The synthetic cannabinoid HU210 induces spatial memory deficits and suppresses hippocampal firing rate in rats

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1 British Journl of Phrmcology (27) 151, & 27 Nture Pulishing Group All rights reserved /7 $3. RESEARCH PAPER The synthetic cnninoid HU21 induces sptil memory deficits nd suppresses hippocmpl firing rte in rts L Roinson 1, AV Goonwrden 1,2, RG Pertwee 1, RE Hmpson 2 nd G Riedel 1 1 Deprtment of Biomedicl Sciences, Institute for Medicl Sciences, University of Aerdeen, Foresterhill, Aerdeen, UK nd 2 Deprtment of Physiology nd Phrmcology, Wke Forest University Helth Sciences, Winston-Slem, NC, USA Bckground nd purpose: Previous work implied tht the hippocmpl cnninoid system ws prticulrly importnt in some forms of lerning, ut direct evidence for this hypothesis is scrce. We therefore ssessed the effects of the synthetic cnninoid HU21 on memory nd hippocmpl ctivity. Experimentl pproch: HU21 (1 mgkg -1 ) ws dministered intrperitonelly to rts under three experimentl conditions. One group of nimls were pre-trined in sptil working memory using delyed-mtching-to-position tsk nd effects of HU21 were ssessed in within-suject design. In nother, rts were injected efore cquisition lerning of sptil reference memory tsk with constnt pltform loction. Finlly, seprte group of nimls ws implnted with electrode undles in CA1 nd CA3 nd single unit responses were isolted, efore nd fter HU21 tretment. Key results: HU21 tretment hd no effect on working or short-term memory. Reltive to its control Tween 8, deficits in cquisition of reference memory version of the wter mze were otined, long with drug-relted effects on nxiety, motor ctivity nd sptil lerning. Deficits were not reversed y the CB 1 receptor ntgonists SR141716A (3 mg kg -1 ) or AM281 (1.5 mg kg -1 ). Single unit recordings from principl neurons in hippocmpl CA3 nd CA1 confirmed HU21-induced ttenution of the overll firing ctivity lowering oth the numer of complex spikes fired nd the occurrence of ursts. Conclusions nd implictions: These dt provide the first direct evidence tht the underlying mechnism for the sptil memory deficits induced y HU21 in rts is the ccompnying normlity in hippocmpl cell firing. British Journl of Phrmcology (27) 151, 688 7; doi:1.138/sj.jp.77273; pulished online 14 My 27 Keywords: cnninoid; HU21; SR141716A; AM281; sptil lerning; wter mze; single unit recording; hippocmpus; rt Arevitions: BLA, solterl mygdl; DMTP, delyed-mtching-to-position; DNMS, delyed-non-mtch-to-smple; FR, men frequency; IBI, inter-urst intervl; i.p., intrperitonelly; ISI, inter-spike intervl; ITI, inter-tril intervl; LC, locus coeruleus; S, surprise; D 9 -THC, D 9 -tetrhydrocnninol Introduction The identifiction of cnninoid receptors nd the development of synthetic cnninoids led to significnt dvnces in ssessing the effects of mrijun on cognitive processes. It enled reserch to develop more specific phrmcologicl tools for endogenous receptors nd confirmed the long-held hypothesis tht mrijun intke leds to receptor-medited specific ltertions in mentl ilities (Riedel nd Dvies, 25). However, etter understnding of these effects on lerning nd memory function is wrrnted, since mrijun is the most widely used recretionl drug nd lso ecuse of its potentil for therpeutic pplictions (Pertwee, 2; Roson, 21). Correspondence: Dr G Riedel, Deprtment of Biomedicl Sciences, Institute of Medicl Sciences, University of Aerdeen, Foresterhill, Aerdeen AB25 2ZD, UK. E-mil: g.riedel@dn.c.uk Received 9 Jnury 27; revised 27 Ferury 27; ccepted 14 Mrch 27; pulished online 14 My 27 Cnninoid CB 1 receptors re widely distriuted throughout the centrl nervous system (CNS), with prticulrly high density in cererl cortex nd hippocmpus. This ntomicl loction correltes well with effects of cnninoids on memory formtion. CB 1 receptor gonists hve een investigted on numerous occsions using different ehviourl prdigms. Both cute nd chronic dministrtion of CB 1 gonists, including the primry psychoctive constituent of mrijun, D 9 -tetrhydrocnninol (D 9 -THC), the endocnninoid nndmide nd the synthetic cnninoids, WIN-55212,2 nd CP5594, induced lerning nd memory impirments in rts nd mice (for review see Dvies et l., 22; Roinson et l., 24; Roinson nd Riedel, 24). Severe impirments occur in the short-term domin of sptil memory (for reviews see Roinson et l., 24; Roinson nd Riedel, 24). Despite its wide use in memory reserch, ppliction of the open-field wter mze to cnninoid reserch is reltively

2 HU21, sptil lerning nd hippocmpl single units L Roinson et l 689 recent. Vrvel et l. (21), for instnce, reported impirments in sptil reference nd working memory fter D 9 -THC dministrtion in mice. In the reference memory version, mice overtrined to the pltform loction were susequently impired t doses of D 9 -THC tht lso produced gross sensory nd motor disturnces (1 mg kg 1 ). By contrst, mice were deficient t much lower dose (3 5 mg kg 1 ), with less severe side effects in working memory version, in which the pltform ws chnged to new position ech dy. Reference nd working memory versions of the wter mze, however, ccess two different memory mechnisms. Overtrining in the sptil reference memory tsk followed y drug dministrtion tests effects of D 9 -THC on recll. Such memory retrievl is insensitive to cnninoid tretment (D Silv nd Tkhshi, 22) nd deficits only occur with drug doses severely impeding motor coordintion (see Vrvel et l., 21). By contrst, despite extensive trining in the working memory tsk, nimls still lern the novel pltform loction nd this encoding of sptil informtion is CB 1 sensitive (Hmpson nd Dedwyler, 1998, 1999, 2, 23). Our work proved tht hippocmpl CA3 nd CA1 neurl firing rtes were not only reduced in the presence of D 9 -THC nd WIN , ut ehviourl tsk-specific firing rte chnges were lso suppressed in rts performing sptil delyed-non-mtch-to-smple (DNMS) tsk. In those studies, hippocmpl principl cell firing incresed 1- to 2-fold over ckground (1 5 Hz) within 71.5 s of the Smple (encoding) nd Nonmtch (recll) phses of DNMS trils. Despite 2 4% decrese in the numer of correct DNMS trils, D 9 -THC nd WIN suppressed this firing pek only for the Smple nd not the Nonmtch phse, suggesting tht cnninoids selectively impired the encoding of tril-specific working memory ut not memory retrievl. The effect of HU21, synthetic nlogue of D 9 -THC, hs not een investigted in detil in lerning prdigms nd physiologicl recordings. HU21 is clssicl cnninoid with high lipophilicity. Clssicl cnninoids re tricyclic dienzopyrn derivtives occurring nturlly in cnnis (D 9 -THC) or synthetic nlogues of these compounds, s is the cse for HU21 (for review see Howlett et l., 22). Efficcy t oth CB 1 nd CB 2 receptors is similr to tht of other cnninoids; however, the ffinity of HU21 for these receptors is higher (Pertwee, 21; Howlett et l., 22). This results in HU21 eing potent cnninoid gonist with long-lsting phrmcologicl effects in vivo. An initil study y Ferrri et l. (1999) reveled drug-induced dosedependent lerning deficit in sptil reference memory tested in the wter mze, ut there ws no effect on visile pltform tsk. The deficit ws interpreted s eing due to sptil lerning impirment, presumly due to CB 1 receptors locted in hippocmpus. Drug effects on prmeters of nxiety were lso presented, inviting the lterntive interprettion tht HU21, similr to D 9 -THC, my hve cused plce version (Cheer et l., 2). A more recent ccount extended these results nd reveled deficit in the cquisition of working memory tsk in the wter mze in rts pretreted with HU21 for 15 consecutive dys (Hill et l., 24). The working memory deficit ws specific for intertril intervl (ITI) of 5 min ut not for 3 s. Since nimls were nïve to the tsk, there ws lso deficit in procedurl lerning, mking it difficult to determine the contriution of hippocmpus nd sptil deficits to the overll cquisition impirment. A more detiled nlysis is wrrnted, which should lso explore whether HU21 effects on sptil lerning re possily medited vi the hippocmpl cnninoid system. Our min ims were first the detiled ssessment of the effects of HU21 on sptil lerning nd memory in rts using the open-field wter mze. Experiment 1 employed working memory tsk using delyed-mtching-to-position (DMTP) prdigm s descried previously (Roloff et l., 22, ). Performnce on DMTP tsk in the wter mze is sensitive to hippocmpl lesions, intr-hippocmpl N-methyl-D-sprtte (NMDA) receptor lockde (Steele nd Morris, 1999), scopolmine tretment, comined -myloid nd scopolmine exposure (Roloff et l., 22), nd D 9 -THC dministrtion to stimulte CB 1 receptors (Vrvel et l., 21; D Silv nd Tkhshi, 22; Fdd et l., 24). As we did not oserve the expected results, experiment 2 repeted the work of Ferrri et l. (1999). This yielded the proposed deficit nd we then ssessed, whether the effect is medited y CB 1 receptors using co-dministrtion of the two CB 1 ntgonists, SR141716A nd AM281, with HU21 in some groups. The second im of the study ws to determine whether the oserved ehviourl deficits in sptil reference memory could hve resulted from ltertions in hippocmpl neurl firing. Recent pulictions y Pistis nd co-workers (Pistis et l., 24; Muntoni et l., 26) provide contrsting results for the effects of cnninoids on firing of locus coeruleus (LC) nd solterl mygdl (BLA) neurons in vivo. WIN nd D 9 -THC incresed firing of LC neurons, while WIN nd HU21 decresed firing of BLA neurones in nesthetized nimls. Given tht we hve shown cnninoid-elicited inhiition of hippocmpl neurl firing in ehving nimls, it is importnt to confirm these effects with HU21. Mterils nd methods Sujects Mle Lister Hooded rts from commercil sources (Rowett Reserch Institute, Aerdeen or Hrln, UK), ged 7 8 weeks nd weighing 25 3 g t the strt of trining were used in ll experiments (exp. 1, N ¼ 8; exp. 2, N ¼ 63; exp. 3, N ¼ 8). Sujects were group housed (four per cge), with free ccess to food nd wter on 12:12 h dy/night cycle (lights on t 7 m). Animls were housed in pthogen-free niml fcility in ccordnce with the Federtion of Europen Lortory Animl Science Associtions (FELASA) guidelines. Recording took plce etween the dylight hours of 1 m nd 4 pm. All experiments were performed under UK Home Office regultions. Drug tretment nd groups Stock solutions of HU21 (5 mg ml 1 ) (Tocris Cookson, Bristol, UK), AM281 (1 mg ml 1 ) (Tocris Cookson, Bristol, British Journl of Phrmcology (27)

3 69 HU21, sptil lerning nd hippocmpl single units L Roinson et l UK) nd SR A (1 mg ml 1 ) (Reserch Tringle Inst., Ntl. Inst. Drug Ause, Cry, NC, USA) in ethnol were used. Drugs were prepred fresh ech dy using these stock solutions in vehicle of Tween 8, evported nd further diluted with sline (.9%) to the finl doses of HU21 1 mgkg 1 ; SR A 3 mg kg 1 ; AM281.5 mg kg 1 nd 1.5 mg kg 1. Tween 8 ws used s the vehicle in this study. Drug doses were selected sed on pulished efficcy of the respective doses in ehviourl terms in Lister Hooded rts nd were dministered intrperitonelly (i.p.) 1 h efore testing t volume of 5 ml kg 1. The ntgonists were dministered immeditely efore HU21. All drugs were dministered t the sme time on ech dy of testing. Behviourl pprtus A circulr white Perspex wter mze (15 cm dimeter, 5 cm depth) ws plced in room surrounded y vrious sptil cues. The pool ws filled with wter (25721C) to depth of 35 cm. A cler Perspex pltform (1 cm dimeter) ws plced t predetermined pltform position pproximtely 1 cm elow the surfce of the wter. Trils were recorded y n overhed video cmer nd trcker with dt video tped nd stored online for lter nlysis using PC-sed softwre (HVS Imge, Hmpton, UK). Behviourl trining nd testing Experiment 1 HU21 nd DMTP performnce. All nimls were fully pretrined in the delyed-mtching-to-smple tsk efore testing in the presence of drug. This trining lsted for t lest 2 weeks (four trils per dy) nd performnce in tril 2 of the DMTP tsk ws elow 2 s ltency to find the pltform. The procedure ws identicl to testing in the presence of drug. Testing comprised of the following protocol: HU21 injection ws followed y ehviourl testing n hour lter. The next dy ws drug nd test-free to llow wsh-out nd we dministered Tween 8 on the following dy nd performed nother test. This ws gin followed y n injection nd test-free dy. A fully counterlnced within-suject design ws employed in which ech niml went through ll drug nd control conditions. Twelve predetermined, rndomly locted pltform positions distriuted in ll prts of the pool were selected. The pltform loction remined constnt within ech session (one session of four trils per dy), ut ws chnged etween sessions. Sujects were relesed fcing the wll of the pool from one of four crdinl relese sites (N, S, E or W) in semi-rndom mnner (ll relese sites used on ech dy). A mximum time llownce of 9 s ws set for rts to locte the sumerged pltform (dimeter 1 cm, 1 cm elow wter surfce), where they remined for 3 s efore eing returned to their cges. These were plced into heting ox to void hypothermi. If rts filed to find the pltform within 9 s, the experimenter guided them to it. DMTP testing involved within-suject design, with ll nimls receiving oth drugs nd lso performing t oth delys in n lternting mnner. The ITI etween trils 1 nd 2 ws either 3 s or 1 h; ll other ITIs were 3 s. It follows tht performnce in tril 2 (pth length, ltency nd swim speed) is of prticulr relevnce, s it reflects sptil short-term memory of tody s pltform loction (Steele nd Morris, 1999; Roloff et l., 22, ). Performnce in tril 4 represents the floor level tht cn e chieved under drug/control conditions. Experiment 2 HU21 nd reference memory. Reference memory ws exmined y following the protocol of Ferrri et l. (1999). The pltform loction ws constnt for ech niml. It ws plced t the centre of one pool qudrnt nd trget qudrnts were counterlnced for ll groups. Animls were nïve t the strt of trining nd were given four trils per dy on four consecutive dys. On ech tril, rts were relesed from one of the four relese sites (N, S, E or W) fcing the wll of the pool nd llowed 9 s to locte the pltform, where they remined for further 3 s. All ITIs were 3 s. During ITIs, nimls styed in the heting ox. If rts filed to locte the pltform within 9 s, the experimenter guided them to it. A proe tril ws dministered 24 h following cquisition trining, which lsted 6 s, with the pltform removed from the pool nd nimls eing relesed opposite to the trget qudrnt. In experiments 1 nd 2, pth length tken to locte the pltform on ech tril, time spent in swim corridor (Whishw, 1984) directly connecting the relese site with the pltform loction nd ltency to trget re were recorded s indices of sptil memory. The trget re ws two times the size of the pltform (2 cm dimeter) nd centres over the pltform position. Pth length ws used s mesure of sptil lerning nd memory, s unlike ltency, it lso tkes into ccount difference in swim speed. Pth length hs een suggested s the most importnt mesure of sptil memory (Lindner, 1997). Swim speed nd thigmotxis (time spent in the outer 1 percent of the pool) were ssessed s mesures of procedurl memory. Experiment 3 surgery. Rts were nesthetized under constnt flow of isoflurne (Aot lortories, IL, USA) nd positioned in stereotxic frme efore unilterl implnttion with multi-electrode recording rry (Neurolinc, NY, USA) consisting of 16 stinless steel electrodes (4 mm) rrnged in two rows (8 mm etween rows), such tht ech row consisted of eight electrodes with 2 mm centre-tocentre spcing. The rry ws constructed with symmetric lengths such tht CA3 electrode tips would e utomticlly positioned 1.2 mm ltero-ventrl to the CA1 electrode tips. The sclp ws incised long the midline, pulled ck to the lterl crnil ridges nd single holes drilled for insertion of 1 mm silver ground wire into the prietl cortex. The centre of the rry ws positioned 3.4 mm posteriorly nd 72.7 mm mediolterlly from Bregm, depending on whether the rry trgeted the left or right hippocmpus. The posterior end of the rry ws ngled lterlly such tht the longitudinl xis ws 31 from midline of the skull. An ovlshped crniotomy ws mde pproximtely 2 mm lrger thn the rry, the dur resected nd the long electrode tips plced in contct with the surfce of the rin. The electrode ws then driven in 5 mm steps to depth of 2.8 mm for the British Journl of Phrmcology (27)

4 HU21, sptil lerning nd hippocmpl single units L Roinson et l 691 CA1 leds nd 4. mm for the CA3 leds, then lowered to finl depth of pproximtely 3. mm for CA1 nd 4.2 mm for CA3, using continuous recording of neurl ctivity to confirm optimum plcement of the electrode tips in the respective cell lyers. Neurl ctivity ws continuously monitored throughout surgery to ensure tht the electrode rry mintined plcement in the pproprite hippocmpl sufields. Following rief settling time (15 2 min) for electrodes to stilize in plce, the electrode depths were djusted, if necessry, nd the crniotomy seled with dentl cement. Results Generl ehviourl oservtions It ws ovious from oservtions on dry lnd surfces tht nimls injected with HU21 presented with some ctleptic symptoms nd, lthough we quntified this using the r test s descent ltencies, HU21-treted nimls were not significntly different from Tween 8 controls (df ¼ 11; t ¼ ; P4.2) nd descent ltencies hrdly exceeded 1 s (dt not shown). The lim positions during swimming were norml nd the typicl forelim inhiition ws seen in ll rts. Electrophysiologicl recording Following stiliztion of the electrode rry, selected principl cells with firing rte of.5 5 Hz were isolted using Multiunit Acquisition Processor (MAP, Plexon Inc., Dlls, TX, USA). The neuronl ensemle ctivities of these preselected CA3/CA1 principl cells were trcked nd recorded following the forementioned tretments. Recording prmeters Single neuron spike trins were nlyzed using Neuroexplorer (Nex Technologies, MA, USA) softwre. The following prmeters were computed for ech selected principl cell cross tretments nd nimls: (1) men frequency of firing (FR, Hz) nd (2) men inter-spike intervls (ISIs). In ddition, ursts of spikes were chrcterized using the Surprise method tht identified sequences of t lest three consecutive spikes with ISIs less thn one-hlf of the men ISI over ll spike occurrences. Bursts were identified y: (1) clculting men FR nd ISI for ll spikes, (2) identifying sequences of three or more consecutive spikes with ISIs, in which ll ISIs were less thn one-hlf the men ISI, (3) computing Surprise (S) such tht S ¼ log 1 (P) where proility tht the sme sequence of spikes could occur in rndom Poisson distriution with the sme men frequency s FR, (4) mximizing S y either dding consecutive spikes to the end of the urst, or removing consecutive spikes from the eginning of the urst. Bursts with S-vlues 41 were then chrcterized ccording to men urst durtion, men interurst intervl (IBI), men numer of spikes in urst, men frequency of ursts in the spike trin, men FR within ursts nd men ISI within ursts. Dt nlysis Behviourl dt were nlyzed using the computer-sed sttistics pckge Grphpd Prism (version 4.1. for windows, Grphpd softwre, Sn Diego, CA, USA). Repeted mesures two-wy nlysis of vrince (ANOVA) with trils, dely nd drug tretment s fctors were employed. Approprite plnned comprisons including further two-wy ANOVAs nd t-tests were performed with the significnce level set to Po.5. Electrophysiologicl dt were nlyzed y Student s t-tests (pired) to compre the mens7s.e.m. etween HU21 nd Tween 8 cross ll prmeters descried ove. Experiment 1 HU21 does not ffect performnce in DMTP in wter mze After 2 weeks of pretrining, ll nimls were competent in performing the DMTP tsk. Reltive to the Tween 8 tretment, there ws no impirment in tril 2 performnce in rts under the influence of HU21. This ws oserved for pth length (Figure 1), corridor nlysis (Figure 1) nd ltency to trget re (Figure 1c). Sttisticl nlysis confirmed relile effects of tril (F-vlues 42.7; PX.5) ut not drug nd dely (ll F-vlues o1.2) on ll these prmeters. Similrly, effects of tril (F-vlues 43.5; Po.2) were otined for procedurl mesures (swim speed nd thigmotxis; Figure 1d nd e), nd the min effect of drug (F(3,112) ¼ 17.1; Po.1) ws relile only for swim speed. Contrry to expecttion (Hill et l., 24), dt from this experiment indicted tht despite differences in swim speed, there ws no sptil working/short-term memory deficit in HU21-treted rts. Previous work (Bnnermn et l., 1995; Cin et l., 1997, 22) using the wter mze suggests tht mny drug tretments led to procedurl deficits in nïve ut not pretrined nimls. We therefore resoned tht pretrining, s conducted here to fmilirize sujects with the tsk requirements, my hve eliminted differences in procedurl prmeters nd lso the sptil memory deficit. Pretrining cn indeed render these lerning nd memory processes insensitive to drug ctions (Cin et l., 1997, 22) or hypoxic insult (Row et l., 23), nd we therefore progressed y testing nïve nimls in sptil reference memory tsk s previously suggested. Experiment 2 cquisition lerning of sptil reference memory tsk is impired y HU21 The filure of HU21 to induce sptil short-term/working memory impirment in the DMTP tsk my e due to the previously reported procedurl memory deficit (Ferrri et l., 1999; Hill et l., 24) nd the pretrining-induced fmiliriztion with the procedurl tsk demnds my thus hve rendered the tsk HU21 insensitive. Therefore, re-exmintion of the effects of HU21 on cquisition lerning of sptil reference memory s reported y Ferrri nd coworkers should revel n overll deficit. At the sme time, we exmined whether HU21-induced memory deficits re medited y CB 1 receptors? Figure 2 depicts the effect of HU21 on pth length required to loclize the pltform British Journl of Phrmcology (27)

5 692 HU21, sptil lerning nd hippocmpl single units L Roinson et l 3s 1h Pth length [cm] s 1h Tween HU21 Tween HU c 75 Time in corridor [% of tril time] Ltency to trget re [s] d Swim speed [cm/s] Tril e Thigmotxis [% of tril time] Tril Figure 1 HU21 did not ffect short-term memory ssessed in delyed-mtching-to-position tsk in pre-trined nimls. Vlues shown in ( e) re mens7s.e.m. HU21 tretment did not led to n overll increse in pth length (), nd recorded vlues did not revel ny sptil (, c) or thigmotxis-relted (e) differences etween groups t either dely (3 s or 1 h). Swim speed ws higher in the HU21 group (d; sterisks indicte Po.1.). Performnce improved on tril 2 independent of drug tretment. Representtive swim trces indicte tril 2 performnce for oth delys. during cquisition lerning in sptil reference memory tsk. Animls treted with HU21 swm longer distnces on most trils compred with Tween 8-treted rts. This impression ws confirmed sttisticlly with min effect of drug tretment (F(1,24) ¼ 39.8; Po.1), tril (F(15,24) ¼ 22.4; Po.1), nd n interction (F(1,24) ¼ 3.2; Po.1) suggesting tht the deficit did not ffect ll trils eqully. Spcing trils over repeted sessions ttests different qulities to trils 1 nd 4 of ech session. Lerning etween dys refers to long-term memory formtion nd includes processes of consolidtion (Riedel nd Micheu, 21), while within-session lerning (from tril 1 to 4 in our cse) my e n index of short-term memory (Kesner et l., 1993). Detiled ehviourl nlysis of ll groups, with focus on trils 1 nd 4, ws conducted in order to distinguish etween these memory processes, nd results re summrized in Figure 3. HU21 ws very effective nd oth ntgonists, AM281 nd SR141716A, did not reverse the deficits. For tril 1, (Figure 3) this ws confirmed y 7 4 fctoril ANOVA, with drug tretment s etween- nd dy s within-suject fctors. Overll, there were significnt effects of drug tretment (F(6,192) ¼ 1.83; Po.1), dy (F(3,192) ¼ 71.48; Po.1), nd n interction (F(18,192) ¼ 1.93; P ¼.1). All drug groups were significntly different to Tween 8 (ll F-vlues 415; Po.3), prt from the AM mg kg 1 group (F ¼ 1.1). Both AM281 lone groups were found to e significntly different to ll other HU21 groups (ll F-vlues 416; Po.1), yet they were not different from ech other (F o4; P4.5). All HU21- contining drug tretments lso did not differ from ech other (ll F-vlues o4; P4.5). It is ovious, however, tht ll groups irrelevnt of drug tretment improved their performnce over dys (ll F-vlues 47.1; Po.1). A qulittively different result ws otined for tril 4 (Figure 3). Some drug groups were initilly impired on dys 1 nd 2, ut then ttined floor levels similr to the Tween 8 group on the fourth dy of trining. It ppered, British Journl of Phrmcology (27)

6 HU21, sptil lerning nd hippocmpl single units L Roinson et l Tween 8 HU21 Pth length [cm] 2 1 Dy Tril Figure 2 HU21 impired sptil lerning in the wter mze. () Pth length cross ll trils of cquisition for oth Tween 8- nd HU21 (1 mgkg 1 )-treted nimls. Vlues shown in () re mens7s.e.m.. HU21 nimls were severely impired nd required significntly longer swim pths to locte the pltform. Asterisks indicte relile differences (Po.5; Po.1) etween groups for the time periods indicted y the horizontl rs. In (), representtive swim pths indicte performnce under the two drugs for the first (dy 1) nd finl dy (dy 4) of trining. Dy 4 Tril 1 Tril 4 3 Tril 1 Tween Pth length [cm] 2 1 HU21 HU+AM.5 mg/kg HU+AM 1.5 mg/kg Tril 4 3 HU+SR Pth length [cm] 2 1 AM.5 mg/kg AM 1.5 mg/kg Dy Figure 3 HU21-induced deficits in sptil lerning were not reversed y cnninoid ntgonists. Vlues shown in () nd () re mens7s.e.m. () Tril 1 performnce (long-term memory etween sessions) of ll drug groups. There ws no reversl of the HU21 deficit with cnninoid ntgonists SR141716A nd AM281. () Tril 4 performnce (short-term memory within ech session) for ll drug groups. HU21 lone induced minor deficit. Co-dministrtion of HU21 nd cnninoid ntgonists led to short-term memory deficits. Swim trces show representtive exmples of swim pths for ll drug groups during trils 1 nd 4 on dy 4. Asterisks indicte significnt differences etween HU21-contining groups nd Tween 8 controls t the specific time points (t-test, Po.5; Po.1). however, tht neither AM281 nor SR141716A could reverse the deficit induced y HU21. Anlysis of ll tretments yielded min effect of drug tretment (F(6,192) ¼ 7.58; Po.1), dy (F(3,192) ¼ 3.3; Po.1) nd lso n interction (F(18,192) ¼ 2.32; P ¼.26). As with tril 1, ll tretment groups contining HU21 were significntly impired in tril 4 reltive to Tween 8 (F-vlues 426; Po.1), prt from oth AM281 lone groups (F-vlues o1.8). Further British Journl of Phrmcology (27)

7 694 HU21, sptil lerning nd hippocmpl single units L Roinson et l comprisons reveled tht co-dministrtion of HU21 with ntgonists impired performnce compred with HU21 lone (F-vlues 46.7; Po.14), ut HU21 þ ntgonist groups did not differ from ech other (F-vlues o1). These dt clerly suggest tht the performnce of nimls treted with HU21 (lone or co-dministered with AM281 or SR141716A) is compromised. Interestingly, ntgonist tretment enhnced the deficit oserved for tril 4. Overll, these dt suggest tht HU21 tretment impirs oth sptil long-term nd short-term memory in rts. Anlysis of ltency to trget re (twice the size of the pltform) for tril 1 (Figure 4) confirmed min effect of drug tretment (P4(6,192) ¼ 3.47; P ¼.1), dy (F(3,192) ¼ 11.91; Po.1) nd n interction (F (18,192) ¼ 2.95;P ¼.1); ll groups treted with HU21 differed from Tween 8 controls (F s44.52; P-vlues o.5), prt from oth AM281 lone groups (F-vlues o2.64; P4.1). This strongly suggests tht oth ntgonists (SR141716A nd AM281) did not reverse the sptil long-term memory deficits induced y HU21. For tril 4 ltency to trget re (Figure 4), nlysis confirmed min effects of drug tretment (F(4,128) ¼ 3.14; P ¼.3), dy (F2,128) ¼ 1.4; Ltency to trget re [s] Tril 1 Tril c Swim speed [cm/s] d HU21 HU+AM.5 mg/kg HU+AM 1.5 mg/kg HU+SR Tween AM.5 mg/kg AM 1.5 mg/kg e 1 f 1 Time thigmotxic [%] Dy Dy Figure 4 Anlysis of sptil nd non-sptil prmeters monitored during cquisition of the reference memory tsk. Vlues shown in ( f) re mens7s.e.m. HU21 tretment impired performnce on the sptil prmeter of ltency to trget re (, ) for trils 1 nd 4. In generl, co-dministrtion of ntgonists incresed the deficit cused y HU21. Swim speed ws incresed in ll HU21-treted groups for tril 1 during the initil trining dys (c); however, no difference ws oserved t tril 4 (d). All HU21-treted nimls presented with severely incresed levels of thigmotxis during tril 1 (e), ut HU21 lone hd only minor effects on thigmotxis t tril 4 (f). However, comined dministrtion especilly with SR141716A cused drmtic rise in nxiety-relted swimming long the edges of the pool. Asterisks indicte relile differences etween HU21-contining groups nd Tween 8 controls t the specific time points (t-test, Po.1). British Journl of Phrmcology (27)

8 HU21, sptil lerning nd hippocmpl single units L Roinson et l 695 Time in Qudrnt [%] HU21 HU+AM.5 mg/kg HU+AM 1.5 mg/kg HU+SR Tween Al Trget Ar Opp AM.5 mg/kg AM 1.5 mg/kg Figure 5 Proe tril conducted 24 h fter the lst trining tril with pltform removed. In (), representtive swim trces of individuls of the different drug groups otined during the proe tril. In (), vlues shown re mens7s.e.m.; horizontl line indictes chnce level; Al, djcent left; Ar, djcent right; Opp, opposite. A sptil is for the trget qudrnt ws only found in nimls treted with Tween 8 or AM281 lone (.5 nd 1.5 mg kg 1 ), wheres ll other groups displyed no retention. Asterisks indicte relile difference etween Tween 8 controls nd HU21-contining groups indicted y the rs (Po.5). Po.1) nd n interction (F(12,128) ¼ 2.65; P ¼.3). The HU þ AM 1.5 mg kg 1 nd AM281 lone groups were the only groups tht did not differ from Tween 8 (F-vlues o1.67; P4.2), while ll other groups were different from Tween nimls (F-vlues 48.75; Pp.1). This suggests tht the higher dose of AM281 reversed the sptil short-term memory deficit induced y HU21. This ws confirmed in tht the group of nimls treted with (HU21 þ AM 1.5 mg kg 1 ) performed significntly etter thn ll other HU21 groups (F-vlues X6.4; Po.2). Anlysis of non-sptil prmeters for trils 1 nd 4 of ech dy re summrized in Figure 4c f. At tril 1, swim speeds (Figure 4c) were not different etween drug tretments (F(4,128) ¼ 1.7; P ¼.17), ut we otined effects of dy (F(3,128) ¼ 17.3; Po.1) nd n interction (F(12,128) ¼ 3.2; P ¼.4). Plnned comprison confirmed tht ll drug groups differed from Tween 8 controls (ll F-vlues 46.8; Po.1), ut not etween ech other (ll F-vlues o1), suggesting tht ntgonists did not reverse the HU21- induced increse in swim speed. For tril 4, swim speeds (Figure 4d) were more stle nd we did not otin relile differences etween dys, drug groups or n interction (ll F-vlues o2; P4.5). Dt for tril 1 (Figure 4e) indicte sustntilly higher thigmotxis in ll groups reltive to Tween 8. Sttisticl comprison confirmed this impression with min effects of drug tretment (F(4,128) ¼ 1.5; Po.1), dy (F(3,128) ¼ 176; Po.1) nd n interction (F(12,128) ¼ 6.7; Po.1). All drug groups, prt from the higher dose of AM281 (1.5 mg kg 1 ) (F ¼ 2.7; P4.1), were significntly different from Tween 8 controls (ll F-vlues 44; Po.1). Furthermore, there were sutle differences etween the drug tretments, nmely etween HU21 nd HU21 þ SR141716A, s well s HU21 þ SR141716A nd HU21 þ AM281 (1.5 mg kg 1 ) (ll F-vlues 413; Pp.1). There ws, however, no reversl of the HU21-induced increse in nxiety. Thigmotxis mesured t tril 4 of ech session (Figure 6d) reveled min effects of drug tretment (F(4,128) ¼ 16.4; Po.1), dy (F(3,128) ¼ 13.1; Po.1) nd n interction (F(12,128) ¼ 2.9; P ¼.2). Controls showed prcticlly no swimming in the outer zone (prt from relese). This ws similr for the HU21 nd AM281 (1.5 mg kg 1 ) lone groups (F-vlues o4.32; P4.5), ut ll drug groups comining HU21 with ntgonists were significntly worse thn Tween 8 nd HU21 treted rts (ll F-vlues 43.2, Po.5); the most pronounced deficit ws oserved for the HU21 þ SR141716A comintion. Retention test Performnce of the groups differed gretly in the proe test conducted 24 h fter the lst trining tril (Figure 5). Compred with Tween 8 controls nd the AM281 lone groups, ll HU21 tretments filed to show sptil is for the trget qudrnt providing evidence for the lck of sptil memory. This ws confirmed sttisticlly in 5 (drug tretment s etween-suject fctors) 4 (qudrnt s within-suject fctor) fctoril ANOVA. We otined highly significnt interction etween these prmeters (F(12, 16) ¼ 7.5; Po.1), nd post hoc pirwise comprison confirmed interctions for ll HU21-treted drug groups reltive to Tween 8 (ll F-vlues 49.5; Po.1), wheres AM281 groups did not differ from controls (F-vlues o1.6; P4.2). This confirms tht despite some lerning during ech session (s indicted in tril 4 performnces) nd smll overll improvements in performnce over dys, HU21-treted nimls did not form sptil memory of the loction of the hidden pltform. HU21 ttenutes spontneous firing ctivity of principle neurons in hippocmpus Extrcellulr ction potentils were recorded from 45 principl cells (N ¼ 8 nimls) in hippocmpl CA3 nd CA1 sufields. Averge firing frequency nd short durtion ursting chrcteristics of the spike trin were nlyzed. Exposure to HU21 produced significnt suppression of verge firing frequency (t ¼ 7.941, Po.1) throughout the spike trins (Figure 6). As result, men ISIs were significntly incresed (t ¼ 3.871, Po.1) following HU21 tretment, confirming tht the decrese in spontneous neurl ctivity ws coupled to prolongtion of the intervl etween ny two spike occurrences in the spike trin. British Journl of Phrmcology (27)

9 696 HU21, sptil lerning nd hippocmpl single units L Roinson et l Frequency (Hz) Frequency (Hz) c Time (min) Tween8 HU21 8 Frequency (Hz) d 3 Inter-spike intervl (s) Time (min) Tween8 Figure 6 Spike firing properties of hippocmpl cells were ttenuted fter HU21 tretment. The rte histogrms (men frequency/1 s ins) depict firing ctivity of preselected principl cell during 2 min recording epoch following Tween 8 () nd HU21 () tretments. Vlues shown in (c) nd (d) re mens7s.e.m. of frequency (c) nd ISIs (d) pooled cross 1 h electrophysiologicl recordings clculted from 45 hippocmpl principl cells locted in the dorsl CA3 nd CA1 sufields. Asterisks indicte Po.1. HU21 Moreover, HU21 tretment reveled mrked reduction (t ¼ 6.57, Po.1) in the verge numer of ursts (Figure 7) nd numer of spikes per ursts (t ¼ 2.274, Po.5; Figure 7f) in comprison to controls (Tween 8) cross ll preselected principl cells. HU21 significntly ttenuted the verge urst durtion (t ¼ 2.49, Po.5; Figure 7) nd IBI (t ¼ 3.848, Po1; Figure 7c) in comprison to controls, suggesting tht not only were spontneous urst occurrence, spikes per urst nd urst durtion reduced, ut the intervl etween successive ursts ws incresed s consequence of HU21 tretment. In ddition, HU21 noticely reduced the verge frequency within ursts (t ¼ 3.816, Po.1; Figure 7d) nd incresed men ISI within ursts (t ¼ 4.22, Po.1; Figure 7e). These findings suggest tht the overll chnges in spike trin firing chrcteristics were replicted in urst ctivity following HU21 tretment. Finlly, we lso ssessed the effects of AM281 ginst HU21-induced suppression of firing ctivity. In line with the ehviourl results, AM281 t either.5 or 1.5 mg kg 1, injected 1 h post-hu21 tretment, did not reverse the reduction in firing frequency or ursting (ll P4.5). Reltive to Tween 8, depression of ctivity persisted (ll Po.5) (dt not shown). Discussion We here report, tht HU21 induced sptil deficit in the wter mze in lerning reference memory tsk in numerous prmeters (Tle 1 for summry) together with ltertions in hippocmpl firing ptterns of single principl neurons. Pretrining in working memory tsk, however, prevented these deficits. HU21 induces sptil nd non-sptil deficits Plnt-derived nd synthetic cnninoids impir working or short-term memory, with more severe effects when nimls re exposed to long delys etween trils (for review see Dedwyler et l., 1995; Roinson nd Riedel, 24), which is consistent with the involvement of the hippocmpus in longer ut not shorter delys (Hmpson et l., 1999). Deficits in sptil lerning fter D 9 -THC dministrtion hve een reported for oth rts (Stiglick nd Klnt, 1982; Nkmur et l., 1991; Lichtmn et l., 1995; Molin-Holgdo et l., 1995; Lichtmn nd Mrtin, 1996; Ferrri et l., 1999; Hernndez-Tristn et l., 2; Mishim et l., 21; Fdd et l., 24) nd mice (Vrvel et l., 21, 25; D Silv nd Tkhshi, 22) tested in rdil or wter mze. Although usully injected i.p., few studies confirm tht cnninoids lso ct vi direct intrhippocmpl dministrtion (Lichtmn et l., 1995; Egshir et l., 22). This suggests tht the hippocmpl CB 1 receptor popultion is crucil for sptil lerning. HU21 is D 9 -THC nlogue, with higher efficcy nd much more potent nd long-lsting effects on CB 1 receptors thn D 9 -THC (Pertwee, 1999). Its ehviourl effects hve not een explored in gret detil. HU21-induced effects on sptil lerning in the wter mze hve een reported in nïve rts (Ferrri et l., 1999; Hill et l., 24). Pretrining nimls in DMTP version of the wter mze (Roloff et l., 22, ), however, enles rts to lern nd consolidte the procedurl elements of the tsk (Sucier et l., 1996; Cin British Journl of Phrmcology (27)

10 HU21, sptil lerning nd hippocmpl single units L Roinson et l Numer of ursts Burst durtion (sec) c 1 Inter-urst intervl (s) Tween8 Tween8 Tween8 HU21 HU21 HU21 d Frequency in ursts (Hz) e Inter-spike intervl in ursts (s) f Spikes per urst Tween8 Tween8 Tween8 HU21 HU21 12 Figure 7 Overll urst nlysis of recordings from hippocmpl cells. Vlues shown in ( f) re mens7s.e.m. of numer of ursts (); urst durtion (); IBI (c); frequency in ursts (d); ISI in ursts (e) nd spikes per urst (f) cross 1 h electrophysiologicl recordings clculted from the sme 45 dorsl hippocmpl principl cells locted in the CA3 nd CA1 sufields, following Tween8 nd HU21 tretment. Reltive to Tween 8, HU21 induced strong reduction in excitility, s evidenced y chnges in urst chrcteristics. Asterisks indicte Po.5; Po.1. HU21 Tle 1 Summry of experimentl results of HU21 on DMTP nd reference memory nd retention in the wter mze, s well s electrophysiologicl responses of single hippocmpl neurons DMTP Reference memory Retention Electrophysiology 3 s 1 h Tril 1 Tril 4 Pth length NS NS m No reversl Ltency to trget re NS NS m No reversl Swim speed m m m No reversl Thigmotxis NS NS m No reversl m No reversl Impirment Frequency m No reversl with AM281 or SR Reversl with AM mg kg 1 m of ursts NS m of spikes HU21 þ AM281 or SR impired No reversl with AM281 Arevitions: DMTP, delyed-mtching-to-position; NS, not significnt. Arrows indicte n increse or decrese in respective mesures, together with sterisks denoting their significnce Po.5. et l., 1997, 22) nd to form gross sptil mp of the environment (O Keefe nd Ndel, 1978). Dily movement of the pltform would require the niml to memorize the novel loction of the pltform nd sptil deficits could e recorded uncontminted of ny drug effects on the lerning procedure. However, we otined no memory deficit in the HU21 group despite implementtion of dely of up to 1 h etween trils 1 nd 2 (exp. 1). This finding is contrry to work y Hill et l. (24), who found HU21-induced deficits in cquisition of DMTP tsk in the wter mze t ITIs of 5 min ut not 3 s following exposure of rts to HU21 (1 mgkg 1 ) dily for 15 consecutive dys. The sence of impirment with HU21 in short-term/ working memory is lso in contrst to previous studies tht British Journl of Phrmcology (27)

11 698 HU21, sptil lerning nd hippocmpl single units L Roinson et l hve used D- 9 THC nd other CB 1 gonists (see Fdd et l., 24, 26). Therefore replicting the work of Ferrri et l. (1999) imed t gining deeper insight into psychophrmcologicl mechnisms underlying the ctions of HU21 nd lso to determine if there ws indeed differentil effect on sptil working nd reference memory s sptil reference memory protocols my ctivte different rin circuits nd/ or different cellulr mechnisms compred to short-term or working memory prdigms (Izquierdo et l., 1999). In the reference memory tsk, we found lerning deficit similr to the one reported y Ferrri nd collegues, nd further nlysis reveled tht oth sptil nd non-sptil prmeters were ffected y HU21. There ws cler increse in swim speed oserved in the reference memory tsk. However, this could not explin the deficit otined in sptil lerning since similr increse in swim speed ws otined in pre-trined nimls in the working memory prdigm (exp.1), yet there ws no lerning or memory impirment. We tke this s evidence for dissocition of cnninoid effects on motor ctivity nd on sptil lerning. They re lso independent of previous experience with the drug, s fster swim speeds were executed on ll drug dys (Figures 1d nd 4c). D 9 -THC hd similr effects in humns, where enhnced speed of performnce correlted with increses in errors (Gison s mze, doule digit cncelltion; Currn et l., 22), indicting trde-off etween speed nd ccurcy. Riskier driving t high speed under the influence of cnnis (Ashton, 21) my indeed e relted to chnges in performnce speed. We otined contrsting results in our niml study with ccurcy eing independent of swim speed. HU21-induced thigmotxis ws high in the reference memory prdigm, ut not different from controls in the working memory protocol suggesting tht nxiety-relted prmeters my ccount for the oserved lerning deficit. Close inspection of Figure 4f, however, indictes tht HU21-treted nimls rech floor level in their wll hugging, while sptil tsk prmeters (pth length (Figure 3) nd ltency to trget re (Figure 4)) remin incresed. This suggests tht the initil increse in nxiety is overcome during dily trining nd cnnot explin the remining sptil within-session lerning deficit. The simplest explntion thus is tht the deficit seen in tril 4 fter HU21 tretment rises from deficits in sptil processing, most likely due to reduced firing of principl neurons in hippocmpus. Memory deficits re not reversed y cnninoid ntgonists Co-dministrtion of HU21 nd AM281 or SR141716A imed t reversing the reference memory deficits. SR141716A is well studied highly potent CB 1 receptor ntgonist tht hs previously een shown to reverse the CB 1 gonist-medited impirments in cognition (Mllet nd Beninger, 1998; Nv et l., 2; Mishim et l., 21; D Silv nd Tkhshi, 22; Hmpson nd Dedwyler, 1998, 1999, 2, 23). AM281 is structurl nlogue of SR141716A. Co-dministrtion of cnninoid gonists nd ntgonists is common wy of ssessing the specificity of drug for the CB 1 receptor nd hs, in the cse of D 9 -THC nd WIN55,212-2, yielded convincing evidence for CB 1 receptormedited ctions (Lichtmn nd Mrtin, 1996; Hmpson nd Dedwyler, 1998, 1999, 2; Mishim et l., 21; Vrvel et l., 21; D Silv nd Tkhshi, 22). We expected similr result in our protocol, ut were unle to reverse deficits with either AM281 or SR141716A. This not only pertined to the sptil prmeters recorded during sptil lerning ut lso to procedurl memory nd motor ctivity. Rther, HU21-induced thigmotxis reflecting heightened nxiety ws excerted y the ntgonists (exp. 2), ut ws reversed y pretrining in the DMTP tsk (exp.1). Anxiety nd procedurl memory-relted deficits thus seem not to e medited vi CB 1 receptors, lthough the doses of AM281 or SR141716A used here were effective in work reported y others (Hmpson nd Dedwyler, 1999; Cosenz et l., 2; Vrvel et l., 21). Together with the excertion in thigmotxis, this strongly implies tht doses of AM281 nd SR141716A were effective. A more specultive explntion my even ssume tht while HU21 my hve cted on non-cb 1 receptors (for review see Howlett et l., 22; Wilson nd Nicoll, 22) or vi non-receptor mechnism, oth ntgonists my hve cted s inverse gonists (Lndsmn et l., 1997; Cosenz et l., 2), therey enhncing some ut reversing the other ehviourl deficits induced y HU21. HU21 cts vi hippocmpl mechnisms Electrophysiologicl recordings from hippocmpl principl cells hve reveled tht cnninoids ffect tsk-relted firing (Hmpson nd Dedwyler, 2; Hmpson et l., 23). In rts trined to perform DNMS tsk, hippocmpl CA3 nd CA1 neurons increse firing in response to one or more ehviourlly relevnt events, such s Smple (encoding) or Non-mtch (recll) lever press responses. WIN nd D 9 -THC dministered efore strting the DNMS tsk suppress firing during the Smple phse ut not the Nonmtch phse. The Smple phse neurl ctivity ws shown to e necessry for correct performnce of the DNMS tsk. Here, we recorded seline firing of CA3/CA1 neurons to determine the effects of HU21, given tht hippocmpl neurl ctivity is essentil for performnce in the wter mze (Hollup et l., 21). Figures 6 nd 7 revel tht HU21 suppresses spontneous firing rtes of CA3 nd CA1 of principl cells, consistent with our previous oservtions of cnninoid effects in freely moving nimls (Hmpson et l., 23). In ddition, the ltertion of spike trin urst firing chrcteristics (Figure 7) re likewise in greement with the reduction of pek firing rtes in the DNMS tsk (Hmpson nd Dedwyler, 2). Cnninoid-induced reductions in seline spike firing hve lso een oserved y others (Pistis et l., 24) in BLA. Exposure to HU21 did not result in complete silencing of hippocmpl neurons; rther, frequency nd durtion of urst firing, s well s firing within ursts, were reduced y pproximtely 5%. Since hippocmpl cells hve previously een shown to encode informtion pertinent to memory formtion y rief episodes (.5 s) of incresed firing rte, the overll diminution of urst firing y HU21 would likewise British Journl of Phrmcology (27)

12 HU21, sptil lerning nd hippocmpl single units L Roinson et l 699 prevent such episode in the wter mze. Thus, reduced seline ctivity nd ttenuted urst firing would lock neurl encoding episodes nd therey ccount for the filure of HU21-treted nimls to cquire sptil elements relevnt to lerning the pltform loction in the wter mze. Conclusions In summry, our results corroorte previous work suggesting role of cnninoids in lerning nd memory. In the sptil reference memory tsk conducted in the open-field wter mze, HU21 impired memory formtion, ut this concerned oth sptil nd non-sptil elements of the tsk. While exposure to the wter mze progressively reduced non-sptil prmeters, sptil deficit still persisted, even in the presence of cnninoid ntgonists. This sptil impirment my e due to HU21 cusing reduction of hippocmpl neurl excittion. Acknowledgements Supported y Component grnt from the MRC to GR nd RGP, nd NIH grnt DA8549 to REH. Conflict of interest The uthors stte no conflict of interest. References Ashton CH 21. Phrmcology, effects of cnnis: rief review. Br J Psychitry 178: Bnnermn DM, Good MA, Butcher SP, Rmsy M, Morris RGM Distinct components of sptil lerning reveled y prior trining, NMDA receptor lockde. Nture 378: Cin DP, Sucier D, Boon F Testing hypotheses of sptil lerning: the role of NMDA receptors, NMDA-medited long-term potentition. Behv Brin Res 84: Cin DP, Finlyson C, Boon F, Beiko J 22. Ethnol impirs ehviorl strtegy use in nïve rts ut does not prevent sptil lerning in the wter mze in pre-trined rts. Psychophrmcology 164: 1 9. Cheer JF, Kendll DA, Mrsden CA 2. Cnninoid receptors, rewrd in the rt: conditioned plce preference study. Psychophrmcology 151: Cosenz M, Gifford AN, Gtely SJ, Pytt B, Liu Q, Mkriynnis A et l. 2. Locomotor ctivity, occupncy of rin cnninoid CB 1 receptors y the ntgonist/inverse gonist AM281. Synpse 38: Currn HV, Brignell C, Fletcher S, Middleton P, Henry J 22. Cognitive, sujective dose response effects of cute orl D 9 - tetrhydrocnninol (THC) in infrequent cnnis users. Psychophrmcology 164: D Silv GE, Tkhshi RN 22. SR A prevents Delt(9) tetrhydrocnninol induced sptil lerning deficit in Morristype wter mze in mice. Prog Neuropsychophrmcol Biol Psychitry 26: Dvies SN, Pertwee RG, Riedel G 22. Functions of cnninoid receptors in the hippocmpus. Neurophrmcology 42: Dedwyler SA, Heyser CJ, Hmpson RE Complete dpttion to the memory disruptive effects of delt- 9 -THC following 35 dys of exposure. Neurosci Res Comm 17: Egshir N, Mishim K, Iwski K, Fujiwr M 22. Intrcererl microinjections of D9-tetrhydrocnninol: serch for the impirment of sptil memory in the eight- rm rdil mze in rts. Brin Res 952: Fdd P, Roinson L, Frtt W, Pertwee RG, Riedel G 24. Differentil effects of THC- or CBD-rich cnnis extrcts on working memory in rts. Neurophrmcology 47: Fdd P, Roinson L, Frtt W, Pertwee RG, Riedel G 26. Scopolmine nd MK81-induced working memory deficits in rts re not reversed y CBD-rich cnnis extrcts. Behv Brin Res 168: Ferrri F, Ottni A, Vivoli R, Giulini D Lerning impirment produced in rts y the cnninoid gonist HU21 in wtermze tsk. Phrmcol Biochem Behv 64: Hmpson RE, Dedwyler SA Role of cnninoid receptors in memory storge. Neuroiol Disese 5: Hmpson RE, Dedwyler SA Cnninoids, hippocmpl function nd memory. Life Sci 65: Hmpson RE, Dedwyler SA 2. Cnninoids revel the necessity of hippocmpl neurl encoding for short-term memory in rts. J Neurosci 2: Hmpson RE, Dedwyler SA 23. Temporl firing chrcteristics nd the effects of clofen on the strtegic role of suiculr neurons in short-term memory. Hippocmpus 13: Hmpson RE, Jrrrd LE, Dedwyler SA Effects of iotente hippocmpl nd extrhippocmpl destruction on delyedmtch nd -nonmtch-to-smple ehvior in rts. J Neurosci 19: Hmpson RE, Simerl JD, Kelly EJ, Dedwyler SA 23. Tolernce to the memory disruptive effects of cnninoids involves dpttion y hippocmpl neurons. Hippocmpus 13: Hernndez-Tristn R, Arevlo C, Cnls S, Leret ML 2. The effects of cute tretment with Delt(9)-THC on explortory ehvior nd memory in the rt. J Physiol Biochem 56: Hill MN, Froc DG, Fox CJ, Gorzlk BB, Christie BR 24. Prolonged cnninoid tretment results in sptil working memory deficits nd impired long-term potentition in the CA1 region of the hippocmpus in vivo. Eur J Neurosci 2: Hollup SA, Molden S, Donnett JG, Moser MB, Moser EI 21. Accumultion of hippocmpl plce fields t the gol loction in n nnulr wtermze tsk. J Neurosci 21: Howlett AC, Brth F, Bonner TI, Crl G, Csells P, Devne WA et l. 22. Interntionl Union of Phrmcology. XXVII. Clssifiction of cnninoid receptors. Phrmcol Rev 54: Izquierdo I, Medin JH, Vinn MRM, Izquierdo LA, Brros DM Seprte mechnisms for short nd long-term memory. Behv Brin Res 13: Kesner RP, Dkis M, Bollnd BL Phencyclidine disrupts longut not short- term memory within sptil lerning tsk. Psychophrmcology 111: Lndsmn RS, Burkey TH, Consroe P, Roeske WR, Ymmur HI SR141716A is n inverse gonist t the humn cnninoid CB 1 receptor. Eur J Phrmcol 334: R1 R2. Lichtmn AH, Mrtin BR Delt9-tetrhydrocnninol impirs sptil memory through cnninoid receptor mechnism. Psychophmcology 126: Lichtmn AH, Dimen KR, Mrtin BR Systemic or intrhippocmpl cnninoid dministrtion impirs sptil memory in rts. Psychophrmcology 119: Lindner MD Reliility, distriution, nd vlidity of ge-relted cognitive deficits in the Morris wter mze. Neuroiol Lern Mem 68: Mllet PE, Beninger RJ The cnninoid CB 1 receptor ntgonist SR141716A ttenutes the memory impirment produced y D 9 -tetrhydrocnninol or nndmide. Psychophrmcology 14: Mishim K, Egshir N, Hirosw N, Fujii M, Mtsumoto Y, Iwski K et l. 21. Chrcteristics of lerning nd memory impirment induced y D 9 -tetrhydrocnninol in rts. Jpn J Phrmcol 87: Molin-Holgdo F, González MI, Leret ML Effect of D 9 -tetrhydrocnninol on short term memory in the rt. Physiol Behv 57: British Journl of Phrmcology (27)