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

Cannabinoid agonist WIN55,212-2 prevents scopolamine-induced impairment of spatial memory in rats Marta Moreno-Rodríguez a,1 , Iker Bengoetxea de Tena a,1 , Jonatan Martínez-Gardeazabal a , Gorka Pereira-Castelo a , Alberto Llorente-Ovejero a , Iv´ an Manuel a,b , Rafael Rodríguez-Puertas a,b,* a Department of Pharmacology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Spain b Neurodegenerative Diseases, BioBizkaia Health Research Institute, Barakaldo, Spain ARTICLE INFO Keywords: Cannabinoid agonist CB 1 receptors Central cholinergic system Scopolamine Memory impairment Autoradiographic studies Sprague-Dawley rats ABSTRACT The endocannabinoid system is involved in diverse processes, like learning and memory, governed by cholinergic neurotransmission. Recent research demonstrates that in a rat model of dementia derived from basal forebrain cholinergic degeneration, WIN55,212-2, a potent cannabinoid receptor agonist, improves cognition through increased cortical choline levels. However, the effect of cannabinoids on cholinergic deficits is still under investigation. In this work, we studied the effect of this treatment in a pharmacological rat model of transient cholinergic hypofunction by the acute administration of the muscarinic antagonist, scopolamine (2 mg/kg), in spatial, recognition and aversive memory tests. Scopolamine induced memory impairment was observed in the three tests and, importantly, the cannabinoid subchronic treatment with low doses of WIN55,212-2 (0.5 mg/kg) prevented this deleterious effect in spatial memory when evaluated in Barnes maze test. Autoradiographic studies indicate that, following the WIN55,212-2 treatment, cannabinoid receptor density increased in the motor and somatosensory cortices. In layers I-V of the motor cortex, the activity of cannabinoid and muscarinic receptors also increased. These results suggest that WIN55,212-2, through the activation of cannabinoid receptors, indirectly elevates the muscarinic tone in key cortical areas for learning and memory, preventing the memory deficits induced by scopolamine specifically in spatial memory. This highlights the importance of the crosstalk between the endocannabinoid and the cholinergic system for learning and memory processes and suggest that cannabinoid agonists might be an alternative for the treatment of cognitive deficits associated with cholinergic dysfunction. 1. Introduction The endocannabinoid system (eCB) regulates relevant physiological processes including pain, appetite, immune responses and cognitive processes (Lu and Mackie, 2021). These functions are predominantly regulated by CB 1 receptors, which are among the most expressed and active G protein-coupled receptors (GPCRs) in the central nervous system (CNS) (Martinez Ramirez et al., 2023). Numerous studies indicate that cannabinoids negatively affect learning and memory in physiologically normal states (Urits et al., 2021) e.g., Δ-9-tetrahydrocannabinol (Δ 9 -THC) consumption has been linked to impairments in various brain functions, including disruptions in attention and cognitive task performance (Stella, 2023). However, growing evidence suggests that the effects of cannabinoids are biphasic and dose-dependent, and are influenced by the preexisting cognitive status. In fact, lower doses of cannabinoids might be beneficial in some contexts, including aging, Alzheimer’s disease (AD) and other dementia-related neurodegenerative conditions (Bilkei-Gorzo et al., 2017; Ozaita and Aso, 2017; Llorente-Ovejero et al., 2018; for review see Pereira-Castelo et al., 2024). Some of these effects result from interactions between the eCB system and other neurotransmission systems in the CNS, such as the cholinergic system, which is essential for memory formation (Gedankien et al., 2023). Presynaptic CB 1 receptors at regulate the expression of aversive memories through the specific control of cholinergic neurotransmission (Soria-G´ omez et al., 2015). In a spatial memory task, the * Corresponding author. Department of Pharmacology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Spain. E-mail address: [email protected] (R. Rodríguez-Puertas). 1 These authors contributed equally to this work and share first authorship. Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar https://doi.org/10.1016/j.ejphar.2025.177612 Received 24 March 2025; Accepted 8 April 2025 European Journal of Pharmacology 998 (2025) 177612 Available online 17 April 2025 0014-2999/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/bync/4.0/ ). cognitive impairment produced by a treatment with the cannabinoid receptors agonist WIN55,212-2 was mediated by cholinergic hypofunction (Robinson et al., 2010) and, notably, both CB 1 and M 2 receptors are preferentially presynaptic and coupled to inhibitory G proteins (Nyíri et al., 2005). Various studies performed using the muscarinic antagonist scopolamine further support the interaction between the cholinergic and the eCB systems, although data concerning the impact of cannabinoid compounds on scopolamine-induced effects is inconclusive. In fact, studies performed with inverse agonists of CB 1 receptors report both a potentiation of the disruptive effects of scopolamine (Nakamura-Palacios et al., 2000), or improved cognitive performance following administration (Dillon et al., 2011). In a passive avoidance task, bilateral microinjection at the basolateral amygdala (BLA) of arachydonilcyclopropylamide (ACPA), a CB 1 receptor agonist, improved scopolamine-induced memory impairment, while co-administration of ineffective scopolamine doses with AM251, a cannabinoid CB 1 receptor antagonist, mimicked the amnesic effect obtained with higher scopolamine doses (Nedaei et al., 2016). A previous study from our group investigated the effect of a subchronic WIN55,212-2 (0.5 mg/kg) treatment on learning and memory in a model of basal forebrain cholinergic hypofunction, and the observed cognitive improvement was mediated by increased levels of acetylcholine (ACh) together with the restoration of choline-containing cortical lipids (Moreno-Rodríguez et al., 2025). On this basis, we hypothesized that WIN55,212-2 could also revert memory deficits deriving from the acute inhibition of the muscarinic system in the same context. Hence, the present study evaluated, in vivo, the effect of the same cannabinoid treatment in preventing the transient amnesia induced by acute scopolamine administration in paradigms related to spatial, recognition and aversive memory, and analyzed the neurochemical correlates behind the observed behaviors using a pharmacological approach by autoradiographic assays. 2. Material and methods 2.1. Reagents and drugs All the compounds necessary for the different procedures were of the highest quality commercially available for the purpose of our studies. [ 3 H]CP55,940 (149 Ci/mmol) and [ 35 S]GTPγS (1250 Ci/mmol) were acquired from Revvity (Waltham, MA, USA). The [ 3 H] microscales and [ 14 C] microscales used as standards in the autoradiographic experiments were purchased from ARC (American Radiolabeled Chemicals, Saint Louis, MO, USA). The β-radiation sensitive films, Kodak Biomax MR, bovine serum albumin (BSA), DL-dithiothreitol (DTT), guanosine 5 ′ - diphosphate (GDP), guanosine 5 ′ -O-3-thiotriphosphate (GTPγS), ketamine and xylazine were acquired from Sigma-Aldrich (St Louis, MO, USA). 2-carbamoyloxyethyl-trimethyl-azanium (Carbachol) and ( α ,S)- α -(Hydroxymethyl)benzeneacetic acid (1 α ,2β,4β,5 α ,7β)-9-methyl-3-oxa9-azatricyclo[3.3.1.02,4]non-7-yl ester hydrobromide (Scopolamine) were acquired from Sigma-Aldrich (St Louis, MO, USA). (11R)-2-Methyl11-[(morpholin-4-yl)methyl]-3-(naphthalene-1-carbonyl)-9-oxa-1 azatricyclo[6.3.1.04,12]dodeca-2,4(12),5,7-tetraene (WIN55,212-2) was acquired from Tocris (Bristol, UK). 2.2. Animals Every effort was made to minimize animal suffering and to use the minimum number of animals possible throughout the whole study. All procedures were performed in accordance with European animal research laws (Directive, 2010/63/EU) and the Spanish National protocols and were approved by the Local Ethical Committee for Animal Research of the University of the Basque Country (CEEA M20-2018-52 and 54 and M20/2023/352). Male Sprague-Dawley rats (n =90) weighing 200–300 g were housed in groups of 3–4 per cage at a temperature of 22 ◦C and in a humiditycontrolled (65 %) room with a 12:12 h light/dark cycle, with access to food and water ad libitum. These rats were administered with scopolamine/vehicle and WIN55,212-2/vehicle and were used for the performance of the different behavioral tests. A representative sample of the brains from all groups of rats which performed the behavioral tests were also used for autoradiographic studies (n =35). After the performance of the behavioral tests and before the autoradiographic studies, rats were anesthetized (i.p. ketamine/xylazine 90/10 mg/kg), sacrificed by decapitation and the brains were carefully removed from the skull before being frozen and stored at −80 ◦C, until use. 2.3. Behavioral tests 2.3.1. Barnes maze test The test was conducted on a white circular platform (130 cm diameter, 1 m above the floor) with 20 equally spaced holes. Only one hole led to a dark escape chamber beneath the platform. Bright lights placed around the platform created an unpleasant atmosphere, encouraging rats to find the target hole. Visual cues on the walls helped with spatial orientation. The test had two phases over five days: acquisition (4 days) and probe trial (day 5). During acquisition, rats were placed on the platform, given 3 min to find the target hole, and were guided to it if they failed. Four trials were conducted daily with 15 min of inter-trial intervals. On the fifth day, the day after the last acquisition trial, a single 180 s probe trial was performed, where memory retention was tested without the escape box present. In this phase, the platform was virtually divided into four quadrants and the time spent by the rats in the target quadrant, where the escape box was previously located, was measured as indicative of spatial memory. A tracking system (SMART, Panlab S.L., Barcelona, Spain) was used to record latency, path length, and speed during the acquisition phase, while quadrant time was used to assess spatial memory during the probe trial. 2.3.2. Novel object recognition test The test was conducted in a white open-field arena (90 ×90 ×50 cm) (Panlab S.L., Barcelona, Spain). It included four phases over five days: habituation (3 days), familiarization, short-term testing (5 h later), and long-term testing (24 h later). Before each phase, rats were handled gently for 1 min. During habituation phase, rats explored the arena for 5 min. In the familiarization phase, two identical objects (object A) were placed in opposite corners of the arena. Rats explored them until a combined 25-s exploration threshold was reached. In short-term testing, rats were presented with the familiar object (A) and a new object (B) for 5 min. Long-term testing, conducted 24 h after the familiarization phase, involved the familiar object (A) and another new object (C). A video camera recorded rat behavior, and exploration times for both familiar and novel objects were measured as indicative of recognition memory. The object discrimination ratio (DR) was calculated, with scores near zero indicating no preference, negative scores indicating a preference for the familiar object, and positive scores indicating preference for the novel object. 2.3.3. Passive avoidance test The behavioral test was conducted in a shuttle box (PanLab S.L. Barcelona, Spain) with two compartments: a larger, brightly lit, white compartment (31 ×31 ×24 cm) and a smaller, dark, black compartment (19.5 ×10.8 ×12 cm), separated by a guillotine door. The test had two phases: acquisition and retention. Before each phase, rats were habituated to the experimental room. In the acquisition phase (day 1), rats explored the white compartment for 30 s before the door to the black compartment opened. Upon instinctively crossing into the black compartment, the door closed, and a mild foot shock (0.4 mA M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 2 for 2 s) was administered. Rats remained in the black compartment for 15 s before being returned to their cage. The shuttle box was sanitized with ethanol (70 %) between trials. Rats that did not cross into the black compartment in this phase were excluded from the test. In the retention phase (day 2), rats were again placed in the white compartment, and after 30 s, the door to the black compartment opened. Rats had 5 min to decide whether to cross; those that crossed received no shock. The behavior of the rats was recorded with a video camera positioned above the shuttle box, with acquisition latency (day 1) and step-through latency (day 2) measured. Longer latencies in the retention phase indicated a passive avoidance response, reflecting positive performance. 2.3.4. Hot plate test The test was performed in a hot plate apparatus (Leica Biosystems, Barcelona, Spain) consisting in a cylindrical see-through Plexiglas wall (19 ×30 cm) located above a plate. The plate warms through an electric resistance and is equipped with a timer and a thermostat. On the top of the cylinder, a metal grid is located to hold the rat when it jumps. Rats were placed on the metal plate, which was previously warmed (55 ±0.5 ◦C). The time spent by the rats until jumping was recorded (jump latency). The latency to paw-licking was also measured. Both parameters, following a noxious thermal stimulus, were used as indicative of nociceptive threshold. 2.3.5. Electrical shock evoked pain threshold The procedure was performed in the same shuttle box used for the PA test. This apparatus has a grid floor and electrical foot shocks of different potency can be delivered. Rats were placed in the white compartment and received mild foot shocks, beginning at 0.0 mA, gradually increasing by 0.05 mA, until the first vocalization was measured, which is an indicative of nociception and discomfort. 2.4. WIN55,212-2 administration in a pharmacological model of muscarinic antagonism Scopolamine was dissolved in saline 0.9 % and was administered intraperitoneally (2 mg/kg), 30 min before the performance of the PA test acquisition phase, the NORT test short and long-term testing phases and the BM probe trial phase. Control group received vehicle without scopolamine. For the evaluation of the effect of a cannabinoid treatment in the amnesic effects elicited by scopolamine, WIN55,212-2 was intraperitoneally administered once daily (0.5 mg/kg), 1 h before every phase of each test. WIN55,212-2 was dissolved in pure DMSO and diluted with kolliphor and 0.9 % saline, in a 1:1:18 proportion. Control group received vehicle without WIN55,212-2. Rats were randomly divided into four groups for each test: vehicle (VEH), WIN55,212-2 (WIN), scopolamine (SCOP) and WIN55,212-2 +scopolamine (WIN +SCOP). 2.5. Autoradiographic studies 2.5.1. Functional [ 35 S]GTPγS autoradiography For the performance of functional autoradiography to study muscarinic and cannabinoid receptors activity, brain sections were air dried for 30 min, followed by two consecutive incubations of 30 min each in an HEPES-based buffer (50 mM HEPES, 100 mM NaCl, 3 mM MgCl 2 , 0.2 mM EGTA and 0.5 % BSA, pH 7.4) at 30 ◦C to remove the endogenous ligands. Slices were then incubated for 2 h at 30 ◦C in the same buffer supplemented with 2 mM GDP, 1 mM DTT and 0.04 nM [ 35 S]GTPγS. Basal binding was determined in two consecutive slices in the absence of the agonist. The agonist-stimulated binding was determined in another consecutive slice in the presence of the corresponding receptor agonists, carbachol (10 μ M) for muscarinic receptors and WIN55,212-2 (10 μ M) for cannabinoid receptors. Non-specific binding was defined by competition with unlabeled GTPγS (10 μ M) in another section. Tissue slices were finally washed twice in cold (4 ◦C) HEPES (50 mM) buffer (pH 7.4), dried and exposed for 48 h to β-radiation sensitive films with a set of [ 14 C] standards calibrated for [ 35 S]. Calibrated films were scanned and quantified using Fiji software. Data was expressed as the % of stimulation over basal. 2.5.2. [ 3 H]CP55,940 receptor autoradiography For the performance of cannabinoid receptors autoradiography, brain sections were air dried for 30 min and then immersed in Coplin jars for preincubation in a buffer containing 50 mM Tris-HCl and 1 % of BSA (pH 7.4) for 30 min, at room temperature, to remove endogenous ligands. Tissue slices were later incubated in the presence of the [ 3 H] CP55,940 radioligand (3 nM) for 2 h at 37 ◦C. Non-specific binding was measured by competition with non-labeled CP55,940 (10 μ M) in another consecutive slice. Following the incubation, tissue slices were washed with an ice-cold preincubation buffer, dipped in distilled water and dried overnight. To generate autoradiograms, dry sections were exposed to β-radiation-sensitive films in hermetically closed cassettes for 21 days at 4 ◦C. For the calibration of the optical densities to fmol/mg tissue equivalent, [ 3 H] microscales were exposed to the films. Calibrated films were scanned and quantified using Fiji software (Fiji, Bethesda, MD, USA). Data was expressed as fmol/g of tissue equivalent (t.e.). 2.6. Statistical analysis Data from behavioural tests was analyzed using a Mann-Whitney test for two groups and Kruskal-Wallis test followed by Dunn’s post hoc test for more than two groups. Repeated measures two-way ANOVA followed by post-hoc test Bonferroni’s was used for repeated measures of the same parameter in different days (total latency, total path length and speed in BM test). Step-through latency times of PA test were represented as Kaplan-Meier survival curves and analyzed using a log-rank/ Mantel–Cox test, as previously described by our group (Llorente-Ovejero et al., 2017; Bengoetxea de Tena et al., 2022). Data from autoradiographic assays was analyzed using a Mann-Whitney test. The threshold for statistical significance was set at p <0.05. Besides that, df, F and H values are provided in the figure legends, where necessary. Statistical analyses and data representation were performed using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA). 3. Results 3.1. WIN55,212-2 prevents scopolamine-induced amnesia in a spatial memory test BM test was performed to evaluate the effect of WIN55,212-2 in preventing scopolamine-induced impairment of spatial memory. Throughout the four days of acquisition, both WIN55,212-2 and vehicletreated groups decreased the total latency and the total path length (Fig. 1A and B). On the first trial, this parameter was significantly higher in the WIN55,212-2-treated group (VEH vs. WIN, p <0.05; Fig. 1A), indicating a slight delay in the learning curve. The average speed increased in each trial for both groups, but WIN55,212-2-treated rats walked more slowly than vehicle-treated ones overall (VEH vs. WIN, p < 0.05; Fig. 1C). On probe trial, after the last training day, the latency in the target quadrant was measured as an indicator of spatial memory in all groups. The administration of WIN55,212-2 to control rats did not produce an impairment in spatial memory (VEH vs. WIN, p >0.05; Fig. 2A). Scopolamine-treated rats spent a significantly lower time in the target quadrant (VEH vs. SCOP, p <0.001; Fig. 2A), indicating spatial memory deficits following its administration. Importantly, a subchronic treatment with WIN55,212-2 prevented the amnesic effect exerted by scopolamine in BM test (SCOP vs. SCOP +WIN, p <0.01; Fig. 2A). Considering the positive effects of a cannabinoid treatment in BM, we analyzed its effect on preventing the amnesic effects induced by M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 3 scopolamine in other memory tests, including the novel object recognition test (NORT) and passive avoidance (PA) test. 3.2. Effect of a cannabinoid treatment on a pharmacological model of muscarinic antagonism in recognition memory Recognition memory was evaluated at two different time points, short (5 h post-familiarization) and long-term (24 h postfamiliarization). In the short-term test, the treatment with WIN55,2122 impaired memory in control rats (VEH vs. WIN, p <0.001; Fig. 3A). Notably, the administration of scopolamine did not significantly decrease the DR (VEH vs. SCOP, p >0.05; Fig. 3A). In the long-term, the subchronic treatment with WIN55,212-2 also showed a deleterious effect on memory in control rats (VEH vs. WIN, p < 0.05; Fig. 3B). In contrast to what was observed in the short-term test, the administration of scopolamine also caused a significant decrease in the DR (VEH vs. SCOP, p <0.01; Fig. 3B), indicating memory impairment following the administration of a muscarinic antagonist in longFig. 1. Effect of WIN55,212-2 on locomotion and learning in BM test. (A) Total latency during the four days of learning of BM test. No significant differences were observed between VEH and WIN groups comparing both curves, but significant differences were observed between the two groups in the first trial (Repeated measures two-way ANOVA, post-hoc test Bonferroni’s, df =1, F =4.51, #p <0.05, VEH vs. WIN). (B) Total path length during the four days of learning of BM test. No significant differences were observed between VEH and WIN groups comparing both curves (Repeated measures two-way ANOVA, post-hoc test Bonferroni’s, df =1, F =1.15). (C) Mean speed during the four days of learning of BM test. Significant differences were observed between VEH and WIN groups comparing both curves (Repeated measures two-way ANOVA, post-hoc test Bonferroni’s, df =1, F =7.24, #p <0.05, VEH vs. WIN). Fig. 2. WIN55,212-2 prevents the scopolamine-induced impairment of spatial memory. (A) Time spent in the target quadrant on the probe trial day of BM test (Kruskal–Wallis’s test, post-hoc test Dunn’s multiple comparison, df =3, H =19.36, ***p <0.001 VEH vs. SCOP, $$ p <0.01 SCOP vs. SCOP +WIN). (B) Representative trajectories of rats from each group during 180 s in the probe trial day of BM test. Note the accumulation of trajectories in the target quadrant (where the target hole is located, depicted in black in the figure) for VEH, WIN and SCOP +WIN groups, as opposed to SCOP group. M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 4 term recognition and working memory, but not in short-term one. The administration of WIN55,212-2 to rats that also received scopolamine only produced a slight increase in the DR and did not prevent or reverse the observed memory deficits (SCOP vs. SCOP +WIN, p >0.05; Fig. 3B). 3.3. Effect of a cannabinoid treatment on a pharmacological model of muscarinic antagonism in an aversive memory test The administration of scopolamine impaired both acquisition latency (VEH vs. SCOP, p <0.01, Fig. 4A) and step-through latency time (VEH vs. SCOP, p <0.001, Fig. 4B), provoking transient memory deficits. The cannabinoid treatment with WIN55,212-2 alone did not affect acquisition latency, but decreased the step-through latency time (VEH vs. WIN, p <0.01, Fig. 4B). The subchronic administration of WIN55,212-2 did not prevent the amnesic effect of scopolamine observed in the stepthrough latency time (SCOP vs. SCOP +WIN, p >0.05, Fig. 4B). Considering the analgesic effects of cannabinoid agonists (Manzanares et al., 2006), we hypothesized that decreased pain sensitivity following the WIN55,212-2 administration could be a bias in the effect of the treatment observed in PA test. Thus, we measured the effect of this treatment on pain response using the hot plate test. We observed that WIN55,212-2 treatment produced analgesia, as indicated by the increases in the jump latency (VEH vs. WIN, p <0.05, Fig. 5A) and the latency to licked paw (VEH vs. WIN, p <0.01, Fig. 5B). We also measured pain response using a protocol designed to evaluate nociception produced by an electrical shock in PA test, the electrical shock evoked pain threshold, and we observed that the group of rats treated with WIN55,212-2 required a higher shock intensity to evoke a first vocalization (VEH 0.0375 ±0.0082 mA vs. WIN 0.0714 ±0.0101 mA, p <0.01), also indicating an analgesic effect of the cannabinoid treatment. These results indicate a possible bias of the results obtained in PA test following the WIN55,212-2 administration. 3.4. The subchronic WIN55,212-2 administration altered muscarinic and cannabinoid receptor activity The effect of WIN55,212-2 administration on muscarinic and cannabinoid receptors was studied to elucidate the neurochemical correlates behind the observed behaviors. The density and activity of muscarinic and cannabinoid receptors was analyzed by means of autoradiography in rats that had performed BM test, given the positive effect exerted by the treatment in this test. Considering that scopolamine was Fig. 3. Effect of WIN55,212-2 in a model of muscarinic antagonism in recognition memory in the short-term and the long-term. (A) Discrimination ratio in the shortterm testing phase of NORT (Kruskal–Wallis’s test, post-hoc test Dunn’s multiple comparison, df =3, H =7.29, ###p <0.001 VEH vs. WIN). (B) Discrimination ratio in the long-term testing phase of NORT (Kruskal–Wallis’s test, post-hoc test Dunn’s multiple comparison, df =3, H =16.56, **p <0.01 VEH vs. SCOP, #p <0.05 VEH vs. WIN). Fig. 4. Treatment with WIN55,212-2 in a model of muscarinic antagonism evaluated in PA test. (A) Acquisition latency times in the learning trial of the PA test (Kruskal–Wallis’s test, post-hoc test Dunn’s multiple comparison, df =3, H =7.47, **p <0.01, VEH vs. SCOP). (B) Step-through latency times of PA test represented as Kaplan-Meier survival curves (log-rank/Mantel–Cox test, ***p <0.001 VEH vs. SCOP, ##p <0.01 VEH vs. WIN, $$$ p <0.001 VEH vs. SCOP +WIN). The median latency was 24.55 s for SCOP group, 256.00 s for WIN group and 48.00 s for SCOP +WIN group. M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 5 administered acutely and given its fast excretion and low bioavailability (Tian et al., 2015), the autoradiography data only represents the effect of the WIN55,212-2 treatment. The [ 35 S]GTPγS binding stimulated by carbachol, an agonist of muscarinic receptors, was measured in brain areas related to learning and memory to localize and determine the activity of G i/o proteins coupled to M 2 /M 4 receptors (Fig. 6). No differences were observed between the groups treated with vehicle and WIN55,212-2 in basal binding, i.e, [ 35 S]GTPγS binding in the absence of the agonist. The G i/o - coupled M 2 /M 4 receptor activity induced by carbachol was increased in the WIN55,212-2-treated group in layers I-V of the motor cortex (VEH vs. WIN, p <0.05; Fig. 6, Table 1), in some of the septal nuclei, the medial septum and the horizontal diagonal band (VEH vs. WIN, p < 0.05; Fig. 6, Table 1), and in the pyramidal layer of the CA3 region of the hippocampus (VEH vs. WIN, p <0.05; Fig. 6, Table 1). The [ 35 S]GTPγS binding stimulated by WIN55,212-2 was measured in brain areas related to learning and memory to localize and determine the activity of cannabinoid receptors (Fig. 7). The activity mediated by G i/o -coupled cannabinoid receptors was increased following the WIN55,212-2 treatment in several areas related to learning and memory processes, such as layers I-V of the motor cortex (VEH vs. WIN, p <0.05; Fig. 7, Table 1), the striatum (VEH vs. WIN, p <0.05; Fig. 7, Table 1), the vertical diagonal band (VEH vs. WIN, p <0.05; Fig. 7, Table 1), the radiatum and oriens layers of the hippocampus CA1 region (VEH vs. WIN, p <0.05; Fig. 7, Table 1), the hippocampus CA3 region (VEH vs. WIN, p <0.05; Fig. 7, Table 1), the radiatum layer of the hippocampus CA3 region (VEH vs. WIN, p <0.05; Fig. 7, Table 1) and the dentate gyrus (VEH vs. WIN, p <0.05; Fig. 7, Table 1). The density of cannabinoid receptors was also studied in the same brain areas by [ 3 H]CP55,940 binding (Fig. 8). The treatment with the cannabinoid agonist WIN55,212-2 increased cannabinoid receptors density in layer VI of the motor cortex (VEH vs. WIN, p <0.05; Fig. 8, Table 2) and in layer VI of the somatosensory cortex (VEH vs. WIN, p < 0.05; Fig. 8, Table 2). In contrast, cannabinoid density decreased in the dentate gyrus (VEH vs. WIN, p <0.05; Fig. 8, Table 2) and in the pyramidal layer of the hippocampus CA1 region (VEH vs. WIN, p <0.05; Fig. 8, Table 2). 4. Discussion Considering the regulatory role of the eCB system over learning and memory tasks controlled by cholinergic neurotransmission, we have evaluated the effect of a subchronic treatment with potent cannabinoid receptor agonist WIN55,212-2 in preventing transitory amnesia caused by scopolamine. As expected, scopolamine impaired memory in three learning and memory behavior tests compared to a saline group (Malikowska-Racia et al., 2018). The acute pharmacological model of cholinergic blockade with scopolamine is well-characterized and used in the study of numerous anti-amnesic drugs (Akinyemi et al., 2017; El-Khadragy et al., 2014; Marisco et al., 2013). Our results are consistent with data reported in the literature; however, we observed that pretreatment with scopolamine did not significantly disrupt short-term recognition memory in NORT. This observation contrasts with other studies indicating that muscarinic antagonists impair the acquisition and performance of various learned behaviors, including short-term memory (Balderas et al., 2012; Fibiger et al., 1991; Palmer et al., 2016). However, our own previous results showed short-term memory was not affected in NORT after a basal cholinergic lesion, when baso-cortical cholinergic neurotransmission was diminished (Moreno-Rodríguez et al., 2025). It is also in line with a study reporting that reduced ACh release delays specifically the consolidation of object recognition memory (De Jaeger et al., 2013). Our findings suggest that blocking the cholinergic signaling is crucial for long-term, but not short-term, recognition memory. WIN55,212-2 alone induced similar cognitive impairment in NORT and PA as scopolamine did, but not in BM. The contradictory effects of a relatively low dose of WIN55,212-2 (0.5 mg/kg) observed in BM, NORT and PA tests suggest different sensitivities to cannabinoid effects depending on the type of memory assessed, i.e. spatial memory, recognition and contextual memory and aversive or fear-related memory. Previous reports are somewhat contradictory, similar doses of WIN55,212-2 facilitated the extinction of contextual fear and spatial memory (Pamplona et al., 2006). However, different sensitivities to the effects mediated by cannabinoid receptors depending on the type of memory have been described (Li and Kim, 2016). Although the detrimental effect of cannabinoid agonism on memory is well studied and proven, the underlying mechanisms remain unclear. Here we demonstrated that subchronic administration of a low dose of WIN55,212-2 (0.5 mg/kg) prevented the amnesic effects of scopolamine in spatial learning and memory in the BM test, but not in NORT test. Importantly, WIN55,212-2 alone, at this dose, did not induce detrimental effects on memory in BM test, but it did in NORT. In other studies, low doses of THC restored age-related cognitive dysfunction by modulating hippocampus-dependent memory processes (Bilkei-Gorzo et al., 2017; Sarne et al., 2018). While data suggest Fig. 5. Analgesic effect of WIN55,212-2 in the hot plate test. (A) Jump latency in the hot plate test (Mann Whitney test, #p <0.05 VEH vs. WIN). (B) Latency to licked paw in the hot plate test (Mann Whitney test, ##p <0.01 VEH vs. WIN). Fig. 6. Representative autoradiograms showing the activity of M 2 /M 4 receptors. Coronal sections corresponding to (A) VEH and (B) WIN groups showing [ 35 S]GTPγS binding stimulated by carbachol in the horizontal diagonal band (HDB), layers I-V of the motor cortex (Mot Cx I-V), medial septum (MS) and the hippocampus (HPC). Scale bar: 4 mm. M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 6 cross-state-dependent learning between WIN55,212-2 and scopolamine (Jamali-Raeufy et al., 2011), the differential sensitivity to cannabinoids may be elucidated by exploring the mechanisms through which scopolamine induces memory loss. Scopolamine is a non-selective muscarinic receptor antagonist, which reduces ACh binding to the muscarinic receptors and leads to memory deficits (Blokland, 1995). It mainly affects cholinergic transmission at the basal forebrain, motor cortex, globus pallidus, hippocampus, perirhinal cortex and amygdala. However, choline acetyltransferase (ChAT) levels seem to be affected specifically in the hippocampus and amygdala (Hescham et al., 2014; Ray et al., 1992). Scopolamine can decrease cortical ACh levels by 52 % and hippocampal ACh levels by 39 % (Spignoli et al., 1987). Several studies have shown that cholinesterase inhibitors, which increase ACh levels at the synapse (Pattanashetti et al., 2017; Sadek et al., 2016; Shin et al., 2018) or natural compounds that also increase ACh levels (Lazarova et al., 2024; Teralı et al., 2024), prevent the amnesic effects of Table 1 [ 35 S]GTPγS binding of carbachol-stimulated and WIN55,212-2-stimulated receptor activity expressed as the percentage of stimulation over basal, in different brain areas related to learning and memory. Carbachol stimulation (% over basal) WIN55,212-2 stimulation (% over basal) Brain region VEH WIN VEH WIN Cerebral cortex             Cingulate 102 ±21 115 ±19 248 ±36 298 ±55 Motor             Layer I-V 88 ±16 154 ±17* 216 ±41 405 ±72* Layer VI 169 ±21 154 ±34 353 ±51 354 ±39 Somatosensory             Layer I-V 156 ±28 164 ±53 156 ±47 259 ±41 Layer VI 178 ±21 194 ±38 353 ±66 378 ±96 Basal ganglia             Globus pallidus 76 ±33 116 ±75 874 ±115 890 ±103 Striatum 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 septum 268 ±27 374 ±43* 206 ±37 356 ±61 Hippocampus             CA1 56 ±17 62 ±17 104 ±27 175 ±22 Oriens 51 ±16 64 ±23 17 ±5 62 ±12* Pyramidal 65 ±15 88 ±46 230 ±41 312 ±93 Radiatum 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* Oriens 64 ±23 67 ±18 58 ±19 57 ±12 Pyramidal 25 ±19 95 ±29* 305 ±110 323 ±130 Radiatum 34 ±9 30 ±8 74 ±21 184 ±44* Dentate gyrus 37 ±7 48 ±13 277 ±51 484 ±66* Granular 38 ±10 42 ±17 318 ±101 398 ±104 Molecular 40 ±8 34 ±11 174 ±38 308 ±90 Polymorphic 27 ±10 27 ±6 324 ±45 338 ±76 Amygdala 98 ±18 75 ±31 213 ±59 407 ±87 HDB: horizontal diagonal band, NBM: nucleus basalis magnocellularis, VDB: vertical diagonal band. Data are expressed as mean ±S.E.M. values from VEH and WIN groups. VEH vs. WIN (*). Mann-Whitney test, *p <0.05. Fig. 7. Representative autoradiograms showing the activity of cannabinoid receptors evoked by WIN55,212-2. Coronal sections corresponding to (A) VEH and (B) WIN groups showing [ 35 S]GTPγS binding stimulated by WIN55,212-2 in the vertical diagonal band (VDB), layers I-V of the motor cortex (Mot Cx IV), the striatum (STR) and the hippocampus (HPC). Scale bar: 4 mm. Fig. 8. Representative autoradiograms showing the density of cannabinoid receptors. Coronal sections corresponding to (A) VEH and (B) WIN groups showing [ 3 H]CP55,940 binding in layers I-V of the motor cortex (Mot Cx I-V) and in layer VI of the somatosensory cortex. Scale bar: 4 mm. M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 7 scopolamine. However, the effectiveness of memory improvement is dependent on the dose of scopolamine administered (Bejar et al., 1999), suggesting that the levels of ACh in the synaptic cleft are important for the effect of scopolamine. In this sense, it is already described that cannabinoids, including WIN55,212-2, modulate ACh release in the hippocampus and cortex (Gessa et al., 1997, 1998; Moreno-Rodríguez et al., 2025; Nava et al., 2001; Tzavara et al., 2003). WIN55,212-2 can also mitigate the toxicity induced on the cholinergic system following acetylcholinesterase inhibition (Nallapaneni et al., 2006, 2008). Therefore, we hypothesise that the WIN55, 212-2-mediated increase in ACh could potentially compensate for the acute reduction in cortical and hippocampal ACh levels induced by scopolamine (Spignoli et al., 1987), as we have previously described in cortical areas (Moreno-Rodríguez et al., 2025). In addition, WIN55, 212–2 may induce memory deficits itself due to an excessive increase in ACh levels, as recent evidence suggests (Huang et al., 2022), among other mechanisms. However, the mechanism by which the same doses of cannabinoid agonists protect against scopolamine-induced cognitive impairment in the BM while showing no effect on NORT remains unclear. While BM and NORT are both hippocampus-dependent tests, BM may require higher modulation of ACh to facilitate precise navigation and spatial memory, whereas NORT may be more sensitive to moderate levels of ACh for the effective processing of visual information and recognition memory (Antunes and Biala, 2012; Haam and Yakel, 2017; Pitts, 2018). The levels of ACh required for recovery from scopolamine-induced impairment in the BM may, thus, be excessive for NORT. Regarding the third test performed in this study, PA, the deleterious effects of cannabinoids on aversive memory are well known and have been previously described (Jamali-Raeufy et al., 2011). However, cannabinoid compounds also affect the ascending and descending pathways that regulate pain perception (Starowicz and Finn, 2017). This is relevant, considering the aversive stimulus (a mild foot shock) inherent to this task (Bengoetxea de Tena et al., 2022). Indeed, our results indicate that WIN55,212-2 at this dose altered pain sensitivity in rats, likely influencing the learning and memory outcomes in the PA test. Finally, our subchronic treatment with WIN55,212-2 also modified motor activity. The animals treated with WIN55,212-2 moved more slowly compared to those treated with vehicle. However, the unchanged total path length during training in BM test suggests that this reduction in movement did not affect spatial acquisition. Cholinergic denervation of the forebrain enhances locomotor activity (Mattsson et al., 2002). Additionally, ACh release in the striatum, hippocampus, and frontal cortex correlates with locomotion (Day et al., 1991), suggesting that the status of the cholinergic system affects motor activity. The opposite effect, a reduction in locomotor activity following WIN55,212-2 administration, has also been described (Compton et al., 1992). The capacity of cannabinoids to modulate motor activity, as well as cognition, may be attributed, at least partly, to alterations in cortical cholinergic neurotransmission. The results from the autoradiographic assays in rats that completed the BM test support this hypothesis. Following the WIN55, 212-2 treatment, the activity of muscarinic M 2 /M 4 receptors specifically increased in parts of the motor cortex and the hippocampus. These results apparently contrast with others that show WIN55,212-2 treatment leading to a decrease in M 2 receptor densities (Schulte et al., 2012) and a down-regulation of M 2 /M 4 receptors in animals with high levels of ACh in the hippocampus (du Bois et al., 2005). In specific layers of these same areas, the density and activity of cannabinoid receptors was also modulated following WIN55,212-2 administration. In this regard, the use of a non-selective cannabinoid receptor agonist does not allow to differentiate whether this modulation is attributed to CB 1 or CB 2 receptors. Nevertheless, the density of the first one is much higher in key brain areas related with learning and memory processes, and particularly in the cortex (Chen et al., 2017; Llorente-Ovejero et al., 2022). Hence, while the contribution of CB 2 receptor subtype to the observed effects cannot be discarded, a majority of it might derive from the most densely expressed subtype, CB 1 . As previously mentioned, the activation of these cannabinoid receptors triggers an increase in ACh levels, by mobilizing specific cholinecontaining phospholipids which could explain the enhanced muscarinic receptor activity (Moreno-Rodríguez et al., 2025). We hypothesise that this increase was enough to counteract the effects of scopolamine in BM test, but it was excessive for NORT. Our group has previously described the positive effects of WIN55,212-2 in the BM. We demonstrated that following the same WIN55,212-2 treatment, ACh levels were increased in a dose-dependent manner in the cortex and restored cortical cholinergic neurotransmission after a basal forebrain cholinergic lesion (Moreno-Rodríguez et al., 2025). Based on the results from this study, where the same protocol for WIN55,212-2 administration and performance in BM was followed, we propose a similar mechanism. Since scopolamine induces an acute reduction of muscarinic receptor activity, the previously elevated ACh levels caused by WIN55,212-2 treatment prevent scopolamine from exerting deleterious cognitive effects. The differential effect observed following the treatment in BM and NORT test might derive from the dose used. Given that this is a different paradigm of muscarinic impairment than the one used in our previous study, the same dose of WIN55,212-2 (0.5 mg/kg) that was enough to revert the deficits in both tests could only revert those deficits in BM in this case. Probably, using a wider dose range in NORT test it could be possible to also detect an effective dose to prevent scopolamine-induced memory impairment. Table 2 Autoradiographic densities of cannabinoid receptors expressed in fmol/g t.e., obtained as specific binding of [ 3 H]CP55,940, in different brain areas related to learning and memory. Specific binding of [3H]CP55,940 (fmol/g t.e.) Brain region VEH WIN Cerebral cortex       Cingulate 272 ±16 299 ±29 Motor       Layer I-V 247 ±13 296 ±13 Layer VI 262 ±34 346 ±13* Somatosensory       Layer I-V 211 ±20 212 ±20 Layer VI 194 ±31 273 ±26* Basal ganglia       Globus pallidus 1274 ±93 1314 ±121 Striatum 586 ±35 589 ±62 Diencephalon       NBM 307 ±41 325 ±41 HDB 189 ±22 172 ±32 VDB 229 ±21 250 ±23 Medial septum 237 ±25 259 ±21 Hippocampus       CA1 413 ±16 394 ±20 Oriens 435 ±29 413 ±22 Pyramidal 388 ±35 301 ±24* Radiatum 475 ±33 447 ±41 CA2 428 ±26 407 ±24 CA3 485 ±18 448 ±23 Oriens 414 ±18 402 ±32 Pyramidal 349 ±22 301 ±30 Radiatum 483 ±33 454 ±31 Dentate gyrus 372 ±21 293 ±15* Granular 116 ±20 117 ±22 Molecular 352 ±13 318 ±21 Polymorphic 450 ±26 438 ±31 Amygdala 202 ±23 206 ±21 HDB: horizontal diagonal band, NBM: nucleus basalis magnocellularis, VDB: vertical diagonal band. Data are expressed as mean ±S.E.M. values from VEH and WIN groups. VEH vs. WIN (*). Mann-Whitney test, *p <0.05. M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 8 5. Conclusions Overall, these results suggest that a treatment with WIN55,212-2 (0.5 mg/kg) can prevent the amnesic effects induced by scopolamine in a spatial learning and memory test like BM, but not in a recognition memory test, such as NORT. The specific mechanisms underlying this effect remain elusive, but results suggest a differential modulation of the crosstalk between the eCB and the cholinergic systems depending on the type of memory assessed in each test. More precisely, the subchronic activation of cannabinoid receptors potentially increased the cholinergic tone in key cortical and hippocampal areas, just enough to overcome the scopolamine-induced cholinergic deficit in BM test. The prospective clinical application of this promising experimental data could be related to the modulation of the eCB system for the treatment of dementias associated with a cholinergic deficit, like is the case of AD. CRediT authorship contribution statement Marta Moreno-Rodríguez: Conceptualization, Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing. Iker Bengoetxea de Tena: Conceptualization, Formal analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing. Jonatan Martínez-Gardeazabal: Investigation, Writing – original draft, Writing – review & editing. Gorka Pereira-Castelo: Investigation, Writing – review & editing. Alberto Llorente-Ovejero: Investigation, Writing – review & editing. Iv´ an Manuel: Writing – review & editing. Rafael Rodríguez-Puertas: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Funding This research was financially supported by grants from the Basque Government to the “Neurochemistry and Neurodegeneration” consolidated research group (IT975-16 and IT1454-22 to R.R-P), by Instituto de Salud Carlos III, co-funded by European Regional Development Fund “A way to make Europe” (PI20/00153 to R.R-P) and by BIOEF funded by Eitb Maratoia (BIO22/ALZ/010 to R.R-P). I.B.d.T was the recipient of an Investigo fellowship funded by the European Union Next Generation. G. P-C was the recipient of a University of the Basque Country predoctoral fellowship. J.M-G. was the recipient of a postdoctoral program at the University of the Basque Country. Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rafael Rodriguez Puertas has patent #Tratamiento de la demencia con agonistas cannabinoides. Spain. 02-03-2017. University of the Basque Country. ES2638057. issued to ES2638057. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. References Akinyemi, A.J., Oboh, G., Oyeleye, S.I., Ogunsuyi, O., 2017. Anti-amnestic effect of curcumin in combination with donepezil, an anticholinesterase drug: involvement of cholinergic system. Neurotox. Res. 31 (4), 560–569. https://doi.org/10.1007/ s12640-017-9701-5. Antunes, M., Biala, G., 2012. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn. Process. 13 (2), 93–110. https://doi.org/ 10.1007/s10339-011-0430-z. Balderas, I., Morin, J.-P., Rodriguez-Ortiz, C.J., Bermudez-Rattoni, F., 2012. Muscarinic receptors activity in the perirhinal cortex and hippocampus has differential involvement in the formation of recognition memory. Neurobiol. Learn. Mem. 97 (4), 418–424. https://doi.org/10.1016/j.nlm.2012.03.006. Bejar, C., Wang, R.H., Weinstock, M., 1999. Effect of rivastigmine on scopolamineinduced memory impairment in rats. Eur. J. Pharmacol. 383 (3), 231–240. https:// doi.org/10.1016/s0014-2999(99)00643-3. Bengoetxea de Tena, I., Moreno-Rodríguez, M., Llorente-Ovejero, A., Monge-Benito, S., Martínez-Gardeazabal, J., Onandia-Hinchado, I., Manuel, I., Gim´ enez-Llort, L., Rodríguez-Puertas, R., 2022. Handling and novel object recognition modulate fear response and endocannabinoid signaling in nucleus basalis magnocellularis. Eur. J. Neurosci. 55 (6), 1532–1546. https://doi.org/10.1111/ejn.15642. Bilkei-Gorzo, A., Albayram, O., Draffehn, A., Michel, K., Piyanova, A., Oppenheimer, H., Dvir-Ginzberg, M., R´ acz, I., Ulas, T., Imbeault, S., Bab, I., Schultze, J.L., Zimmer, A., 2017. A chronic low dose of Δ9-tetrahydrocannabinol (THC) restores cognitive function in old mice. Nat. Med. 23 (6), 782–787. https://doi.org/10.1038/nm.4311. Blokland, A., 1995. Acetylcholine: a neurotransmitter for learning and memory? Brain Res. Rev. 21 (3), 285–300. https://doi.org/10.1016/0165-0173(95)00016-X. Chen, D., Gao, M., Gao, F., Su, Q., Wu, J., 2017. Brain cannabinoid receptor 2: expression, function and modulation. Acta Pharmacol. Sin. 38 (3), 312–316. https:// doi.org/10.1038/aps.2016.149. Compton, D.R., Gold, L.H., Ward, S.J., Balster, R.L., Martin, B.R., 1992. Aminoalkylindole analogs: cannabimimetic activity of a class of compounds structurally distinct from Delta 9-tetrahydrocannabinol. J. Pharmacol. Exp. Therapeut. 263 (3), 1118–1126. Day, J., Damsma, G., Fibiger, H.C., 1991. Cholinergic activity in the rat hippocampus, cortex and striatum correlates with locomotor activity: an in vivo microdialysis study. Pharmacol. Biochem. Behav. 38 (4), 723–729. https://doi.org/10.1016/00913057(91)90233-R. De Jaeger, X., Cammarota, M., Prado, M.A.M., Izquierdo, I., Prado, V.F., Pereira, G.S., 2013. Decreased acetylcholine release delays the consolidation of object recognition memory. Behav. Brain Res. 238, 62–68. https://doi.org/10.1016/j.bbr.2012.10.016. Dillon, G.M., Lubbers, L.S., Ferguson, M.T., Lao, J.Z., Huang, R.-R.C., Xiao, J.C., Fong, T. M., Hale, J.J., Rupprecht, K., Miao, S., Rowe, B.A., Kornecook, T.J., Dodart, J.-C., 2011. MK-7128, a novel CB1 receptor inverse agonist, improves scopolamineinduced learning and memory deficits in mice. Behav. Pharmacol. 22 (2), 91–100. https://doi.org/10.1097/FBP.0b013e3283423d7e. du Bois, T.M., Bell, W., Deng, C., Huang, X.-F., 2005. A high n-6 polyunsaturated fatty acid diet reduces muscarinic M2/M4 receptor binding in the rat brain. J. Chem. Neuroanat. 29 (4), 282–288. https://doi.org/10.1016/j.jchemneu.2005.03.005. El-Khadragy, M., Al-Olayan, E., Moneim, A., 2014. Neuroprotective effects of citrus reticulata in scopolamine-induced dementia oxidative stress in rats. CNS Neurol. Disord. - Drug Targets 13 (4), 684–690. https://doi.org/10.2174/ 1871527313666140618105404. Fibiger, H.C., Damsma, G., Day, J.C., 1991. Behavioral pharmacology and biochemistry of central cholinergic neurotransmission. https://doi.org/10.1007/978-1-47570145-6_23. Gedankien, T., Tan, R.J., Qasim, S.E., Moore, H., McDonagh, D., Jacobs, J., Lega, B., 2023. Acetylcholine modulates the temporal dynamics of human theta oscillations during memory. Nat. Commun. 14 (1), 5283. https://doi.org/10.1038/s41467-02341025-y. Gessa, G.L., Casu, M.A., Carta, G., Mascia, M.S., 1998. Cannabinoids decrease acetylcholine release in the medial-prefrontal cortex and hippocampus, reversal by SR 141716A. Eur. J. Pharmacol. 355 (2–3), 119–124. https://doi.org/10.1016/ s0014-2999(98)00486-5. Gessa, G.L., Mascia, M.S., Casu, M.A., Carta, G., 1997. Inhibition of hippocampal acetylcholine release by cannabinoids: reversal by SR 141716A. Eur. J. Pharmacol. 327 (1), R1–R2. https://doi.org/10.1016/s0014-2999(97)89683-5. Haam, J., Yakel, J.L., 2017. Cholinergic modulation of the hippocampal region and memory function. J. Neurochem. 142 (2), 111–121. https://doi.org/10.1111/ jnc.14052. Hescham, S., Temel, Y., Casaca-Carreira, J., Arslantas, K., Yakkioui, Y., Blokland, A., Jahanshahi, A., 2014. A neuroanatomical analysis of the effects of a memory impairing dose of scopolamine in the rat brain using cytochrome c oxidase as principle marker. J. Chem. Neuroanat. 59–60, 1–7. https://doi.org/10.1016/j. jchemneu.2014.04.001. Huang, Q., Liao, C., Ge, F., Ao, J., Liu, T., 2022. Acetylcholine bidirectionally regulates learning and memory. J. Neurorestoratol. 10 (2), 100002. https://doi.org/10.1016/ j.jnrt.2022.100002. Jamali-Raeufy, N., Nasehi, M., Ebrahimi-Ghiri, M., Zarrindast, M.R., 2011. Cross statedependency of learning between WIN55, 212-2 and scopolamine in rat dorsal hippocampus. Neurosci. Lett. 491 (3), 227–231. https://doi.org/10.1016/j. neulet.2011.01.056. Lazarova, M., Stefanova, M., Denev, P., Taseva, T., Vassileva, V., Tasheva, K., 2024. Neuroprotective effect of Marrubium vulgare extract in scopolamine-induced cognitive impairment in rats: behavioral and biochemical approaches. Biology 13 (6). https://doi.org/10.3390/biology13060426. M. Moreno-Rodríguez et al. European Journal of Pharmacology 998 (2025) 177612 9