Cannabinoid type 1 receptors in the mice prefrontal cortex regulate object location memory acquisition via GABAergic neurons

Tokutake et al. Behavioral and Brain Functions https://doi.org/10.1186/s12993-025-00306-w (2025) 21:36 Behavioral and Brain Functions Open Access RESEARCH Cannabinoid type 1 receptors in the mice prefrontal cortex regulate object location memory acquisition via GABAergic neurons Tomohiro Tokutake1, Jun Yokose1, Yusuke Yano1, Yuki Shigetsura2, Shin-Ichi Muramatsu3 and Atsumi Nitta1* Abstract Background Adverse psychiatric symptoms caused by cannabis are a significant concern, and Δ9-tetrahydrocannabinol (THC) has been identified as a key contributor to these symptoms. THC binds to cannabinoid type 1 receptors (CB1Rs), which are abundant in the brain and associated with cognition. The prefrontal cortex (PFC) is crucial for cognitive functions. However, the functions of CB1Rs in the PFC in cognition processes remain unclear. Here, we injected arachidonylcyclopropylamide (ACPA), a CB1Rs agonist, into the PFC of male C57BL/6J mice via the cannula and conducted cognitive tests, including the novel object recognition test and object location test (OLT). Results These tests assessed memory in three stages: acquisition, consolidation, and retrieval. ACPA was administered immediately before each stage, and its intra-PFC administration specifically impaired memory acquisition in the OLT. In addition, in vivo microdialysis revealed that ACPA reduced extracellular GABA levels within the PFC. Next, we produced an adeno-associated virus with a glutamic acid decarboxylase promoter and an hM3Dqencording chemogenic activator to activate GABAergic neurons in the PFC. Subsequently, deschloroclozapine (DCZ), an hM3Dq agonist, restored the memory acquisition impaired by ACPA. Conclusion Our findings suggest that CB1Rs in the PFC are involved in memory acquisition through the regulation of GABA release, offering new insights into how cannabis use lead to cognitive impairment. Keywords Cannabis, Cannabinoid type 1 receptors, Object location test, Memory, Prefrontal cortex, GABA *Correspondence: Atsumi Nitta 1 Department of Pharmaceutical Therapy & Neuropharmacology, Faculty of Pharmaceutical Sciences, University of Toyama, Toyama, Japan 2 Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Kyoto, Japan 3 Division of Neurological Gene Therapy, Open Innovation Center, Jichi Medical University, Shimotsuke, Japan Background Cannabis is a regulated substance widely used worldwide; however, its use is a major social concern [1]. Cannabis has been reported to negatively affect cognitive functions, including memory, in animals and humans [2, 3]. Notably, memory impairment is a well-known acute effect of cannabis [3]. Cannabinoid type 1 receptors (CB1Rs) in the brain are thought to be involved in the mechanism underlying memory impairment [4, 5]. These receptors have been identified as specific binding sites for Δ9-tetrahydrocannabinol (THC) [6]. CB1Rs are Gi/o protein-coupled receptors possessing seven © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creati vecommons.org/licenses/by-nc-nd/4.0/. Tokutake et al. Behavioral and Brain Functions (2025) 21:36 transmembrane domains [7, 8]. They are highly expressed in the brain [9, 10], with GABAergic interneurons in the cortex expressing more CB1Rs than excitatory neurons in axon terminals [11]. A major function of CB1Rs is to inhibit the release of neurotransmitters, including glutamate and GABA, when stimulated by an agonist [12]. However, the functions of CB1Rs in each brain region are poorly known, and effective treatment for cannabisrelated memory impairment has not been developed yet. Elucidating the mechanisms of action of CB1Rs can aid in the development of new treatments. Here, we focused on the prefrontal cortex (PFC), which is central to executive functions, including decision- making and memory [13, 14]. Several studies conducted in rodents have used recognition memory tests to measure cognitive functions, and the PFC has been identified to be tightly involved in recognition memory [15–17]. We examined the functions of CB1Rs in recognition memory in the PFC and the therapeutic targets for cannabis-caused memory impairment. The mice were administered with arachidonylcyclopropylamide (ACPA), a CB1Rs agonist, into their PFC and subjected to a recognition memory test. The stages of memory include acquisition, consolidation, and retrieval [18, 19]. We defined training as the acquisition stage, immediately after training as the consolidation stage, and test as the retrieval stage in the recognition memory tests. We administered ACPA into mice PFC right before each stage. Our study may shed light on the mechanisms of cannabis-induced memory impairment and offer a new therapeutic strategy. Methods Animals Male adult C57BL/6J mice (7–8 weeks old) of 20–25 g were purchased from Nihon SLC (Hamamatsu, Japan) and housed under controlled temperature (23 °C ± 2 °C) and humidity (50% ± 5%), and a 12 h/12 h light/dark cycle (7:00–19:00). The mice were provided standard rodent chow (CE-2, CLEA Japan, Inc., Tokyo, Japan) and water ad libitum. All mice were allowed to acclimatize to the environment for 1–2 weeks. The animal experimental protocols were approved by the Animal Care and Use Committee of the University of Toyama (Approval Number: A2023PHA14), and experiments were conducted in accordance with the Institutional Animal Experiment Handling Rules of the University of Toyama. The number of animals used was carefully estimated and kept to the minimum necessary for meaningful data interpretation. Drugs ACPA was purchased from Funakoshi (Tokyo, Japan) and dissolved in saline. ACPA (10 µg/ml) or saline was bilaterally administered into the PFC of mice 5 min before behavioral tests at 1 µl/mouse (0.5 µl/min/each side = 10 Page 2 of 11 ng/mouse) for 1 min using a micro syringe pump (ESP64; Eicom, Kyoto, Japan). The ACPA infusion dose and rate/speed were based on a previous study [20]. Deschloroclozapine (DCZ) was purchased from Med Chem Express (Monmouth Junction, NJ, USA) and dissol (...truncated)