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Adapter protein 2-modulated μ-opioid receptor trafficking in paraventricular thalamus contributes to fentanyl contextual addiction memory in mice

Yu-jie Li1, Ruo-song Chen2, Gui-ying Zan3, Ying-cai Song4, Xue-ying Huang5, Bing Zhang4, Wei-jia Du4, Ti-fei Yuan6,7, Zhi-qiang Liu4
1 Department of Anesthesiology, Shanghai Key Laboratory of Maternal Fetal Medicina, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai 200092, China
2 Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Key Laboratory of Acupuncture and Neurobiology of Zhejiang Province, Hangzhou 310053, China
3 CAS Key Laboratory of Receptor Research and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
4 Department of Anesthesiology, Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Lab of Reproduction and Development, Shanghai Key Lab of Female Reproductive Endocrine Related Diseases, Shanghai 200433, China
5 Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai 200092, China
6 Shanghai Key Laboratory of Psychotic Disorders, Brain Health Institute, National Center of Mental Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine and School of Psychlogy, Shanghai 200030, China
7 Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226019, China
Correspondence to: Wei-jia Du: duweijia10317@fckyy.org.cn, Ti-fei Yuan: ytf0707@126.com, Zhi-qiang Liu: drliuzhiqiang@163.com,
DOI: 10.1038/s41401-025-01684-7
Received: 25 May 2025
Accepted: 24 September 2025
Advance online: 1 December 2025

Abstract

Fentanyl and its analogues are the most commonly used synthetic opioid analgesics in clinical practice, but their abuse is a significant concern. Drug-paired environmental cues often trigger memory retrieval, leading to relapse, complicating treatment and overdose prevention. In this study we investigated μ-opioid receptor-related molecular mechanisms underlying the retrieval of fentanyl contextual addiction memory in mice. A conditional place preference (CPP) model was established in mice by citrate injections of fentanyl (0.1 mg/kg) for 4 days. By performing whole-brain screening using c-Fos immunofluorescence staining, we found that the paraventricular thalamus (PVT) was dramatically activated. We conducted Western blotting, co- immunoprecipitation and proteomics to evaluate the proteins interacting with μ-opioid receptors on the membrane, and found marked externalization of μ-opioid receptors on the membrane in PVT neurons. We revealed that μ-opioid receptors trafficking in PVT was regulated by the extent of binding of Ap2a1 to the membrane μ-opioid receptors. By conditional knockdown and chemogenetic manipulation, we demonstrated the contribution of μ-opioid receptors to the retrieval of fentanyl contextual memory via modulating the neuronal activity in PVT. In conclusion, this study suggests that Ap2a1-mediated trafficking of μ-opioid receptors underlies the retrieval of fentanyl contextual addiction memory through regulating the neuronal activity in PVT.
Keywords: fentanyl; contextual addiction memory; paraventricular thalamus; μ-opioid receptor trafficking; Ap2a1

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