, Upmnster, UK). Slces were transferred nto standard acsf soluton at 37 C for h and then mantaned at room temperature ( 4 C). The standard acsf contaned NaCl 4 mm, KCl 3 mm, NaHCO 3 6 mm, NaH - PO 4.5 mm, MgSO 4 mm, CaCl mm, D-glucose mm, mantaned at ph 7.3 by bubblng wth 95% O /5% CO. The sucrose-based soluton used for dssecton and slcng was dentcal to standard acsf, wth the excepton that NaCl was replaced by sosmotc sucrose. Sngle-cell electrophysologcal recordng Slces were placed n a recordng chamber at room temperature and perfused at 4 ml mn wth standard carboxygenated acsf. Indvdual neurones were vsualzed va a 6 water mmerson lens wth nfrared dfferental nterference contrast optcs usng an uprght Olympus BX5WI mcroscope (Olympus, Tokyo, Japan). Whole-cell recordngs were performed usng an EPC-9 patch-clamp amplfer (HEKA Electronk, Lambrecht, Germany), controlled by Pulse software (HEKA) wth a Macntosh G4 computer. Patch electrodes were made from boroslcate glass (GC5-F, Harvard Apparatus, Kent, UK) and, when flled wth an ntracellular soluton contanng CsCl 4 mm, MgCl mm, CaCl mm, EGTA mm, MgATP 4 mm, NaGTP.4 mm, HEPES mm, ph 7.3, had resstances between 3 and 7 MO. Seres resstance was typcally 5 MO and was montored and compensated by 7 9% throughout. Data were sampled at 7 khz and fltered at one-thrd of the samplng frequency. Spontaneous mnature nhbtory postsynaptc currents (mipscs), recorded n the presence of mm tetrodotoxn (TTX), were dentfed as rapdly actvatng, nward currents at a holdng potental of 7 mv. Agents were appled n the presence of the non-nmda glutamate receptor antagonst 6-ntro-7- sulphamoylbenzo(f)qunoxalne- 3-done (NBQX) and the GABA B receptor antagonst CGP Data were analysed usng Pulseft (HEKA), Axograph (Molecular Devces, Sunnydale, CA, USA), Igor (Wavemetrcs, Lake Oswego, OR, USA), Orgn (Mcrocal, Northampton, MA, USA) and Excel (Mcrosoft, Redmond, WA, USA) software. Cumulatve frequency plots were constructed for mipsc nter-event ntervals (IEIs) usng 5 ms bns. Data are presented as mean value±s.e.mean, where n ¼ number of cells. Statstcal sgnfcance was determned usng a Student s pared t-test or a one-way ANOVA followed by Tukey s HSD test, as approprate. Cumulatve frequency plots were analysed by Kolmogorov Smrnov tests. Brtsh Journal of Pharmacology (8)

3 6 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al MEA electrophysologcal recordng Spontaneous spke actvty was recorded from cerebellar slces (produced as descrbed above) usng substrate-ntegrated MEAs (Mult Channel Systems, Reutlngen, Germany) (Egert et al., a; Stett et al., 3). MEAs comprsed of 6 electrodes of 3 mm dameter, arranged n an 8 8 array wth mm spacng between electrodes (Fgure 5a()). MEAs were cleaned before each recordng usng seres treatments of Terg- A-Zyme (Cole-Palmer, UK), methanol and, fnally, dstlled water before ar dryng and coatng. Slces were adhered to the MEA surface usng an appled (B4 ml) and evaporated cellulose ntrate soluton n methanol (.4% w/v; Fsher Scentfc, Loughborough, UK) ensurng maxmum contact between the tssue and recordng electrodes. Slce poston upon the MEA was determned by observaton at magnfcaton 4wtha Nkon TS-5 (Nkon, Tokyo, Japan) nverted mcroscope and maged va a Bresser Mkro-Okular camera (Meade, Rhede, Germany). Once placed, slces were mantaned at room temperature, contnuously superfused ( 4 ml mn ) wth carboxygenated acsf and allowed to stablze for at least mn before recordngs. Only data acqured from electrodes vsually confrmed as proxmal to the PC layer were used for analyss. Sgnals were amplfed ( gan), band-passfltered ( 3 Hz) by a 6-channel amplfer (MEA6 System, Mult Channel Systems) and smultaneously sampled at 5 khz per channel on all 6 channels. Data acquston was to a PC usng MC_Rack software (Mult Channel Systems) to montor and record data for offlne analyss. Data analyss was performed usng Neuroexplorer3 (Nex Technologes, Lttleton, MA, USA) and MATLAB (Mathworks Inc., Natck, MA, USA) plus MEA Tools (Egert et al., b). Negatve spke events were dentfed by threshold detecton (mnmum ampltude, 5 mv; typcal nose level, p±5 mv; threshold determned as X3s, where s represents the s.d. of the mean of data sets wthout spke actvty (Egert et al., a)) n MC_Rack from contnuously recorded data sets of not less than 3 s duraton. Spke data were exported to Neuroexplorer3 and spke rate hstograms constructed usng ms bns. Mean spke frng rates were calculated and are presented as normalzed mean±s.e.mean. Inevtable dead cell debrs on the slce surface and PC depth from recordng electrodes produced some varablty n the apparent spontaneous frng frequency n the PC layer (typcal range, 8 Hz; see also Egert et al., a). Consequently, drugnduced changes are presented as percentage changes n frng frequency vs control frng frequency per experment to provde normalzed measures for pooled data. n numbers for MEA recordngs are shown as n ¼ total number of electrodes (total number of slces). Statstcal sgnfcance was determned by a non-parametrc Mann Whtney U-test. In all cases, Po.5 was consdered sgnfcant. Materals The followng agents were used: N-(pperdn--yl)-- (,4-dchlorophenyl)-5-(4-odophenyl)-4-methyl-H-multpyrazole-3-carboxamde (AM5), CGP 55845, NBQX, -, (R)-( þ )-[,3-dhydro-5-methyl-3-(4-morpholnylmethyl)pyrrolo-[,,3-de]-,4-benzoxazn-6-yl]--naphthalenylmethanone mesylate (Tocrs Cookson, Brstol, UK) and bcucullne methodde (BMI; Sgma, UK). TTX was from Alomone (Jerusalem, Israel). Rmonabant was kndly provded by NIDA/NIH, Bethesda, USA. D 9 -THCV was a knd gft of GW Pharmaceutcals (Salsbury, Wlts, UK). All drugs, except D 9 - THCV, whch was suppled as a 58 mm stock soluton n ethanol and stored at 4 C, were made up as stock solutons n dstlled water or dmethyl sulphoxde (AM5, CGP 55845, ) and stored at C. Solvent was present at a maxmum fnal concentraton of.%; solvent, appled alone at equvalent expermental concentratons, had no effect on synaptc responses n ths preparaton (see Bardo et al., and the Results secton). Alquots were thawed and dssolved n carboxygenated acsf mmedately before use. In all experments, drugs were bath-appled for a mnmum of mn to acheve steady-state effects. Results Effect of cannabnods on nhbtory synaptc transmsson at IN-PC synapses Whole-cell recordngs were made from PCs n acute cerebellar bran slces n the presence of mm TTX (plus 5 mm NBQX and 5 mm CGP 55845) to solate acton potentalndependent GABAergc mipscs. Drugs were bath-appled for a mnmum of mn to acheve steady-state effects. The CB /CB agonst ( and 5 mm) caused a clear decrease n mean mipsc frequency (Fgures a, b, d, e). At 5 mm, caused a steady-state reducton from 5.±.8 to 3.3±.5 Hz, equvalent to 63.±3.% of control values (n ¼ 8; Po.; pared t-test); ths was reflected by an ncrease n the mipsc IEI by (Fgure d; n ¼ 8; each replcant Po.5; Kolmogorov Smrnov). had no effect on mean mipsc ampltude (74.5±.7 7.4±.7 pa; n ¼ 8; P ¼.34; pared t-test), as reflected n normalzed data (Fgure c). The selectve CB antagonst AM5 ( mm) reversed the -nduced decrease n mean mipsc frequency (Fgures a, b, e); ths was to 7.±. Hz, equvalent to 37.7±9.6% of control values (n ¼ 8; Po.; ANOVA) and reflected by a decrease n the mipsc IEI by AM5 that exceeded control levels (Fgure d; n ¼ 8; each replcant Po.5; Kolmogorov Smrnov). AM5 had no effect on mean mipsc ampltude (84.±4.8 pa; n ¼ 8; P ¼.9; ANOVA), as reflected n normalzed data (Fgure c). In all subsequent experments, no clear changes n mean mipsc ampltude were seen. Reductons n mpsc frequency n the absence of effects on event ampltude are consstent wth a reducton n quantal transmtter release from presynaptc stes. Although CB receptor mmunohstochemcal labellng has recently been descrbed n the rat cerebellum (Ashton et al., 6), there s, as yet, lttle evdence for functonal CB receptor actvty n ths regon. Accordngly, the selectve CB agonst JWH-33 ( mm) showed no effects on mean mipsc frequency or ampltude (n ¼ 3; data not shown); these data were consstent wth a lack of functonal CB receptors at IN-PC synapses. Overall, these data confrm prevous studes showng that IN-PC synapses n mouse cerebellum possess functonal presynaptc CB receptors; moreover, the above results descrbng observed AM5 effects suggest that CB antagonsts act to ncrease nhbtory neurotransmsson at these synapses. Brtsh Journal of Pharmacology (8)

4 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al 7 pa 5 ms /AM5 Normalsed mipsc frequency AM Tme (mn) Normalsed mipsc ampltude Tme (mn) AM5 Cumulatve frequency IEI (s) /AM5 Normalsed mipsc frequency µm 5 µm 5 µm + µm AM5 Fgure IN-PC synapses possess functonal presynaptc CB receptors. (a) Contnuous data traces showng effects of (5 mm) and subsequent applcaton of AM5 ( mm) n the contnued presence of (5 mm) on control () mipscs (holdng potental (V H ) ¼ 7 mv). Note that decreased mipsc frequency, whereas AM5 caused ncreases beyond basal levels. Tme course for effect of (5 mm) and AM5 ( mm) on normalzed mipsc frequency (b) and normalzed mipsc ampltude (c) (both n ¼ 8±s.e.mean). (d) Cumulatve frequency plots for effect of (5 mm) and subsequent applcaton of AM5 ( mm) on control () mipsc IEI (bn wdths 5 ms) (all n ¼ 8±s.e.mean). (e) Bar graph showng summarzed effects of ( mm; n ¼ 6±s.e.mean and 5 mm; n ¼ 8±s.e.mean) and of subsequent applcaton of AM5 ( mm, n ¼ 8±s.e.mean) n the contnued presence of (5 mm) on control (, n ¼ 8±s.e.mean) normalzed mipsc frequency; Po.5, Po. (pared t-test or ANOVA). Phytocannabnods have recently attracted nterest as potental therapeutc agents. Here, we examned functonal effects of D 9 -THCV on neuronal exctablty for the frst tme. D 9 -THCV reversed the -nduced decrease n mean mipsc frequency, causng ncreases n mean mipsc frequency, whch were beyond basal levels (Fgure a). The actons of D 9 -THCV (5 58 mm), each concentraton for a mnmum of mn to acheve steady-state effects, were concentraton related (Fgures a c). D 9 -THCV had no effect on mean mipsc ampltude dstrbuton at any concentraton tested. When appled alone, AM5 ( mm: 43±8.7% of control values; n ¼ 6; Po.; Fgure 3a), another synthetc CB antagonst rmonabant (3 mm: 6±%; n ¼ 6; Po.5; Fgure 3b) or D 9 -THCV ( mm: 54±%; n ¼ 6; Po.; Fgure 3c) all caused sgnfcant steady-state ncreases n mean mipsc frequency vs control levels over the tme perods shown (pared t-test used for each). In all cases, no changes n mean mipsc ampltude dstrbuton were seen. These data ndcate that D 9 -THCV acts to ncrease quantal GABA release at IN-PC synapses n a smlar manner to selectve CB antagonsts and, moreover, that such effects were ndependent of prevous expermental actvaton of presynaptc CB receptors. We next determned the effects of applyng a sngle, maxmal concentraton of D 9 -THCV (Ma et al., 6) before applcaton. D 9 -THCV (58 mm) caused the expected ncrease n mean mipsc frequency (Fgures 4a and b); ths ncrease was from.5±.6 to 4.±.8 Hz ( ¼ 76±% of control values; n ¼ 5; Po. Brtsh Journal of Pharmacology (8)

5 8 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al pa 5 ms / 9 -THCV ( µm) / 9 -THCV (4 µm) mipsc frequency (Hz) 9 -THCV µm µm 4 µm Tme (mn) Normalsed mipsc frequency Control Wn 5 µm THCV 5 µm THCV µm THCV µm THCV 4 µm THCV 58 µm Fgure -nduced nhbton at IN-PC synapses s reversed by D 9 -THCV. (a) Contnuous data traces showng effects of (5 mm) and subsequent applcaton of D 9 -THCV ( and 4 mm) n the contnued presence of (5 mm) on control () mipscs; V H ¼ 7 mv. D 9 -THCV reversed -nduced nhbton and ncreased mipsc frequency beyond basal levels. (b) Example of tme course for effects of (5 mm) and subsequent applcaton of D 9 -THCV ( 4 mm) on mipsc frequency. (c) Bar graph showng summarzed effects of (5 mm, n ¼ 8±s.e.mean) and subsequent applcaton of D 9 -THCV (5 58 mm; n ¼ 3 8±s.e.mean) n the contnued presence of (5 mm) on control (, n ¼ 8±s.e.mean) normalzed mipsc frequency; Po.5, Po. vs, or vs as shown (pared t-test or ANOVA). (pared t-test)). In the contnued presence of D 9 -THCV, the -nduced nhbton was now blocked (Fgures 4a and b; 4.±.9 Hz ( ¼ 99±% of D 9 -THCV values) n ¼ 5; P ¼.8; ANOVA). No changes n mean mipsc ampltude dstrbuton were seen. These data ndcate that pre-ncubaton wth D 9 -THCV blocks acton at IN-PC synapses. Effect of cannabnods on PC output n cerebellar bran slces Havng demonstrated that D 9 -THCV and synthetc CB antagonsts act to ncrease nhbtory neurotransmsson at IN-PC synapses, we next used MEA recordngs from acute cerebellar bran slces to determne whether D 9 -THCV could also affect spontaneous PC output under condtons of basal synaptc transmsson. Bran slces were cut n a sagttal plane, whch mantans PC soma and dendrtc tree structure and allows recordng from ntact PCs close to the slce surface (Egert et al., a, b). Recordngs were made only from electrodes proxmal to the PC layer (Fgure 5a()) to correlate wth ntracellular recordngs from sngle PCs (Fgures 4). Here, spkes of negatve polarty, representatve of sngle-unt or multple sngle-unt exctatory events (Egert et al., a), were dentfed by threshold detecton. Under these condtons, we recorded robust, hgh-frequency Brtsh Journal of Pharmacology (8)

6 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al 9 AM5 pa 5 ms Normalsed mipsc frequency AM Tme (mn) rmonabant pa 5 ms Normalsed mipsc frequency rmonabant Tme (mn) 9 -THCV pa 5 ms Normalsed mipsc frequency 9 -THCV Tme (mn) Fgure 3 CB -selectve antagonsts and D 9 -THCV appled alone ncrease nhbtory neurotransmsson at IN-PC synapses. Contnuous data traces showng effects of (a()) AM5 ( mm), (b()) rmonabant (3 mm) and (c()) D 9 - THCV ( mm) on control () mipscs; n all cases, V H ¼ 7 mv. Note that each cannabnod acted to ncrease mipsc frequency. Correspondng tme courses for effects of (a()) AM5 ( mm; n ¼ 6±s.e.mean), (b()) rmonabant (3 mm; n ¼ 5±s.e.mean) and (c()) D 9 -THCV ( mm; n ¼ 6±s.e.mean). spontaneous electrcal actvty from PCs (typcal range, 8 Hz; n ¼ 34 (from sx separate slces); Fgure 5a()). All spontaneous actvty recorded by the MEA was abolshed n the presence of mm TTX (n ¼ 3; data not shown), and applcaton of the vehcle (dmethyl sulphoxde) at concentratons used for drug dluton (B.% v/v) revealed no observable effects upon spke frng (n ¼ 3; data not shown). Bath applcaton of (5 mm) caused a sgnfcant ncrease n normalzed spontaneous mean spke frng rate (6±% of control values; n ¼ 8 (6); Po.5; Mann Whtney U-test; Fgures 5a(), b(), (), c, Table ). Subsequent applcaton of D 9 -THCV (5 4 mm), each concentraton for a mnmum of mn to acheve steady-state effects, caused clear, concentraton-dependent decreases n PC spontaneous spke frng (for example, 58 mm: 49±7% of control values; n ¼ 8 (6); Po.5; Mann Whtney; Fgures 5a(), b(), c, Table ). These data demonstrate that - nduced ncreases n PC spontaneous spke frng were opposed by D 9 -THCV. We next examned the effects of cannabnods wthout pre-treatment wth the CB receptor agonst. AM5 ( mm) alone caused a sgnfcant decrease n PC spontaneous spke frng rate (68±4% of control values; n ¼ 4 (6); Po.5; Mann Whtney; Fgures 6a c). Smlarly, D 9 -THCV (5 mm) alone also decreased PC output (56±4% of control values; n ¼ 3 (6); Po.5; Mann Whtney; Fgure 6c). MEA recordngs demonstrate that -nduced CB receptor actvaton caused an ncrease n PC spontaneous exctatory dscharge, whereas D 9 -THCV and a CB receptor antagonst acted to lmt exctaton. A clear possblty s that these data reflect modulaton of nhbtory neurotransmsson at IN-PC synapses (Fgures 4). To test ths hypothess, we examned the effects of cannabnods n the presence of the GABA A receptor antagonst BMI. Bath applcaton of BMI tself caused expected large ncreases n spontaneous spke frng frequency, at mm (Po.5; Mann Whtney; Fgure 7a(), Table ) and at 3 mm (Po.5; Mann Whtney; Fgure 7b(), Table ). These data demonstrate that functonal nhbtory nput modulates spontaneous actvty of PCs and hence cerebellar output. We next examned the effects of GABA A receptor blockade on cannabnod-nduced changes n PC output. In the presence of mm BMI, (5 mm) Brtsh Journal of Pharmacology (8)

7 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al pa 5 ms n GABA release can affect PC output and hence that ths phytocannabnod has the potental to modulate the output of the cerebellar cortex. 9 -THCV 9 -THCV/ Normalsed mipsc frequency THCV Tme (mn) Fgure 4 D 9 -THCV blocks -nduced nhbton at IN-PC synapses. (a) Contnuous data traces showng effects of D 9 -THCV (58 mm) and of subsequent applcaton of (5 mm) n the contnued presence of D 9 -THCV on control () mipscs; V H ¼ 7 mv. D 9 -THCV ncreased mipsc frequency and blocked -nduced nhbton. (b) Tme course for effects of D 9 -THCV (58 mm; n ¼ 5±s.e.mean) and of subsequent applcaton of (5 mm; n ¼ 5±s.e.mean) n the contnued presence of D 9 -THCV on normalzed mipsc frequency. stll caused an ncrease n PC spontaneous spke frng (Mann Whtney; Fgure 7a(), (), Table ); however, effects were now sgnfcantly reduced n comparson wth prevous control levels (6±% vs pre-bmi control compared wth 55±4% vs mm BMI control; Po.5; Mann Whtney). In the presence of mm BMI, D 9 -THCV stll caused an overall decrease n PC frng rate, whch was sgnfcant for D 9 -THCV concentratons X mm (all Po.5 vs BMI control; Mann Whtney; Fgure 7a(); Table ). However, consstent wth data, D 9 -THCV-nduced nhbton of PC frng rate n the presence of mm BMI was also sgnfcantly attenuated (for example, mm D 9 -THCV: 5±8% vs pre-bmi control compared wth 47±3% vs mm BMI control; Po.5; Mann Whtney). In the presence of 3 mm BMI, cannabnod effects were now abolshed: no further changes n PC spontaneous frng rates were seen upon subsequent addton of (5 mm ¼ 4±3% vs 3 mm BMI control; P ¼.67; Mann Whtney; Fgures 7b(), (), Table ) or D 9 -THCV (5 4 mm) (for example, mm ¼ ±4% vs 3 mm BMI control; P ¼.73; Mann Whtney; Fgures 7b(), (), Table ). The senstvty of D 9 -THCV and effects on spontaneous PC output to GABA A receptor blockade ndcate that, under the expermental condtons used, cannabnod effects are medated by ther acton on nhbtory neurotransmsson. More specfcally, we show that D 9 -THCV-nduced ncreases Dscusson and conclusons We have correlated patch-clamp and MEA recordngs to demonstrate that D 9 -THCV acts n a manner smlar to standard, selectve CB receptor antagonsts to modulate nhbtory neurotransmsson at a cellular (IN-PC synapses) and network (spontaneous PC output) level n the mouse cerebellum n vtro. We provde evdence that a CB receptor agonst reduces GABA release onto PCs leadng to synaptc dsnhbton, whereas D 9 -THCV acts to produce opposng effects, promotng an nhbton of spontaneous PC output. D 9 -THCV modulates nhbtory neurotransmsson at IN-PC synapses We have recorded from IN-PC synapses n mouse cerebellar cortex as a sutable system to study modulaton of nhbtory neurotransmsson by cannabnods. The CB receptor agonst nhbted quantal GABA release; actons were consstent wth a presynaptc effect downstream of Ca þ entry, possbly va a drect acton on the exocytotc release machnery, as proposed for other presynaptc GPCR agonsts at IN-PC synapses (Harvey and Stephens, 4). Standard CB receptor antagonsts reversed -nduced nhbton and, when appled alone, they caused an ncrease n GABA release over basal levels. Smlar effects at IN-PC synapses have been reported n some studes (Dana et al. ; Kretzer et al., ; Galante and Dana, 4; Yamasak et al., 6), but not others (Szabo et al., 4). CB antagonsts also ncrease synaptc transmsson at other synapses (Mels et al., 4; Hentges et al., 5; Zhu and Lovnger, 5), suggestng that mechansms of presynaptc regulaton may be conserved between certan central synapses. Ths study provdes the frst evdence that D 9 -THCV modulates neuronal exctablty n the CNS. D 9 -THCV shared propertes wth synthetc CB antagonsts, reversng - nduced nhbton of GABA release and ncreasng release over basal levels when appled alone; n addton, prencubaton wth D 9 -THCV blocked subsequent - nduced nhbton. These data were consstent wth D 9 -THCV actng as a functonal CB receptor antagonst n the CNS. CB antagonst effects most lkely nvolve removal of endocannabnergc tone, as proposed for studes at IN-PC and other central, synapses (Kretzer et al., ; Galante and Dana, 4; Mels et al., 4; Hentges et al., 5; Zhu and Lovnger, 5; Neu et al., 7). In partcular, Kretzer et al. () reported that AM5 caused an ncrease n nterneurone frng n cerebellar bran slces, suggestng basal nhbton due to CB receptor actvty. Thus, although dfferences n cannabnod acton are reported between synapses, our data confrm that IN-PC synapses are lkely to be under strong endocannabnergc nhbton. In ths regard, t has recently been shown that the major endocannabnod -arachdonoyl glycerol s released from PCs to Brtsh Journal of Pharmacology (8)

8 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al 5 µv ms GL ML PC 4 µm / 9 -THCV Spke frng rate (spkes/ ms) Spke frng rate (spkes/ ms) Spke frng rate (spkes/ ms) Tme (s) Tme (s) / 9 -THCV 3 Tme (s) % mean frng rate vs control Control 5 µm WIN-55 5 µm 9 -THCV µm 9 -THCV µm 9 -THCV 4 µm 9 -THCV + Fgure 5 Cannabnods modulate PC spontaneous exctatory spke frng frequency n cerebellar bran slces. (a()) Mcrograph showng a cerebellar slce adherng to an MEA. Specfc cerebellar layers: GL ¼ granular layer, ML ¼ molecular layer and PC ¼ Purknje cell layer. The flled whte crcle shows a recordng electrode poston n the PC layer. (a()) Examples of contnuous MEA recordngs from a sngle electrode showng effects of (5 mm) and subsequent applcaton of D 9 -THCV (5 mm) n the contnued presence of (5 mm) on PC control () spontaneous exctatory spke frng frequency; steady-state effects, drugs appled for a mnmum of mn. Note the ncrease n PC frng rate upon applcaton of and consequental decrease below control levels upon subsequent applcaton of D 9 -THCV. (b) PC spke frng rate hstograms (mnmum 5 mn contnuous recordng, ms bn sze) for a representatve electrode for () control, () (5 mm) and () subsequent applcaton of D 9 -THCV (5 mm). (c) Bar graph showng summarzed (5 mm) and D 9 -THCV (5 4 mm; all n ¼ 8 (6)±s.e.mean) n the contnued presence of (5 mm) effects on PC control (n ¼ 36 (6)±s.e.mean) spontaneous exctatory spke frng rates; Po.5 vs control; Mann Whtney U-test. Table Effects of cannabnods on spontaneous PC exctatory spke propertes PC frng rate (%) BMI þ 5 mm þ 5 mm D 9 -THCV þ mm D 9 -THCV þ mm D 9 -THCV þ 4 mm D 9 -THCV (n ¼ 8 (6)) 6± 49±7 7±5 5±8 4±8 mm BMI (n ¼ 38 (6)) 346±46 536±83 4±86 5±7 w 6± w 35± w 3 mm BMI (n ¼ 35 (6)) 564±6 587±7 a 598±8 a 58±7 a 6±45 a 66±3 a Abbrevatons: BMI ¼ bcucullne methodde; ¼ control; PC ¼ Purknje cell; D 9 -THCV ¼ D 9 -tetrahydrocannabvarn; ¼ -, (R)-( þ )-[,3-dhydro-5- methyl-3-(4-morpholnylmethyl)pyrrolo-[,,3-de]-,4-benzoxazn-6-yl]--naphthalenylmethanone mesylate. PC frng rate (expressed as a percentage of control frng rate) calculated usng Neuroexplorer3 (Nex Technologes), MATLAB (Mathworks Inc.) plus MEA Tools (Egert et al., b) as descrbed n Methods. Note: D 9 -THCV results were obtaned n the contnued presence of 5 mm. a NS ¼ no sgnfcant dfference vs BMI control, Mann Whtney U-test. [3] Po.5 vs control, wpo.5 vs BMI control. Brtsh Journal of Pharmacology (8)

9 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al AM5 Spke frng rate (spkes/ ms) 5 µv ms Control 3 Tme (s) % mean frng rate vs control (%) Control Spke frng rate (spkes/ ms) AM5 3 Tme (s) act retrogradely at presynaptc CB receptors (Szabo et al., 6). Other putatve mechansms assocated wth receptor antagonsm are less lkely. The use of TTX n ths study argues aganst mechansms such as decreases n the rate of falures of acton potental-elcted transmtter release (Neu et al., 7). D 9 -THCV and the synthetc analogue O-4394 have been reported varously to antagonze acton n solated mouse vas deferens and D 9 -tetrahydrocannabnol-nduced ant-noccepton and hypotherma n vvo (Thomas et al., 5; Pertwee et al., 7; Pertwee, 8). [ 35 S]GTPg S-bndng studes n mouse whole bran membranes have supported an antagonst acton, wth an apparent K B o nm reported for D 9 -THCV actng at CB receptors (Thomas et al., 5); we report broadly smlar K B values and a rghtward shft n concentraton response curves nduced by D 9 -THCV n solated cerebellar (and prform µm AM5 µm 9 -THCV Fgure 6 AM5 reduces PC spontaneous exctatory spke frng frequency n cerebellar bran slces. (a) Examples of contnuous MEA recordngs from a sngle electrode showng effects of AM5 ( mm) on control () responses; steady-state effects, drugs appled for a mnmum of mn. Note the decrease n PC spke frng rate upon applcaton of AM5. (b) PC spke frng rate hstograms (mnmum 5 mn contnuous recordng; ms bn sze) for a representatve electrode n control () and () AM5 ( mm). (c) Bar graph showng summarzed effects of AM5 ( mm; n ¼ 3 (6)) on PC control (n ¼ 36 (6)) spontaneous exctatory spke frng rates. Also shown are effects of D 9 -THCV alone (5 mm; n ¼ 4 (6)); Po.5 vs control; Mann Whtney U-test. cortcal) membranes (Denns et al., 7). We have also seen that hgher concentratons of D 9 -THCV (and AM5) can reduce [ 35 S]GTPgS bndng to cerebellar membranes (Denns et al., 7), whch may suggest nverse agonsm (MacLennan et al., 998). Accordngly, there s evdence that CB -selectve compounds AM5 and rmonabant can both act as nverse agonsts and hence that CB receptors may possess consttutve actvty (Bouaboula et al., 997; Pan et al., 998; Pertwee, 5b). However, n our hands, decreases n [ 35 S]GTPgS bndng occurred ndependently of CB receptor expresson. Although we cannot fully rule out that D 9 -THCV actons here are medated, at least n part, by a CB receptor-ndependent mechansm, ths pathway would also have to be affected by both and AM5. In ths regard, t s also mportant to note that drug concentratons necessary to elct measurable responses n bran slce preparatons are dffcult to relate to equlbrum-bndng constants. For example, despte nanomolar affntes for CB receptors, clear effects requred mllmolar concentratons here and n other studes (Brown et al., 4). Concentraton-dependence ssues are exacerbated for hghly lpophlc cannabnods, whch are lkely to partton nto lpd membranes n bran slce preparatons (Howlett et al., 989; Bloom et al., 997). Moreover, the end pont of our assay (nhbton of vescular GABA release) s several steps far from receptor bndng/gtpgs turnover. Interestngly, hgher D 9 -THCV doses have also been reported to have agonst actons n vvo, ndcatng a complex pharmacology (Pertwee, 8). In ths study, D 9 -THCV had no agonst effects at IN-PC synapses; n contrast, D 9 -THCV acted n a manner opposte to the CB receptor agonst, but smlar to standard CB antagonsts, to ncrease nhbtory neurotransmsson. Cannabnods modulate spontaneous PC output n the cerebellum The cerebellar cortex has a dstnct, well-defned archtecture and mcro-crcutry (Ramon y Cajal, 9; Eccles et al., 967), affordng an deal opportunty to study spatotemporal network actvty. We have used MEA recordngs to correlate D 9 -THCV effects at IN-PC synapses wth those on spontaneous PC output. Frstly, our data confrm that mouse PCs exhbt robust spontaneous actvty under condtons that mantan synaptc connectvty (Egert et al., a; Mapell and D Angelo, 7). Ths s substantated by PC frng rates measured here beng n good agreement wth those recorded n vvo (Hausser and Clark, 997; Raman and Bean, 999), suggestng that MEA recordng n cerebellar bran slces represents an approprate model to nvestgate PC output. Ths actvty s lkely to reflect spontaneous PC dscharge, modulated by ntegrated nput from spontaneously dschargng nhbtory INs and recurrent PC collaterals (Palay and Chan-Palay, 974; Hausser and Clark, 997; Mann-Metzer and Yarom, 999). We extend prevous studes to provde the frst examnaton of drug effects on spontaneous PC actvty usng MEA methods. caused clear ncreases n exctatory spke frng rate consstent wth a decrease n GABA release at IN-PC synapses leadng to synaptc dsnhbton. Conversely, D 9 -THCV reversed effects and when appled alone t decreased Brtsh Journal of Pharmacology (8)

10 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al µv µv ms ms BMI ( µm) BMI (3 µm) + + +/ THCV +/ THCV µm BMI 3 µm BMI NS % mean frng rate vs control µm BMI 5 µm WIN-55 5 µm 9 -THCV µm 9 -THCV µm 9 -THCV 4 µm 9 -THCV + +BMI % mean frng rate vs control µm BMI 5 µm WIN-55 5 µm 9 -THCV µm 9 -THCV µm 9 -THCV 4 µm 9 -THCV + Fgure 7 The GABA A antagonst BMI attenuates cannabnod effects on PC spontaneous exctatory spke frng frequency. Examples of contnuous MEA recordngs from a sngle electrode showng effects of BMI at (a) mm and (b) 3mM on control () responses and on (5 mm)- and D 9 -THCV(5 mm)-nduced changes n PC spontaneous exctatory spke frng frequency; steady-state responses, drugs appled for a mnmum of mn. (a()) Control frng rate was ncreased by mm BMI and was further ncreased by n the contnued presence of BMI; subsequent applcaton of D 9 -THCV decreased frng rate to below control levels. (a()) Bar graph showng summarzed effects of (5 mm) and of D 9 -THCV (5 4 mm) on PC spontaneous exctatory spke frng rates n the contnued presence of mm BMI (all n ¼ 38 (6); ¼ Po.5 vs control, w ¼ Po.5 vs mm BMI as normalzed control; Mann Whtney U-test. (b()) BMI (3 mm ) caused an ncrease n control spke frng rate, subsequent applcaton of (5 mm) and then D 9 -THCV (5 mm) n the contnued presence of BMI caused no further changes. (b()) Bar graph showng summarzed effects of (5 mm) and of D 9 -THCV (5 4 mm) on PC spontaneous exctatory spke frng rates n the contnued presence of 3 mm BMI; (all n ¼ 35 (6)). Po.5 vs control, NS ¼ no sgnfcant dfference vs 3 mm BMI as normalzed control; Mann Whtney U-test. +BMI exctatory spke frng; these data were consstent wth D 9 -THCV causng an ncreased GABAergc nhbton, manfest as an overall decrease n spontaneous PC actvty. Agan, D 9 -THCV effects on PC output were smlar to those seen for the CB antagonst AM5. Drect CB receptor-medated postsynaptc effects (such as apparent reductons n frng rate due to spke ampltude fallng below the detecton threshold) are unlkely as PCs do not express sgnfcant CB receptor numbers (Matsuda et al., 993; Egertova and Elphck, ; Freund et al., 3). Actvaton of presynaptc CB receptors leads to reducton n transmtter release; the overall ncrease n exctablty at a network level s consstent wth cannabnod-medated modulaton of nhbtory neurotransmsson at IN-PC synapses beng reflected n spontaneous PC output. Ths hypothess s supported by the concentraton-dependent abolton of and D 9 -THCV effects by the GABA A receptor antagonst BMI, whch blocks phasc and tonc GABA currents at mouse IN-PC synapses (Harvey et al., 6). Interestngly, although cannabnod actons were reduced by mm BMI, t was necessary to use 3 mm BMI to ensure complete block; however, ths concentraton s well wthn ranges commonly Brtsh Journal of Pharmacology (8)

11 4 D 9 -THCV modulaton of GABAergc transmsson Y-L Ma et al used n bran slce experments. Overall, our data support a mechansm whereby D 9 -THCV ncreases GABA release at nhbtory termnals (ncludng IN-PC synapses) to cause a decrease n spontaneous PC output; thus, we demonstrate that D 9 -THCV has the potental to modulate network actvty wthn the cerebellar cortex. Functonal relevance We show that D 9 -THCV (and the CB receptor antagonst AM5) acts to ncrease nhbtory neurotransmsson to cause any overall decrease n PC output under the condtons used. In contrast, the CB agonst decreased GABA release and caused synaptc dsnhbton of PCs. PCs project nhbtory nnervaton to deep cerebellar nucle (DCN) to control ther ntrnsc frng rate (Gauck and Jaeger, ); n turn, DCN supply areas such as the vestbular nucleus and motor cortex to coordnate movement, balance and posture. Abnormally hgh spkng actvty can damage neurones and lead to degeneratve dsease. Moreover, excessve PC actvaton has been proposed to ncrease nhbton of DCN and lead to cerebellar dysfuncton (Patel and Hllard, ). Importantly, CB receptor agonsts have been shown to promote cerebellar dysfuncton n behavoural tests, n partcular causng severe motor ncoordnaton (DeSanty and Dar, ; Patel and Hllard, ). Therefore, decreases n GABA release at IN-PC synapses due to excessve endocannabnod release would be expected to result n PC dsnhbton and a correspondng ncreased nhbton of DCN, whch may then precptate cerebellar dysfuncton. Agents that ncrease nhbtory neurotransmsson, such as D 9 -THCV, wll cause opposng effects, reducng PC output and ultmately facltatng the control of posture and movement by DCN. These studes suggest that D 9 -THCV, alongsde standard CB receptor antagonsts, has therapeutc potental to combat dseases nvolvng cerebellar dysfuncton and hyperexctablty. For example, our prelmnary studes suggest that D 9 -THCV may be ant-convulsant n a developmental model of eplepsy (Weston et al., 6; see Pertwee, 8). Our data support recent proposals that phytocannabnods may represent mportant, but neglected, therapeutc agents (Mechoulam, 5). It wll be of nterest n future studes to nvestgate how dfferent phytocannabnods may smlarly modulate dsease states n the CNS. Acknowledgements We would lke to thank GW Pharmaceutcals for the gft of D 9 -THCV. We would also lke to thank Dr Andrew Constant (School of Pharmacy, Unversty of London) for constructve crtcal comments on the manuscrpt. Ths work was supported by The Wellcome Trust (GJS). Conflct of nterest The authors state no conflct of nterest. References Alexander SPH, Mathe A, Peters JA (7). Gude to receptors and channels (GRAC), nd edton (7 revson). Br J Pharmacol 5 (Suppl. ): S S68. Ashton JC, Frberg D, Darlngton CL, Smth PF (6). Expresson of the cannabnod CB receptor n the rat cerebellum: an mmunohstochemcal study. Neurosc Lett 396: 3 6. Bardo S, Robertson B, Stephens GJ (). Presynaptc nternal Ca þ stores contrbute to nhbtory neurotransmtter release onto mouse cerebellar Purknje cells. Br J Pharmacol 37: Bloom AS, Edgemond WS, Moldvan JC (997). Nonclasscal and endogenous cannabnods: effects on the orderng of bran membranes. Neurochem Res : Bouaboula M, Perrachon S, Mllgan L, Canat X, Rnald-Carmona M, Porter M et al. (997). A selectve nverse agonst for central cannabnod receptor nhbts mtogen-actvated proten knase actvaton stmulated by nsuln or nsuln-lke growth factor. J Bol Chem 7: Brown SP, Safo PK, Regehr WG (4). Endocannabnods nhbt transmsson at granule cell to Purknje cell synapses by modulatng three types of presynaptc calcum channels. J Neurosc 4: Denns I, Whalley BJ, Stephens GJ (7). Effects of cannabnods on [ 35 S]GTPgS bndng n specfc regons of the mouse bran. 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