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Adapter protein 2-modulated \({{{\rm{\mu }}}}\)-opioid receptor trafficking in paraventricular thalamus contributes to fentanyl contextual addiction memory in mice

Acta Pharmacologica Sinica volume 47, pages 827–835 (2026)Cite this article

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.

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Fig. 1: Mu-opioid receptors (MORs) in the paraventricular thalamus (PVT) were increased on the neuronal membrane after the fentanyl CPP post-test.
Fig. 2: Proteomic analysis of differentially expressed membrane MOR-binding proteins after fentanyl CPP post-test.
Fig. 3: Binding of Ap2a1 to the MORs on the PVT cell membrane was decreased after the fentanyl CPP post-test.
Fig. 4: Antagonism or knockdown of MOR increased fentanyl contextual addiction memory retrieval.
Fig. 5: The binding sites are predicted by Alphafold4.
Fig. 6: Schematic showing the hypothesis of the Ap2a1-mediated MOR trafficking in fentanyl contextual addiction memory retrieval.

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Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (82371533, 82301677); the Natural Science Foundation of Shanghai (22ZR1449300); and the Program of Shanghai Academic/Technology Research Leader (22XD1402400).

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Author notes

  1. These authors contributed equally: Yu-jie Li, Ruo-song Chen

Authors and Affiliations

  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

    Yu-jie Li

  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

    Ruo-song Chen

  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

    Gui-ying Zan

  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

    Ying-cai Song, Bing Zhang, Wei-jia Du & Zhi-qiang Liu

  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

    Xue-ying Huang

  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

    Ti-fei Yuan

  7. Co-innovation Center of Neuroregeneration, Nantong University, Nantong, 226019, China

    Ti-fei Yuan

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Contributions

YJL, RSC, TFY, and ZQL designed the experiments; YJL, XYH, and YCS performed the experiments. Statistical data analysis was performed by WJD and XYH. YJL prepared the figures. WJD, YJL, and RSC wrote the manuscript. WJD, GYZ, and BZ provided critical comments on the study design. GYZ, BZ, and ZQL reviewed the manuscript. TFY and ZQL supervised the study.

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Correspondence to Wei-jia Du, Ti-fei Yuan or Zhi-qiang Liu.

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Li, Yj., Chen, Rs., Zan, Gy. et al. Adapter protein 2-modulated \({{{\rm{\mu }}}}\)-opioid receptor trafficking in paraventricular thalamus contributes to fentanyl contextual addiction memory in mice. Acta Pharmacol Sin 47, 827–835 (2026). https://doi.org/10.1038/s41401-025-01684-7

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