Cannabinoid agonist WIN55,212-2 prevents scopolamine-induced impairment of spatial memory in rats

Cannabinoid agonis WIN55,212-2 p e en s scopolamine-induced impai men o spa ial memo y in a s Ma a Mo eno-Rod íguez a,1 , Ike Bengoe xea de Tena a,1 , Jona an Ma ínez-Ga deazabal a , Go ka Pe ei a-Cas elo a , Albe o Llo en e-O eje o a , I ´ an Manuel a,b , Ra ael Rod íguez-Pue as a,b,* a Depa men o Pha macology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain b Neu odegene a i e Diseases, BioBizkaia Heal h Resea ch Ins i u e, Ba akaldo, Spain ARTICLE INFO Keywo ds: Cannabinoid agonis CB 1 ecep o s Cen al choline gic sys em Scopolamine Memo y impai men Au o adiog aphic s udies Sp ague-Dawley a s ABSTRACT The endocannabinoid sys em is in ol ed in di e se p ocesses, like lea ning and memo y, go e ned by choline gic neu o ansmission. Recen esea ch demons a es ha in a a model o demen ia de i ed om basal o eb ain choline gic degene a ion, WIN55,212-2, a po en cannabinoid ecep o agonis , imp o es cogni ion h ough inc eased co ical choline le els. Howe e , he e ec o cannabinoids on choline gic de ici s is s ill unde in es iga ion. In his wo k, we s udied he e ec o his ea men in a pha macological a model o ansien choline gic hypo unc ion by he acu e adminis a ion o he musca inic an agonis , scopolamine (2 mg/kg), in spa ial, ecogni ion and a e si e memo y es s. Scopolamine induced memo y impai men was obse ed in he h ee es s and, impo an ly, he cannabinoid subch onic ea men wi h low doses o WIN55,212-2 (0.5 mg/kg) p e en ed his dele e ious e ec in spa ial memo y when e alua ed in Ba nes maze es . Au o adiog aphic s udies indica e ha , ollowing he WIN55,212-2 ea men , cannabinoid ecep o densi y inc eased in he mo o and soma osenso y co ices. In laye s I-V o he mo o co ex, he ac i i y o cannabinoid and musca inic e- cep o s also inc eased. These esul s sugges ha WIN55,212-2, h ough he ac i a ion o cannabinoid ecep o s, indi ec ly ele a es he musca inic one in key co ical a eas o lea ning and memo y, p e en ing he memo y de ici s induced by scopolamine speci ically in spa ial memo y. This highligh s he impo ance o he c oss alk be ween he endocannabinoid and he choline gic sys em o lea ning and memo y p ocesses and sugges ha cannabinoid agonis s migh be an al e na i e o he ea men o cogni i e de ici s associa ed wi h choline gic dys unc ion. 1. In oduc ion The endocannabinoid sys em (eCB) egula es ele an physiological p ocesses including pain, appe i e, immune esponses and cogni i e p ocesses (Lu and Mackie, 2021). These unc ions a e p edominan ly egula ed by CB 1 ecep o s, which a e among he mos exp essed and ac i e G p o ein-coupled ecep o s (GPCRs) in he cen al ne ous sys- em (CNS) (Ma inez Rami ez e al., 2023). Nume ous s udies indica e ha cannabinoids nega i ely a ec lea ning and memo y in physiologically no mal s a es (U i s e al., 2021) e.g., Δ-9- e ahyd ocannabinol (Δ 9 -THC) consump ion has been linked o impai men s in a ious b ain unc ions, including dis up ions in a en ion and cogni i e ask pe o mance (S ella, 2023). Howe e , g owing e idence sugges s ha he e ec s o cannabinoids a e biphasic and dose-dependen , and a e in luenced by he p eexis ing cogni i e s a us. In ac , lowe doses o cannabinoids migh be bene icial in some con ex s, including aging, Alzheime ’s disease (AD) and o he demen ia- ela ed neu odegene a i e condi ions (Bilkei-Go zo e al., 2017; Ozai a and Aso, 2017; Llo en e-O eje o e al., 2018; o e iew see Pe ei a-Cas elo e al., 2024). Some o hese e ec s esul om in e ac ions be ween he eCB sys- em and o he neu o ansmission sys ems in he CNS, such as he choline gic sys em, which is essen ial o memo y o ma ion (Gedankien e al., 2023). P esynap ic CB 1 ecep o s a egula e he exp ession o a e si e memo ies h ough he speci ic con ol o choline gic neu o- ansmission (So ia-G´ omez e al., 2015). In a spa ial memo y ask, he * Co esponding au ho . Depa men o Pha macology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Leioa, Spain. E-mail add ess: [email p o ec ed] (R. Rod íguez-Pue as). 1 These au ho s con ibu ed equally o his wo k and sha e i s au ho ship. Con en s lis s a ailable a ScienceDi ec Eu opean Jou nal o Pha macology jou nal homepage: www.else ie .com/loca e/ejpha h ps://doi.o g/10.1016/j.ejpha .2025.177612 Recei ed 24 Ma ch 2025; Accep ed 8 Ap il 2025 Eu opean Jou nal o Pha macology 998 (2025) 177612 A ailable online 17 Ap il 2025 0014-2999/© 2025 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC license ( h p://c ea i ecommons.o g/licenses/by- nc/4.0/ ). cogni i e impai men p oduced by a ea men wi h he cannabinoid ecep o s agonis WIN55,212-2 was media ed by choline gic hypo- unc ion (Robinson e al., 2010) and, no ably, bo h CB 1 and M 2 ecep o s a e p e e en ially p esynap ic and coupled o inhibi o y G p o eins (Nyí i e al., 2005). Va ious s udies pe o med using he musca inic an agonis scopol- amine u he suppo he in e ac ion be ween he choline gic and he eCB sys ems, al hough da a conce ning he impac o cannabinoid com- pounds on scopolamine-induced e ec s is inconclusi e. In ac , s udies pe o med wi h in e se agonis s o CB 1 ecep o s epo bo h a po en i- a ion o he dis up i e e ec s o scopolamine (Nakamu a-Palacios e al., 2000), o imp o ed cogni i e pe o mance ollowing adminis a ion (Dillon e al., 2011). In a passi e a oidance ask, bila e al mic oinjec ion a he basola e al amygdala (BLA) o a achydonilcyclop opylamide (ACPA), a CB 1 ecep o agonis , imp o ed scopolamine-induced memo y impai men , while co-adminis a ion o ine ec i e scopolamine doses wi h AM251, a cannabinoid CB 1 ecep o an agonis , mimicked he amnesic e ec ob ained wi h highe scopolamine doses (Nedaei e al., 2016). A p e ious s udy om ou g oup in es iga ed he e ec o a sub- ch onic WIN55,212-2 (0.5 mg/kg) ea men on lea ning and memo y in a model o basal o eb ain choline gic hypo unc ion, and he obse ed cogni i e imp o emen was media ed by inc eased le els o ace ylcho- line (ACh) oge he wi h he es o a ion o choline-con aining co ical lipids (Mo eno-Rod íguez e al., 2025). On his basis, we hypo hesized ha WIN55,212-2 could also e e memo y de ici s de i ing om he acu e inhibi ion o he musca inic sys em in he same con ex . Hence, he p esen s udy e alua ed, in i o, he e ec o he same cannabinoid ea men in p e en ing he ansien amnesia induced by acu e scopolamine adminis a ion in pa adigms ela ed o spa ial, ecogni ion and a e si e memo y, and analyzed he neu ochemical co ela es behind he obse ed beha io s using a pha macological app oach by au o adiog aphic assays. 2. Ma e ial and me hods 2.1. Reagen s and d ugs All he compounds necessa y o he di e en p ocedu es we e o he highes quali y comme cially a ailable o he pu pose o ou s udies. [ 3 H]CP55,940 (149 Ci/mmol) and [ 35 S]GTPγS (1250 Ci/mmol) we e acqui ed om Re i y (Wal ham, MA, USA). The [ 3 H] mic oscales and [ 14 C] mic oscales used as s anda ds in he au o adiog aphic expe imen s we e pu chased om ARC (Ame ican Radiolabeled Chemicals, Sain Louis, MO, USA). The β- adia ion sensi i e ilms, Kodak Biomax MR, bo ine se um albumin (BSA), DL-di hio h ei ol (DTT), guanosine 5 ′ - diphospha e (GDP), guanosine 5 ′ -O-3- hio iphospha e (GTPγS), ke a- mine and xylazine we e acqui ed om Sigma-Ald ich (S Louis, MO, USA). 2-ca bamoyloxye hyl- ime hyl-azanium (Ca bachol) and ( α ,S)- α -(Hyd oxyme hyl)benzeneace ic acid (1 α ,2β,4β,5 α ,7β)-9-me hyl-3-oxa- 9-aza icyclo[3.3.1.02,4]non-7-yl es e hyd ob omide (Scopolamine) we e acqui ed om Sigma-Ald ich (S Louis, MO, USA). (11R)-2-Me hyl- 11-[(mo pholin-4-yl)me hyl]-3-(naph halene-1-ca bonyl)-9-oxa-1 aza icyclo[6.3.1.04,12]dodeca-2,4(12),5,7- e aene (WIN55,212-2) was acqui ed om Toc is (B is ol, UK). 2.2. Animals E e y e o was made o minimize animal su e ing and o use he minimum numbe o animals possible h oughou he whole s udy. All p ocedu es we e pe o med in acco dance wi h Eu opean animal esea ch laws (Di ec i e, 2010/63/EU) and he Spanish Na ional p o- ocols and we e app o ed by he Local E hical Commi ee o Animal Resea ch o he Uni e si y o he Basque Coun y (CEEA M20-2018-52 and 54 and M20/2023/352). Male Sp ague-Dawley a s (n =90) weighing 200–300 g we e housed in g oups o 3–4 pe cage a a empe a u e o 22 ◦C and in a humidi y- con olled (65 %) oom wi h a 12:12 h ligh /da k cycle, wi h access o ood and wa e ad libi um. These a s we e adminis e ed wi h scopol- amine/ ehicle and WIN55,212-2/ ehicle and we e used o he pe - o mance o he di e en beha io al es s. A ep esen a i e sample o he b ains om all g oups o a s which pe o med he beha io al es s we e also used o au o adiog aphic s udies (n =35). A e he pe o - mance o he beha io al es s and be o e he au o adiog aphic s udies, a s we e anes he ized (i.p. ke amine/xylazine 90/10 mg/kg), sac i iced by decapi a ion and he b ains we e ca e ully emo ed om he skull be o e being ozen and s o ed a −80 ◦C, un il use. 2.3. Beha io al es s 2.3.1. Ba nes maze es The es was conduc ed on a whi e ci cula pla o m (130 cm diame e , 1 m abo e he loo ) wi h 20 equally spaced holes. Only one hole led o a da k escape chambe benea h he pla o m. B igh ligh s placed a ound he pla o m c ea ed an unpleasan a mosphe e, encou aging a s o ind he a ge hole. Visual cues on he walls helped wi h spa ial o ien a ion. The es had wo phases o e i e days: acquisi ion (4 days) and p obe ial (day 5). Du ing acquisi ion, a s we e placed on he pla o m, gi en 3 min o ind he a ge hole, and we e guided o i i hey ailed. Fou ials we e conduc ed daily wi h 15 min o in e - ial in e als. On he i h day, he day a e he las acquisi ion ial, a single 180 s p obe ial was pe o med, whe e memo y e en ion was es ed wi hou he escape box p esen . In his phase, he pla o m was i ually di ided in o ou quad an s and he ime spen by he a s in he a ge quad an , whe e he escape box was p e iously loca ed, was measu ed as indica- i e o spa ial memo y. A acking sys em (SMART, Panlab S.L., Ba ce- lona, Spain) was used o eco d la ency, pa h leng h, and speed du ing he acquisi ion phase, while quad an ime was used o assess spa ial memo y du ing he p obe ial. 2.3.2. No el objec ecogni ion es The es was conduc ed in a whi e open- ield a ena (90 ×90 ×50 cm) (Panlab S.L., Ba celona, Spain). I included ou phases o e i e days: habi ua ion (3 days), amilia iza ion, sho - e m es ing (5 h la e ), and long- e m es ing (24 h la e ). Be o e each phase, a s we e handled gen ly o 1 min. Du ing habi ua ion phase, a s explo ed he a ena o 5 min. In he amilia - iza ion phase, wo iden ical objec s (objec A) we e placed in opposi e co ne s o he a ena. Ra s explo ed hem un il a combined 25-s explo- a ion h eshold was eached. In sho - e m es ing, a s we e p esen ed wi h he amilia objec (A) and a new objec (B) o 5 min. Long- e m es ing, conduc ed 24 h a e he amilia iza ion phase, in ol ed he amilia objec (A) and ano he new objec (C). A ideo came a eco ded a beha io , and explo a ion imes o bo h amilia and no el objec s we e measu ed as indica i e o ecogni ion memo y. The objec disc imina ion a io (DR) was calcula ed, wi h sco es nea ze o indi- ca ing no p e e ence, nega i e sco es indica ing a p e e ence o he amilia objec , and posi i e sco es indica ing p e e ence o he no el objec . 2.3.3. Passi e a oidance es The beha io al es was conduc ed in a shu le box (PanLab S.L. Ba celona, Spain) wi h wo compa men s: a la ge , b igh ly li , whi e compa men (31 ×31 ×24 cm) and a smalle , da k, black compa men (19.5 ×10.8 ×12 cm), sepa a ed by a guillo ine doo . The es had wo phases: acquisi ion and e en ion. Be o e each phase, a s we e habi ua ed o he expe imen al oom. In he acquisi ion phase (day 1), a s explo ed he whi e compa men o 30 s be o e he doo o he black compa men opened. Upon ins inc i ely c ossing in o he black compa men , he doo closed, and a mild oo shock (0.4 mA M. Mo eno-Rod íguez e al. Eu opean Jou nal o Pha macology 998 (2025) 177612 2 o 2 s) was adminis e ed. Ra s emained in he black compa men o 15 s be o e being e u ned o hei cage. The shu le box was sani ized wi h e hanol (70 %) be ween ials. Ra s ha did no c oss in o he black compa men in his phase we e excluded om he es . In he e en ion phase (day 2), a s we e again placed in he whi e compa men , and a e 30 s, he doo o he black compa men opened. Ra s had 5 min o decide whe he o c oss; hose ha c ossed ecei ed no shock. The beha io o he a s was eco ded wi h a ideo came a posi ioned abo e he shu le box, wi h acquisi ion la ency (day 1) and s ep- h ough la- ency (day 2) measu ed. Longe la encies in he e en ion phase indi- ca ed a passi e a oidance esponse, e lec ing posi i e pe o mance. 2.3.4. Ho pla e es The es was pe o med in a ho pla e appa a us (Leica Biosys ems, Ba celona, Spain) consis ing in a cylind ical see- h ough Plexiglas wall (19 ×30 cm) loca ed abo e a pla e. The pla e wa ms h ough an elec ic esis ance and is equipped wi h a ime and a he mos a . On he op o he cylinde , a me al g id is loca ed o hold he a when i jumps. Ra s we e placed on he me al pla e, which was p e iously wa med (55 ±0.5 ◦C). The ime spen by he a s un il jumping was eco ded (jump la ency). The la ency o paw-licking was also measu ed. Bo h pa ame e s, ollowing a noxious he mal s imulus, we e used as indic- a i e o nocicep i e h eshold. 2.3.5. Elec ical shock e oked pain h eshold The p ocedu e was pe o med in he same shu le box used o he PA es . This appa a us has a g id loo and elec ical oo shocks o di e en po ency can be deli e ed. Ra s we e placed in he whi e compa men and ecei ed mild oo shocks, beginning a 0.0 mA, g adually inc easing by 0.05 mA, un il he i s ocaliza ion was measu ed, which is an indica i e o nocicep ion and discom o . 2.4. WIN55,212-2 adminis a ion in a pha macological model o musca inic an agonism Scopolamine was dissol ed in saline 0.9 % and was adminis e ed in ape i oneally (2 mg/kg), 30 min be o e he pe o mance o he PA es acquisi ion phase, he NORT es sho and long- e m es ing phases and he BM p obe ial phase. Con ol g oup ecei ed ehicle wi hou scopolamine. Fo he e alua ion o he e ec o a cannabinoid ea men in he amnesic e ec s elici ed by scopolamine, WIN55,212-2 was in ape i o- neally adminis e ed once daily (0.5 mg/kg), 1 h be o e e e y phase o each es . WIN55,212-2 was dissol ed in pu e DMSO and dilu ed wi h kollipho and 0.9 % saline, in a 1:1:18 p opo ion. Con ol g oup ecei ed ehicle wi hou WIN55,212-2. Ra s we e andomly di ided in o ou g oups o each es : ehicle (VEH), WIN55,212-2 (WIN), scopolamine (SCOP) and WIN55,212-2 +scopolamine (WIN +SCOP). 2.5. Au o adiog aphic s udies 2.5.1. Func ional [ 35 S]GTPγS au o adiog aphy Fo he pe o mance o unc ional au o adiog aphy o s udy musca- inic and cannabinoid ecep o s ac i i y, b ain sec ions we e ai d ied o 30 min, ollowed by wo consecu i e incuba ions o 30 min each in an HEPES-based bu e (50 mM HEPES, 100 mM NaCl, 3 mM MgCl 2 , 0.2 mM EGTA and 0.5 % BSA, pH 7.4) a 30 ◦C o emo e he endogenous ligands. Slices we e hen incuba ed o 2 h a 30 ◦C in he same bu e supplemen ed wi h 2 mM GDP, 1 mM DTT and 0.04 nM [ 35 S]GTPγS. Basal binding was de e mined in wo consecu i e slices in he absence o he agonis . The agonis -s imula ed binding was de e mined in ano he consecu i e slice in he p esence o he co esponding ecep o agonis s, ca bachol (10 μ M) o musca inic ecep o s and WIN55,212-2 (10 μ M) o cannabinoid ecep o s. Non-speci ic binding was de ined by compe i ion wi h unlabeled GTPγS (10 μ M) in ano he sec ion. Tissue slices we e inally washed wice in cold (4 ◦C) HEPES (50 mM) bu e (pH 7.4), d ied and exposed o 48 h o β- adia ion sensi i e ilms wi h a se o [ 14 C] s anda ds calib a ed o [ 35 S]. Calib a ed ilms we e scanned and quan i ied using Fiji so wa e. Da a was exp essed as he % o s imula ion o e basal. 2.5.2. [ 3 H]CP55,940 ecep o au o adiog aphy Fo he pe o mance o cannabinoid ecep o s au o adiog aphy, b ain sec ions we e ai d ied o 30 min and hen imme sed in Coplin ja s o p eincuba ion in a bu e con aining 50 mM T is-HCl and 1 % o BSA (pH 7.4) o 30 min, a oom empe a u e, o emo e endogenous ligands. Tissue slices we e la e incuba ed in he p esence o he [ 3 H] CP55,940 adioligand (3 nM) o 2 h a 37 ◦C. Non-speci ic binding was measu ed by compe i ion wi h non-labeled CP55,940 (10 μ M) in ano he consecu i e slice. Following he incuba ion, issue slices we e washed wi h an ice-cold p eincuba ion bu e , dipped in dis illed wa e and d ied o e nigh . To gene a e au o adiog ams, d y sec ions we e exposed o β- adia ion-sensi i e ilms in he me ically closed casse es o 21 days a 4 ◦C. Fo he calib a ion o he op ical densi ies o mol/mg issue equi alen , [ 3 H] mic oscales we e exposed o he ilms. Calib a ed ilms we e scanned and quan i ied using Fiji so wa e (Fiji, Be hesda, MD, USA). Da a was exp essed as mol/g o issue equi alen ( .e.). 2.6. S a is ical analysis Da a om beha iou al es s was analyzed using a Mann-Whi ney es o wo g oups and K uskal-Wallis es ollowed by Dunn’s pos hoc es o mo e han wo g oups. Repea ed measu es wo-way ANOVA ol- lowed by pos -hoc es Bon e oni’s was used o epea ed measu es o he same pa ame e in di e en days ( o al la ency, o al pa h leng h and speed in BM es ). S ep- h ough la ency imes o PA es we e ep e- sen ed as Kaplan-Meie su i al cu es and analyzed using a log- ank/ Man el–Cox es , as p e iously desc ibed by ou g oup (Llo en e-O eje o e al., 2017; Bengoe xea de Tena e al., 2022). Da a om au o adio- g aphic assays was analyzed using a Mann-Whi ney es . The h eshold o s a is ical signi icance was se a p <0.05. Besides ha , d , F and H alues a e p o ided in he igu e legends, whe e necessa y. S a is ical analyses and da a ep esen a ion we e pe o med using G aphPad P ism 9 (G aphPad So wa e, Bos on, MA, USA). 3. Resul s 3.1. WIN55,212-2 p e en s scopolamine-induced amnesia in a spa ial memo y es BM es was pe o med o e alua e he e ec o WIN55,212-2 in p e en ing scopolamine-induced impai men o spa ial memo y. Th oughou he ou days o acquisi ion, bo h WIN55,212-2 and ehicle- ea ed g oups dec eased he o al la ency and he o al pa h leng h (Fig. 1A and B). On he i s ial, his pa ame e was signi ican ly highe in he WIN55,212-2- ea ed g oup (VEH s. WIN, p <0.05; Fig. 1A), indica ing a sligh delay in he lea ning cu e. The a e age speed inc eased in each ial o bo h g oups, bu WIN55,212-2- ea ed a s walked mo e slowly han ehicle- ea ed ones o e all (VEH s. WIN, p < 0.05; Fig. 1C). On p obe ial, a e he las aining day, he la ency in he a ge quad an was measu ed as an indica o o spa ial memo y in all g oups. The adminis a ion o WIN55,212-2 o con ol a s did no p oduce an impai men in spa ial memo y (VEH s. WIN, p >0.05; Fig. 2A). Scopolamine- ea ed a s spen a signi ican ly lowe ime in he a ge quad an (VEH s. SCOP, p <0.001; Fig. 2A), indica ing spa ial memo y de ici s ollowing i s adminis a ion. Impo an ly, a subch onic ea - men wi h WIN55,212-2 p e en ed he amnesic e ec exe ed by scopolamine in BM es (SCOP s. SCOP +WIN, p <0.01; Fig. 2A). Conside ing he posi i e e ec s o a cannabinoid ea men in BM, we analyzed i s e ec on p e en ing he amnesic e ec s induced by M. Mo eno-Rod íguez e al. Eu opean Jou nal o Pha macology 998 (2025) 177612 3 scopolamine in o he memo y es s, including he no el objec ecog- ni ion es (NORT) and passi e a oidance (PA) es . 3.2. E ec o a cannabinoid ea men on a pha macological model o musca inic an agonism in ecogni ion memo y Recogni ion memo y was e alua ed a wo di e en ime poin s, sho (5 h pos - amilia iza ion) and long- e m (24 h pos - amilia iza ion). In he sho - e m es , he ea men wi h WIN55,212- 2 impai ed memo y in con ol a s (VEH s. WIN, p <0.001; Fig. 3A). No ably, he adminis a ion o scopolamine did no signi ican ly dec ease he DR (VEH s. SCOP, p >0.05; Fig. 3A). In he long- e m, he subch onic ea men wi h WIN55,212-2 also showed a dele e ious e ec on memo y in con ol a s (VEH s. WIN, p < 0.05; Fig. 3B). In con as o wha was obse ed in he sho - e m es , he adminis a ion o scopolamine also caused a signi ican dec ease in he DR (VEH s. SCOP, p <0.01; Fig. 3B), indica ing memo y impai - men ollowing he adminis a ion o a musca inic an agonis in long- Fig. 1. E ec o WIN55,212-2 on locomo ion and lea ning in BM es . (A) To al la ency du ing he ou days o lea ning o BM es . No signi ican di e ences we e obse ed be ween VEH and WIN g oups compa ing bo h cu es, bu signi ican di e ences we e obse ed be ween he wo g oups in he i s ial (Repea ed measu es wo-way ANOVA, pos -hoc es Bon e oni’s, d =1, F =4.51, #p <0.05, VEH s. WIN). (B) To al pa h leng h du ing he ou days o lea ning o BM es . No signi ican di e ences we e obse ed be ween VEH and WIN g oups compa ing bo h cu es (Repea ed measu es wo-way ANOVA, pos -hoc es Bon e oni’s, d =1, F =1.15). (C) Mean speed du ing he ou days o lea ning o BM es . Signi ican di e ences we e obse ed be ween VEH and WIN g oups compa ing bo h cu es (Repea ed measu es wo-way ANOVA, pos -hoc es Bon e oni’s, d =1, F =7.24, #p <0.05, VEH s. WIN). Fig. 2. WIN55,212-2 p e en s he scopolamine-induced impai men o spa ial memo y. (A) Time spen in he a ge quad an on he p obe ial day o BM es (K uskal–Wallis’s es , pos -hoc es Dunn’s mul iple compa ison, d =3, H =19.36, ***p <0.001 VEH s. SCOP, $$ p <0.01 SCOP s. SCOP +WIN). (B) Rep esen a i e ajec o ies o a s om each g oup du ing 180 s in he p obe ial day o BM es . No e he accumula ion o ajec o ies in he a ge quad an (whe e he a ge hole is loca ed, depic ed in black in he igu e) o VEH, WIN and SCOP +WIN g oups, as opposed o SCOP g oup. M. Mo eno-Rod íguez e al. Eu opean Jou nal o Pha macology 998 (2025) 177612 4 e m ecogni ion and wo king memo y, bu no in sho - e m one. The adminis a ion o WIN55,212-2 o a s ha also ecei ed scopolamine only p oduced a sligh inc ease in he DR and did no p e en o e e se he obse ed memo y de ici s (SCOP s. SCOP +WIN, p >0.05; Fig. 3B). 3.3. E ec o a cannabinoid ea men on a pha macological model o musca inic an agonism in an a e si e memo y es The adminis a ion o scopolamine impai ed bo h acquisi ion la ency (VEH s. SCOP, p <0.01, Fig. 4A) and s ep- h ough la ency ime (VEH s. SCOP, p <0.001, Fig. 4B), p o oking ansien memo y de ici s. The cannabinoid ea men wi h WIN55,212-2 alone did no a ec acquisi- ion la ency, bu dec eased he s ep- h ough la ency ime (VEH s. WIN, p <0.01, Fig. 4B). The subch onic adminis a ion o WIN55,212-2 did no p e en he amnesic e ec o scopolamine obse ed in he s ep- h ough la ency ime (SCOP s. SCOP +WIN, p >0.05, Fig. 4B). Conside ing he analgesic e ec s o cannabinoid agonis s (Manzana es e al., 2006), we hypo hesized ha dec eased pain sensi- i i y ollowing he WIN55,212-2 adminis a ion could be a bias in he e ec o he ea men obse ed in PA es . Thus, we measu ed he e ec o his ea men on pain esponse using he ho pla e es . We obse ed ha WIN55,212-2 ea men p oduced analgesia, as indica ed by he inc eases in he jump la ency (VEH s. WIN, p <0.05, Fig. 5A) and he la ency o licked paw (VEH s. WIN, p <0.01, Fig. 5B). We also measu ed pain esponse using a p o ocol designed o e alua e noci- cep ion p oduced by an elec ical shock in PA es , he elec ical shock e oked pain h eshold, and we obse ed ha he g oup o a s ea ed wi h WIN55,212-2 equi ed a highe shock in ensi y o e oke a i s ocaliza ion (VEH 0.0375 ±0.0082 mA s. WIN 0.0714 ±0.0101 mA, p <0.01), also indica ing an analgesic e ec o he cannabinoid ea men . These esul s indica e a possible bias o he esul s ob ained in PA es ollowing he WIN55,212-2 adminis a ion. 3.4. The subch onic WIN55,212-2 adminis a ion al e ed musca inic and cannabinoid ecep o ac i i y The e ec o WIN55,212-2 adminis a ion on musca inic and cannabinoid ecep o s was s udied o elucida e he neu ochemical co - ela es behind he obse ed beha io s. The densi y and ac i i y o musca inic and cannabinoid ecep o s was analyzed by means o au o- adiog aphy in a s ha had pe o med BM es , gi en he posi i e e ec exe ed by he ea men in his es . Conside ing ha scopolamine was Fig. 3. E ec o WIN55,212-2 in a model o musca inic an agonism in ecogni ion memo y in he sho - e m and he long- e m. (A) Disc imina ion a io in he sho - e m es ing phase o NORT (K uskal–Wallis’s es , pos -hoc es Dunn’s mul iple compa ison, d =3, H =7.29, ###p <0.001 VEH s. WIN). (B) Disc imina ion a io in he long- e m es ing phase o NORT (K uskal–Wallis’s es , pos -hoc es Dunn’s mul iple compa ison, d =3, H =16.56, **p <0.01 VEH s. SCOP, #p <0.05 VEH s. WIN). Fig. 4. T ea men wi h WIN55,212-2 in a model o musca inic an agonism e alua ed in PA es . (A) Acquisi ion la ency imes in he lea ning ial o he PA es (K uskal–Wallis’s es , pos -hoc es Dunn’s mul iple compa ison, d =3, H =7.47, **p <0.01, VEH s. SCOP). (B) S ep- h ough la ency imes o PA es ep esen ed as Kaplan-Meie su i al cu es (log- ank/Man el–Cox es , ***p <0.001 VEH s. SCOP, ##p <0.01 VEH s. WIN, $$$ p <0.001 VEH s. SCOP +WIN). The median la ency was 24.55 s o SCOP g oup, 256.00 s o WIN g oup and 48.00 s o SCOP +WIN g oup. M. Mo eno-Rod íguez e al. Eu opean Jou nal o Pha macology 998 (2025) 177612 5 adminis e ed acu ely and gi en i s as exc e ion and low bioa ailabili y (Tian e al., 2015), he au o adiog aphy da a only ep esen s he e ec o he WIN55,212-2 ea men . The [ 35 S]GTPγS binding s imula ed by ca bachol, an agonis o musca inic ecep o s, was measu ed in b ain a eas ela ed o lea ning and memo y o localize and de e mine he ac i i y o G i/o p o eins coupled o M 2 /M 4 ecep o s (Fig. 6). No di e ences we e obse ed be- ween he g oups ea ed wi h ehicle and WIN55,212-2 in basal bind- ing, i.e, [ 35 S]GTPγS binding in he absence o he agonis . The G i/o - coupled M 2 /M 4 ecep o ac i i y induced by ca bachol was inc eased in he WIN55,212-2- ea ed g oup in laye s I-V o he mo o co ex (VEH s. WIN, p <0.05; Fig. 6, Table 1), in some o he sep al nuclei, he medial sep um and he ho izon al diagonal band (VEH s. WIN, p < 0.05; Fig. 6, Table 1), and in he py amidal laye o he CA3 egion o he hippocampus (VEH s. WIN, p <0.05; Fig. 6, Table 1). The [ 35 S]GTPγS binding s imula ed by WIN55,212-2 was measu ed in b ain a eas ela ed o lea ning and memo y o localize and de e mine he ac i i y o cannabinoid ecep o s (Fig. 7). The ac i i y media ed by G i/o -coupled cannabinoid ecep o s was inc eased ollowing he WIN55,212-2 ea men in se e al a eas ela ed o lea ning and memo y p ocesses, such as laye s I-V o he mo o co ex (VEH s. WIN, p <0.05; Fig. 7, Table 1), he s ia um (VEH s. WIN, p <0.05; Fig. 7, Table 1), he e ical diagonal band (VEH s. WIN, p <0.05; Fig. 7, Table 1), he adia um and o iens laye s o he hippocampus CA1 egion (VEH s. WIN, p <0.05; Fig. 7, Table 1), he hippocampus CA3 egion (VEH s. WIN, p <0.05; Fig. 7, Table 1), he adia um laye o he hippocampus CA3 egion (VEH s. WIN, p <0.05; Fig. 7, Table 1) and he den a e gy us (VEH s. WIN, p <0.05; Fig. 7, Table 1). The densi y o cannabinoid ecep o s was also s udied in he same b ain a eas by [ 3 H]CP55,940 binding (Fig. 8). The ea men wi h he cannabinoid agonis WIN55,212-2 inc eased cannabinoid ecep o s densi y in laye VI o he mo o co ex (VEH s. WIN, p <0.05; Fig. 8, Table 2) and in laye VI o he soma osenso y co ex (VEH s. WIN, p < 0.05; Fig. 8, Table 2). In con as , cannabinoid densi y dec eased in he den a e gy us (VEH s. WIN, p <0.05; Fig. 8, Table 2) and in he py- amidal laye o he hippocampus CA1 egion (VEH s. WIN, p <0.05; Fig. 8, Table 2). 4. Discussion Conside ing he egula o y ole o he eCB sys em o e lea ning and memo y asks con olled by choline gic neu o ansmission, we ha e e alua ed he e ec o a subch onic ea men wi h po en cannabinoid ecep o agonis WIN55,212-2 in p e en ing ansi o y amnesia caused by scopolamine. As expec ed, scopolamine impai ed memo y in h ee lea ning and memo y beha io es s compa ed o a saline g oup (Malikowska-Racia e al., 2018). The acu e pha macological model o choline gic blockade wi h scopolamine is well-cha ac e ized and used in he s udy o nume ous an i-amnesic d ugs (Akinyemi e al., 2017; El-Khad agy e al., 2014; Ma isco e al., 2013). Ou esul s a e consis en wi h da a epo ed in he li e a u e; howe e , we obse ed ha p e- ea men wi h scopolamine did no signi ican ly dis up sho - e m ecogni ion memo y in NORT. This obse a ion con as s wi h o he s udies indica ing ha musca inic an agonis s impai he acquisi ion and pe o mance o a ious lea ned beha io s, including sho - e m memo y (Balde as e al., 2012; Fibige e al., 1991; Palme e al., 2016). Howe e , ou own p e ious esul s showed sho - e m memo y was no a ec ed in NORT a e a basal choline gic lesion, when baso-co ical choline gic neu o ansmission was diminished (Mo eno-Rod íguez e al., 2025). I is also in line wi h a s udy epo ing ha educed ACh elease delays speci ically he consolida ion o objec ecogni ion memo y (De Jaege e al., 2013). Ou indings sugges ha blocking he choline gic signaling is c ucial o long- e m, bu no sho - e m, ecogni ion memo y. WIN55,212-2 alone induced simila cogni i e impai men in NORT and PA as scopolamine did, bu no in BM. The con adic o y e ec s o a ela i ely low dose o WIN55,212-2 (0.5 mg/kg) obse ed in BM, NORT and PA es s sugges di e en sen- si i i ies o cannabinoid e ec s depending on he ype o memo y assessed, i.e. spa ial memo y, ecogni ion and con ex ual memo y and a e si e o ea - ela ed memo y. P e ious epo s a e somewha con- adic o y, simila doses o WIN55,212-2 acili a ed he ex inc ion o con ex ual ea and spa ial memo y (Pamplona e al., 2006). Howe e , di e en sensi i i ies o he e ec s media ed by cannabinoid ecep o s depending on he ype o memo y ha e been desc ibed (Li and Kim, 2016). Al hough he de imen al e ec o cannabinoid agonism on memo y is well s udied and p o en, he unde lying mechanisms emain unclea . He e we demons a ed ha subch onic adminis a ion o a low dose o WIN55,212-2 (0.5 mg/kg) p e en ed he amnesic e ec s o scopol- amine in spa ial lea ning and memo y in he BM es , bu no in NORT es . Impo an ly, WIN55,212-2 alone, a his dose, did no induce de imen al e ec s on memo y in BM es , bu i did in NORT. In o he s udies, low doses o THC es o ed age- ela ed cogni i e dys unc ion by modula ing hippocampus-dependen memo y p ocesses (Bilkei-Go zo e al., 2017; Sa ne e al., 2018). While da a sugges Fig. 5. Analgesic e ec o WIN55,212-2 in he ho pla e es . (A) Jump la ency in he ho pla e es (Mann Whi ney es , #p <0.05 VEH s. WIN). (B) La ency o licked paw in he ho pla e es (Mann Whi ney es , ##p <0.01 VEH s. WIN). Fig. 6. Rep esen a i e au o adiog ams showing he ac i i y o M 2 /M 4 e- cep o s. Co onal sec ions co esponding o (A) VEH and (B) WIN g oups showing [ 35 S]GTPγS binding s imula ed by ca bachol in he ho izon al diagonal band (HDB), laye s I-V o he mo o co ex (Mo Cx I-V), medial sep um (MS) and he hippocampus (HPC). Scale ba : 4 mm. M. Mo eno-Rod íguez e al. Eu opean Jou nal o Pha macology 998 (2025) 177612 6 c oss-s a e-dependen lea ning be ween WIN55,212-2 and scopolamine (Jamali-Raeu y e al., 2011), he di e en ial sensi i i y o cannabinoids may be elucida ed by explo ing he mechanisms h ough which scopolamine induces memo y loss. Scopolamine is a non-selec i e musca inic ecep o an agonis , which educes ACh binding o he musca inic ecep o s and leads o memo y de ici s (Blokland, 1995). I mainly a ec s choline gic ansmission a he basal o eb ain, mo o co ex, globus pallidus, hippocampus, pe i hinal co ex and amygdala. Howe e , choline ace yl ans e ase (ChAT) le els seem o be a ec ed speci ically in he hippocampus and amygdala (Hescham e al., 2014; Ray e al., 1992). Scopolamine can dec ease co ical ACh le els by 52 % and hippocampal ACh le els by 39 % (Spignoli e al., 1987). Se e al s udies ha e shown ha cholines e ase inhibi o s, which inc ease ACh le els a he synapse (Pa anashe i e al., 2017; Sadek e al., 2016; Shin e al., 2018) o na u al compounds ha also inc ease ACh le els (Laza o a e al., 2024; Te alı e al., 2024), p e en he amnesic e ec s o Table 1 [ 35 S]GTPγS binding o ca bachol-s imula ed and WIN55,212-2-s imula ed ecep o ac i i y exp essed as he pe cen age o s imula ion o e basal, in di e en b ain a eas ela ed o lea ning and memo y. Ca bachol s imula ion (% o e basal) WIN55,212-2 s imula ion (% o e basal) B ain egion VEH WIN VEH WIN Ce eb al co ex             Cingula e 102 ±21 115 ±19 248 ±36 298 ±55 Mo o             Laye I-V 88 ±16 154 ±17* 216 ±41 405 ±72* Laye VI 169 ±21 154 ±34 353 ±51 354 ±39 Soma osenso y             Laye I-V 156 ±28 164 ±53 156 ±47 259 ±41 Laye VI 178 ±21 194 ±38 353 ±66 378 ±96 Basal ganglia             Globus pallidus 76 ±33 116 ±75 874 ±115 890 ±103 S ia um 221 ±22 188 ±26 452 ±57 738 ±143* Diencephalon             NBM 129 ±26 195 ±35 231 ±51 277 ±46 HDB 139 ±23 244 ±37* 230 ±40 257 ±48 VDB 247 ±22 250 ±36 224 ±58 613 ±179* Medial sep um 268 ±27 374 ±43* 206 ±37 356 ±61 Hippocampus             CA1 56 ±17 62 ±17 104 ±27 175 ±22 O iens 51 ±16 64 ±23 17 ±5 62 ±12* Py amidal 65 ±15 88 ±46 230 ±41 312 ±93 Radia um 39 ±7 49 ±8 137 ±23 350 ±87* CA2 50 ±9 47 ±11 199 ±45 234 ±49 CA3 48 ±5 36 ±6 276 ±42 425 ±58* O iens 64 ±23 67 ±18 58 ±19 57 ±12 Py amidal 25 ±19 95 ±29* 305 ±110 323 ±130 Radia um 34 ±9 30 ±8 74 ±21 184 ±44* Den a e gy us 37 ±7 48 ±13 277 ±51 484 ±66* G anula 38 ±10 42 ±17 318 ±101 398 ±104 Molecula 40 ±8 34 ±11 174 ±38 308 ±90 Polymo phic 27 ±10 27 ±6 324 ±45 338 ±76 Amygdala 98 ±18 75 ±31 213 ±59 407 ±87 HDB: ho izon al diagonal band, NBM: nucleus basalis magnocellula is, VDB: e ical diagonal band. Da a a e exp essed as mean ±S.E.M. alues om VEH and WIN g oups. VEH s. WIN (*). Mann-Whi ney es , *p <0.05. Fig. 7. Rep esen a i e au o adiog ams showing he ac i i y o cannabinoid ecep o s e oked by WIN55,212-2. Co onal sec ions co esponding o (A) VEH and (B) WIN g oups showing [ 35 S]GTPγS binding s imula ed by WIN55,212-2 in he e ical diagonal band (VDB), laye s I-V o he mo o co ex (Mo Cx I- V), he s ia um (STR) and he hippocampus (HPC). Scale ba : 4 mm. Fig. 8. Rep esen a i e au o adiog ams showing he densi y o cannabinoid ecep o s. Co onal sec ions co esponding o (A) VEH and (B) WIN g oups showing [ 3 H]CP55,940 binding in laye s I-V o he mo o co ex (Mo Cx I-V) and in laye VI o he soma osenso y co ex. Scale ba : 4 mm. M. Mo eno-Rod íguez e al. Eu opean Jou nal o Pha macology 998 (2025) 177612 7 scopolamine. Howe e , he e ec i eness o memo y imp o emen is dependen on he dose o scopolamine adminis e ed (Beja e al., 1999), sugges ing ha he le els o ACh in he synap ic cle a e impo an o he e ec o scopolamine. In his sense, i is al eady desc ibed ha cannabinoids, including WIN55,212-2, modula e ACh elease in he hippocampus and co ex (Gessa e al., 1997, 1998; Mo eno-Rod íguez e al., 2025; Na a e al., 2001; Tza a a e al., 2003). WIN55,212-2 can also mi iga e he oxici y induced on he cholin- e gic sys em ollowing ace ylcholines e ase inhibi ion (Nallapaneni e al., 2006, 2008). The e o e, we hypo hesise ha he WIN55, 212-2-media ed inc ease in ACh could po en ially compensa e o he acu e educ ion in co ical and hippocampal ACh le els induced by scopolamine (Spignoli e al., 1987), as we ha e p e iously desc ibed in co ical a eas (Mo eno-Rod íguez e al., 2025). In addi ion, WIN55, 212–2 may induce memo y de ici s i sel due o an excessi e inc ease in ACh le els, as ecen e idence sugges s (Huang e al., 2022), among o he mechanisms. Howe e , he mechanism by which he same doses o cannabinoid agonis s p o ec agains scopolamine-induced cogni i e impai men in he BM while showing no e ec on NORT emains un- clea . While BM and NORT a e bo h hippocampus-dependen es s, BM may equi e highe modula ion o ACh o acili a e p ecise na iga ion and spa ial memo y, whe eas NORT may be mo e sensi i e o mode a e le els o ACh o he e ec i e p ocessing o isual in o ma ion and ecogni ion memo y (An unes and Biala, 2012; Haam and Yakel, 2017; Pi s, 2018). The le els o ACh equi ed o eco e y om scopolamine-induced impai men in he BM may, hus, be excessi e o NORT. Rega ding he hi d es pe o med in his s udy, PA, he dele e ious e ec s o cannabinoids on a e si e memo y a e well known and ha e been p e iously desc ibed (Jamali-Raeu y e al., 2011). Howe e , cannabinoid compounds also a ec he ascending and descending pa hways ha egula e pain pe cep ion (S a owicz and Finn, 2017). This is ele an , conside ing he a e si e s imulus (a mild oo shock) inhe en o his ask (Bengoe xea de Tena e al., 2022). Indeed, ou e- sul s indica e ha WIN55,212-2 a his dose al e ed pain sensi i i y in a s, likely in luencing he lea ning and memo y ou comes in he PA es . Finally, ou subch onic ea men wi h WIN55,212-2 also modi ied mo o ac i i y. The animals ea ed wi h WIN55,212-2 mo ed mo e slowly compa ed o hose ea ed wi h ehicle. Howe e , he unchanged o al pa h leng h du ing aining in BM es sugges s ha his educ ion in mo emen did no a ec spa ial acquisi ion. Choline gic dene a ion o he o eb ain enhances locomo o ac i i y (Ma sson e al., 2002). Addi ionally, ACh elease in he s ia um, hippocampus, and on al co ex co ela es wi h locomo ion (Day e al., 1991), sugges ing ha he s a us o he choline gic sys em a ec s mo o ac i i y. The opposi e e - ec , a educ ion in locomo o ac i i y ollowing WIN55,212-2 admin- is a ion, has also been desc ibed (Comp on e al., 1992). The capaci y o cannabinoids o modula e mo o ac i i y, as well as cogni ion, may be a ibu ed, a leas pa ly, o al e a ions in co ical choline gic neu o- ansmission. The esul s om he au o adiog aphic assays in a s ha comple ed he BM es suppo his hypo hesis. Following he WIN55, 212-2 ea men , he ac i i y o musca inic M 2 /M 4 ecep o s speci - ically inc eased in pa s o he mo o co ex and he hippocampus. These esul s appa en ly con as wi h o he s ha show WIN55,212-2 ea - men leading o a dec ease in M 2 ecep o densi ies (Schul e e al., 2012) and a down- egula ion o M 2 /M 4 ecep o s in animals wi h high le els o ACh in he hippocampus (du Bois e al., 2005). In speci ic laye s o hese same a eas, he densi y and ac i i y o cannabinoid ecep o s was also modula ed ollowing WIN55,212-2 adminis a ion. In his ega d, he use o a non-selec i e cannabinoid ecep o agonis does no allow o di e en ia e whe he his modula ion is a ibu ed o CB 1 o CB 2 e- cep o s. Ne e heless, he densi y o he i s one is much highe in key b ain a eas ela ed wi h lea ning and memo y p ocesses, and pa icu- la ly in he co ex (Chen e al., 2017; Llo en e-O eje o e al., 2022). Hence, while he con ibu ion o CB 2 ecep o sub ype o he obse ed e ec s canno be disca ded, a majo i y o i migh de i e om he mos densely exp essed sub ype, CB 1 . As p e iously men ioned, he ac i a ion o hese cannabinoid e- cep o s igge s an inc ease in ACh le els, by mobilizing speci ic choline- con aining phospholipids which could explain he enhanced musca inic ecep o ac i i y (Mo eno-Rod íguez e al., 2025). We hypo hesise ha his inc ease was enough o coun e ac he e ec s o scopolamine in BM es , bu i was excessi e o NORT. Ou g oup has p e iously desc ibed he posi i e e ec s o WIN55,212-2 in he BM. We demons a ed ha ollowing he same WIN55,212-2 ea men , ACh le els we e inc eased in a dose-dependen manne in he co ex and es o ed co ical cholin- e gic neu o ansmission a e a basal o eb ain choline gic lesion (Mo eno-Rod íguez e al., 2025). Based on he esul s om his s udy, whe e he same p o ocol o WIN55,212-2 adminis a ion and pe o - mance in BM was ollowed, we p opose a simila mechanism. Since scopolamine induces an acu e educ ion o musca inic ecep o ac i i y, he p e iously ele a ed ACh le els caused by WIN55,212-2 ea men p e en scopolamine om exe ing dele e ious cogni i e e ec s. The di e en ial e ec obse ed ollowing he ea men in BM and NORT es migh de i e om he dose used. Gi en ha his is a di e en pa adigm o musca inic impai men han he one used in ou p e ious s udy, he same dose o WIN55,212-2 (0.5 mg/kg) ha was enough o e e he de ici s in bo h es s could only e e hose de ici s in BM in his case. P obably, using a wide dose ange in NORT es i could be possible o also de ec an e ec i e dose o p e en scopolamine-induced memo y impai men . Table 2 Au o adiog aphic densi ies o cannabinoid ecep o s exp essed in mol/g .e., ob ained as speci ic binding o [ 3 H]CP55,940, in di e en b ain a eas ela ed o lea ning and memo y. Speci ic binding o [3H]CP55,940 ( mol/g .e.) B ain egion VEH WIN Ce eb al co ex       Cingula e 272 ±16 299 ±29 Mo o       Laye I-V 247 ±13 296 ±13 Laye VI 262 ±34 346 ±13* Soma osenso y       Laye I-V 211 ±20 212 ±20 Laye VI 194 ±31 273 ±26* Basal ganglia       Globus pallidus 1274 ±93 1314 ±121 S ia um 586 ±35 589 ±62 Diencephalon       NBM 307 ±41 325 ±41 HDB 189 ±22 172 ±32 VDB 229 ±21 250 ±23 Medial sep um 237 ±25 259 ±21 Hippocampus       CA1 413 ±16 394 ±20 O iens 435 ±29 413 ±22 Py amidal 388 ±35 301 ±24* Radia um 475 ±33 447 ±41 CA2 428 ±26 407 ±24 CA3 485 ±18 448 ±23 O iens 414 ±18 402 ±32 Py amidal 349 ±22 301 ±30 Radia um 483 ±33 454 ±31 Den a e gy us 372 ±21 293 ±15* G anula 116 ±20 117 ±22 Molecula 352 ±13 318 ±21 Polymo phic 450 ±26 438 ±31 Amygdala 202 ±23 206 ±21 HDB: ho izon al diagonal band, NBM: nucleus basalis magnocellula is, VDB: e - ical diagonal band. Da a a e exp essed as mean ±S.E.M. alues om VEH and WIN g oups. VEH s. WIN (*). Mann-Whi ney es , *p <0.05. M. Mo eno-Rod íguez e al. Eu opean Jou nal o Pha macology 998 (2025) 177612 8 5. Conclusions O e all, hese esul s sugges ha a ea men wi h WIN55,212-2 (0.5 mg/kg) can p e en he amnesic e ec s induced by scopolamine in a spa ial lea ning and memo y es like BM, bu no in a ecogni ion memo y es , such as NORT. The speci ic mechanisms unde lying his e ec emain elusi e, bu esul s sugges a di e en ial modula ion o he c oss alk be ween he eCB and he choline gic sys ems depending on he ype o memo y assessed in each es . Mo e p ecisely, he subch onic ac i a ion o cannabinoid ecep o s po en ially inc eased he choline gic one in key co ical and hippocampal a eas, jus enough o o e come he scopolamine-induced choline gic de ici in BM es . The p ospec i e clinical applica ion o his p omising expe imen al da a could be ela ed o he modula ion o he eCB sys em o he ea men o demen ias associa ed wi h a choline gic de ici , like is he case o AD. CRediT au ho ship con ibu ion s a emen Ma a Mo eno-Rod íguez: Concep ualiza ion, Da a cu a ion, Fo mal analysis, In es iga ion, Valida ion, Visualiza ion, W i ing – o iginal d a , W i ing – e iew & edi ing. Ike Bengoe xea de Tena: Concep ualiza ion, Fo mal analysis, In es iga ion, Valida ion, Visuali- za ion, W i ing – o iginal d a , W i ing – e iew & edi ing. Jona an Ma ínez-Ga deazabal: In es iga ion, W i ing – o iginal d a , W i ing – e iew & edi ing. Go ka Pe ei a-Cas elo: In es iga ion, W i ing – e iew & edi ing. Albe o Llo en e-O eje o: In es iga ion, W i ing – e iew & edi ing. I ´ an Manuel: W i ing – e iew & edi ing. Ra ael Rod íguez-Pue as: Concep ualiza ion, Da a cu a ion, Funding acqui- si ion, In es iga ion, Me hodology, P ojec adminis a ion, Resou ces, Supe ision, Valida ion, W i ing – o iginal d a , W i ing – e iew & edi ing. Da a a ailabili y The da a ha suppo he indings o his s udy a e a ailable om he co esponding au ho upon easonable eques . Funding This esea ch was inancially suppo ed by g an s om he Basque Go e nmen o he “Neu ochemis y and Neu odegene a ion” consoli- da ed esea ch g oup (IT975-16 and IT1454-22 o R.R-P), by Ins i u o de Salud Ca los III, co- unded by Eu opean Regional De elopmen Fund “A way o make Eu ope” (PI20/00153 o R.R-P) and by BIOEF unded by Ei b Ma a oia (BIO22/ALZ/010 o R.R-P). I.B.d.T was he ecipien o an In es igo ellowship unded by he Eu opean Union Nex Gene a ion. G. P-C was he ecipien o a Uni e si y o he Basque Coun y p edoc o al ellowship. J.M-G. was he ecipien o a pos doc o al p og am a he Uni e si y o he Basque Coun y. Decla a ion o compe ing in e es The au ho s decla e he ollowing inancial in e es s/pe sonal e- la ionships which may be conside ed as po en ial compe ing in e es s: Ra ael Rod iguez Pue as has pa en #T a amien o de la demencia con agonis as cannabinoides. Spain. 02-03-2017. Uni e si y o he Basque Coun y. ES2638057. issued o ES2638057. 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