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Epac2-mediated synaptic insertion of Ca2+-permeable AMPARs in the nucleus accumbens contributes to incubation of cocaine craving

Neuropsychopharmacology volume 50, pages 620–629 (2025)Cite this article

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Abstract

The accumulation of GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) in the medium spiny neurons (MSNs) of the nucleus accumbens (NAc) is required for the expression of incubation of cocaine craving. The exchange protein directly activated by cAMP (Epac) is an intracellular effector of cAMP and a guanine nucleotide exchange factor for the small GTPase Rap1. Epac2 has been implicated in the trafficking of AMPA receptors at central synapses. We tested the hypothesis that Epac2 activation contributes to the accumulation of CP-AMPARs in NAc MSNs and incubation of cocaine craving. Here we demonstrate that the selective Epac2 agonist S-220 facilitated the synaptic insertion of GluA2-lacking CP-AMPARs at excitatory synapses onto NAc MSNs. In addition, prolonged abstinence from cocaine self-administration in rats resulted in elevated Rap1-GTP levels in the NAc, implying that Epac2 is activated during incubation. Importantly, we show that AAV-mediated shRNA knockdown of Epac2 in the NAc core attenuated the accumulation of CP-AMPARs and cue-induced drug-seeking behavior after prolonged abstinence from cocaine self-administration. In contrast, acute pharmacological inhibition of Epac2 with the selective Epac2 inhibitor ESI-05 did not alter CP-AMPARs that had already accumulated during incubation, and intra-NAc application of ESI-05 did not significantly affect cue-induced drug seeking following prolonged abstinence. Taken together, these results suggest that Epac2 activation during the period of incubation, but not during cue-induced drug seeking, leads to the accumulation of CP-AMPARs in NAc MSNs, which in turn contributes to incubation of cocaine craving.

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Fig. 1: The Epac2 agonist S-220 increased CP-AMPARs in MSNs in the NAc core.
Fig. 2: Prolonged abstinence from cocaine IVSA increased Rap1-GTP levels in the NAc.
Fig. 3: Prolonged abstinence from cocaine IVSA increased CP-AMPARs in MSNs of the NAc core.
Fig. 4: Verification of shRNA knockdown of Epac2 in the NAc core.
Fig. 5: shRNA knockdown of Epac2 in the NAc core attenuated incubation-induced cocaine craving and accumulating of CP-APMARs.

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References

  1. Volkow ND, Li TK. Drug addiction: the neurobiology of behaviour gone awry. Nature reviews. 2004;5:963–70.

    CAS  PubMed  Google Scholar 

  2. Parvaz MA, Moeller SJ, Goldstein RZ. Incubation of Cue-Induced Craving in Adults Addicted to Cocaine Measured by Electroencephalography. JAMA Psychiatry. 2016;73:1127–34.

    PubMed  PubMed Central  Google Scholar 

  3. Bedi G, Preston KL, Epstein DH, Heishman SJ, Marrone GF, Shaham Y, et al. Incubation of cue-induced cigarette craving during abstinence in human smokers. Biol Psychiatry. 2011;69:708–11.

    PubMed  PubMed Central  Google Scholar 

  4. Wang G, Shi J, Chen N, Xu L, Li J, Li P, et al. Effects of length of abstinence on decision-making and craving in methamphetamine abusers. PLoS One. 2013;8:e68791.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Li P, Wu P, Xin X, Fan YL, Wang GB, Wang F, et al. Incubation of alcohol craving during abstinence in patients with alcohol dependence. Addict Biol. 2015;20:513–22.

    PubMed  Google Scholar 

  6. Grimm JW, Hope BT, Wise RA, Shaham Y. Neuroadaptation. Incubation of cocaine craving after withdrawal. Nature. 2001;412:141–2.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Wolf ME. Synaptic mechanisms underlying persistent cocaine craving. Nature reviews. 2016;17:351–65.

    CAS  PubMed  Google Scholar 

  8. Lu L, Grimm JW, Hope BT, Shaham Y. Incubation of cocaine craving after withdrawal: a review of preclinical data. Neuropharmacology. 2004;47:214–26.

    CAS  PubMed  Google Scholar 

  9. Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacological reviews. 2010;62:405–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Greger IH, Watson JF, Cull-Candy SG. Structural and Functional Architecture of AMPA-Type Glutamate Receptors and Their Auxiliary Proteins. Neuron. 2017;94:713–30.

    CAS  PubMed  Google Scholar 

  11. Conrad KL, Tseng KY, Uejima JL, Reimers JM, Heng LJ, Shaham Y, et al. Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving. Nature. 2008;454:118–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Loweth JA, Scheyer AF, Milovanovic M, LaCrosse AL, Flores-Barrera E, Werner CT, et al. Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving. Nat Neurosci. 2014;17:73–80.

    CAS  PubMed  Google Scholar 

  13. Kawa AB, Hwang EK, Funke JR, Zhou H, Costa-Mattioli M, Wolf ME. Positive Allosteric Modulation of mGlu(1) Reverses Cocaine-Induced Behavioral and Synaptic Plasticity Through the Integrated Stress Response and Oligophrenin-1. Biological psychiatry. 2022;92:871–79.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Terrier J, Lüscher C, Pascoli V. Cell-Type Specific Insertion of GluA2-Lacking AMPARs with Cocaine Exposure Leading to Sensitization, Cue-Induced Seeking, and Incubation of Craving. Neuropsychopharmacology. 2016;41:1779–89.

    CAS  PubMed  Google Scholar 

  15. Loweth JA, Tseng KY, Wolf ME. Adaptations in AMPA receptor transmission in the nucleus accumbens contributing to incubation of cocaine craving. Neuropharmacology. 2014;76:287–300.

    CAS  PubMed  Google Scholar 

  16. McCutcheon JE, Loweth JA, Ford KA, Marinelli M, Wolf ME, Tseng KY. Group I mGluR activation reverses cocaine-induced accumulation of calcium-permeable AMPA receptors in nucleus accumbens synapses via a protein kinase C-dependent mechanism. J Neurosci. 2011;31:14536–41.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Loweth JA, Reimers JM, Caccamise A, Stefanik MT, Woo KKY, Chauhan NM, et al. mGlu1 tonically regulates levels of calcium-permeable AMPA receptors in cultured nucleus accumbens neurons through retinoic acid signaling and protein translation. The European journal of neuroscience. 2019;50:2590–601.

    PubMed  Google Scholar 

  18. Wunsch AM, Hwang EK, Funke JR, Baker R, Moutier A, Milovanovic M, et al. Retinoic acid-mediated homeostatic plasticity in the nucleus accumbens core contributes to incubation of cocaine craving. Psychopharmacology. 2024;241:1983–2001.

    CAS  PubMed  Google Scholar 

  19. Scheyer AF, Wolf ME, Tseng KY. A protein synthesis-dependent mechanism sustains calcium-permeable AMPA receptor transmission in nucleus accumbens synapses during withdrawal from cocaine self-administration. J Neurosci. 2014;34:3095–100.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Tsubokawa H, Oguro K, Masuzawa T, Nakaima T, Kawai N. Effects of a spider toxin and its analogue on glutamate-activated currents in the hippocampal CA1 neuron after ischemia. J Neurophysiol. 1995;74:218–25.

    CAS  PubMed  Google Scholar 

  21. Lee BR, Ma YY, Huang YH, Wang X, Otaka M, Ishikawa M, et al. Maturation of silent synapses in amygdala-accumbens projection contributes to incubation of cocaine craving. Nat Neurosci. 2013;16:1644–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ma YY, Lee BR, Wang X, Guo C, Liu L, Cui R, et al. Bidirectional modulation of incubation of cocaine craving by silent synapse-based remodeling of prefrontal cortex to accumbens projections. Neuron. 2014;83:1453–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Grimm JW, Lu L, Hayashi T, Hope BT, Su TP, Shaham Y. Time-dependent increases in brain-derived neurotrophic factor protein levels within the mesolimbic dopamine system after withdrawal from cocaine: implications for incubation of cocaine craving. J Neurosci. 2003;23:742–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Li X, DeJoseph MR, Urban JH, Bahi A, Dreyer JL, Meredith GE, et al. Different roles of BDNF in nucleus accumbens core versus shell during the incubation of cue-induced cocaine craving and its long-term maintenance. J Neurosci. 2013;33:1130–42.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. de Rooij J, Zwartkruis FJT, Verheijen MHG, Cool RH, Nijman SMB, Wittinghofer A, et al. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature. 1998;396:474–7.

    PubMed  Google Scholar 

  26. Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, et al. A family of cAMP-binding proteins that directly activate Rap1. Science. 1998;282:2275–9.

    CAS  PubMed  Google Scholar 

  27. Woolfrey KM, Srivastava DP, Photowala H, Yamashita M, Barbolina MV, Cahill ME, et al. Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines. Nat Neurosci. 2009;12:1275–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhu JJ, Qin Y, Zhao M, Van Aelst L, Malinow R. Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Cell. 2002;110:443–55.

    CAS  PubMed  Google Scholar 

  29. Liu X, Chen Y, Tong J, Reynolds AM, Proudfoot SC, Qi J, et al. Epac Signaling Is Required for Cocaine-Induced Change in AMPA Receptor Subunit Composition in the Ventral Tegmental Area. J Neurosci. 2016;36:4802–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mu L, Liu X, Yu H, Hu M, Friedman V, Kelly TJ, et al. Ibudilast attenuates cocaine self-administration and prime- and cue-induced reinstatement of cocaine seeking in rats. Neuropharmacology. 2021;201:108830.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Ting JT, Lee BR, Chong P, Soler-Llavina G, Cobbs C, Koch C, et al. Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method. Journal of visualized experiments : JoVE. 2018.

  32. Mu L, Liu X, Yu H, Vickstrom CR, Friedman V, Kelly TJ, et al. cAMP-mediated upregulation of HCN channels in VTA dopamine neurons promotes cocaine reinforcement. Molecular psychiatry. 2023.

  33. Liu X, Li Y, Yu L, Vickstrom CR, Liu QS. VTA mTOR Signaling Regulates Dopamine Dynamics, Cocaine-Induced Synaptic Alterations, and Reward. Neuropsychopharmacology. 2018;43:1066–77.

    CAS  PubMed  Google Scholar 

  34. Koh DS, Burnashev N, Jonas P. Block of native Ca(2+)-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification. J Physiol. 1995;486:305–12.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Isaac JT, Ashby MC, McBain CJ. The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity. Neuron. 2007;54:859–71.

    CAS  PubMed  Google Scholar 

  36. Schwede F, Bertinetti D, Langerijs CN, Hadders MA, Wienk H, Ellenbroek JH, et al. Structure-guided design of selective Epac1 and Epac2 agonists. PLoS Biol. 2015;13:e1002038.

    PubMed  PubMed Central  Google Scholar 

  37. Collingridge GL, Isaac JT, Wang YT. Receptor trafficking and synaptic plasticity. Nature reviews. 2004;5:952–62.

    CAS  PubMed  Google Scholar 

  38. Daw MI, Chittajallu R, Bortolotto ZA, Dev KK, Duprat F, Henley JM, et al. PDZ proteins interacting with C-terminal GluR2/3 are involved in a PKC-dependent regulation of AMPA receptors at hippocampal synapses. Neuron. 2000;28:873–86.

    CAS  PubMed  Google Scholar 

  39. Ster J, de Bock F, Bertaso F, Abitbol K, Daniel H, Bockaert J, et al. Epac mediates PACAP-dependent long-term depression in the hippocampus. J Physiol. 2009;587:101–13.

    CAS  PubMed  Google Scholar 

  40. van Triest M, de Rooij J, Bos JL. Measurement of GTP-bound Ras-like GTPases by activation-specific probes. Methods in enzymology. 2001;333:343–8.

    PubMed  Google Scholar 

  41. Swanson GT, Kamboj SK, Cull-Candy SG. Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition. J Neurosci. 1997;17:58–69.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Wilson CJ, Kawaguchi Y. The origins of two-state spontaneous membrane potential fluctuations of neostriatal spiny neurons. J Neurosci. 1996;16:2397–410.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Mameli M, Bellone C, Brown MT, Luscher C. Cocaine inverts rules for synaptic plasticity of glutamate transmission in the ventral tegmental area. Nat Neurosci. 2011;14:414–6.

    CAS  PubMed  Google Scholar 

  44. Bellone C, Luscher C. Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression. Nat Neurosci. 2006;9:636–41.

    CAS  PubMed  Google Scholar 

  45. Anderson SM, Pierce RC. Cocaine-induced alterations in dopamine receptor signaling: implications for reinforcement and reinstatement. Pharmacology & therapeutics. 2005;106:389–403.

    CAS  Google Scholar 

  46. Nestler EJ. Molecular basis of long-term plasticity underlying addiction. Nature reviews. 2001;2:119–28.

    CAS  PubMed  Google Scholar 

  47. Self DW. Regulation of drug-taking and -seeking behaviors by neuroadaptations in the mesolimbic dopamine system. Neuropharmacology. 2004;47:242–55.

    CAS  PubMed  Google Scholar 

  48. Nestler EJ. Reflections on: "A general role for adaptations in G-Proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function". Brain research. 2016;1645:71–4.

    CAS  PubMed  Google Scholar 

  49. Wolf ME, Tseng KY. Calcium-permeable AMPA receptors in the VTA and nucleus accumbens after cocaine exposure: when, how, and why? Frontiers in molecular neuroscience. 2012;5:72.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Bock A, Annibale P, Konrad C, Hannawacker A, Anton SE, Maiellaro I, et al. Optical Mapping of cAMP Signaling at the Nanometer Scale. Cell. 2020;182:1519–30.e17.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhang JZ, Lu TW, Stolerman LM, Tenner B, Yang JR, Zhang JF, et al. Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling. Cell. 2020;182:1531–44.e15.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Anton SE, Kayser C, Maiellaro I, Nemec K, Möller J, Koschinski A, et al. Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling. Cell. 2022;185:1130–42.e11.

    CAS  PubMed  Google Scholar 

  53. Sartre C, Peurois F, Ley M, Kryszke MH, Zhang W, Courilleau D, et al. Membranes prime the RapGEF EPAC1 to transduce cAMP signaling. Nature communications. 2023;14:4157.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Esteban JA, Shi SH, Wilson C, Nuriya M, Huganir RL, Malinow R. PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity. Nat Neurosci. 2003;6:136–43.

    CAS  PubMed  Google Scholar 

  55. Banke TG, Bowie D, Lee H, Huganir RL, Schousboe A, Traynelis SF. Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase. J Neurosci. 2000;20:89–102.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Man HY, Sekine-Aizawa Y, Huganir RL. Regulation of {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking through PKA phosphorylation of the Glu receptor 1 subunit. Proc Natl Acad Sci USA. 2007;104:3579–84.

    PubMed  PubMed Central  Google Scholar 

  57. Laurent AC, Breckler M, Berthouze M, Lezoualc'h F. Role of Epac in brain and heart. Biochemical Society transactions. 2012;40:51–7.

    CAS  PubMed  Google Scholar 

  58. Cahill ME, Bagot RC, Gancarz AM, Walker DM, Sun H, Wang ZJ, et al. Bidirectional Synaptic Structural Plasticity after Chronic Cocaine Administration Occurs through Rap1 Small GTPase Signaling. Neuron. 2016;89:566–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Weber SJ, Kawa AB, Beutler MM, Kuhn HM, Moutier AL, Westlake JG, et al. Dopamine transmission at D1 and D2 receptors in the nucleus accumbens contributes to the expression of incubation of cocaine craving. Neuropsychopharmacology. 2024 Online ahead of print https://doi.org/10.1038/s41386-024-01992-2.

  60. Schmidt HD, Pierce RC. Cooperative activation of D1-like and D2-like dopamine receptors in the nucleus accumbens shell is required for the reinstatement of cocaine-seeking behavior in the rat. Neuroscience. 2006;142:451–61.

    CAS  PubMed  Google Scholar 

  61. Savell KE, Tuscher JJ, Zipperly ME, Duke CG, Phillips RA 3rd, Bauman AJ, et al. A dopamine-induced gene expression signature regulates neuronal function and cocaine response. Science advances. 2020;6:eaba4221.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Xi ZX, Li X, Li J, Peng XQ, Song R, Gaal J, et al. Blockade of dopamine D3 receptors in the nucleus accumbens and central amygdala inhibits incubation of cocaine craving in rats. Addiction biology. 2013;18:665–77.

    CAS  PubMed  Google Scholar 

  63. Lu L, Dempsey J, Liu SY, Bossert JM, Shaham Y. A single infusion of brain-derived neurotrophic factor into the ventral tegmental area induces long-lasting potentiation of cocaine seeking after withdrawal. J Neurosci. 2004;24:1604–11.

    CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by National Institutes of Health Grants R01DA035217 and R01DA047269 (QSL) and F31DA054759 (to VF). TJK is member of the Medical Scientist Training Program at MCW, which is partially supported by a training grant from NIGMS T32-GM080202.

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  1. Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA

    Xiaojie Liu, Yao Huang, Lianwei Mu, Vladislav Friedman, Thomas J. Kelly, Ying Hu, Dong Yuan & Qing-song Liu

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  1. Xiaojie Liu
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Contributions

XL, YH, LM, and QSL made contributions to the conception and design of the work, drafting, and revising the manuscript, and analysis and interpretation of data. XL, YH, LM, VF, TJK, YH, and DY performed experiments, analyzed the data, and contributed to writing the manuscript.

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Correspondence to Xiaojie Liu or Qing-song Liu.

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Liu, X., Huang, Y., Mu, L. et al. Epac2-mediated synaptic insertion of Ca2+-permeable AMPARs in the nucleus accumbens contributes to incubation of cocaine craving. Neuropsychopharmacol. 50, 620–629 (2025). https://doi.org/10.1038/s41386-024-02030-x

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