Cue-induced methamphetamine (METH) craving intensifies over weeks of abstinence (incubation of craving), and its expression depends on accumulation of Ca2+-permeable AMPA receptors (CP-AMPARs) at nucleus accumbens core synapses. A 2019 study of METH incubation found that GluA1 translation is elevated in accumbens core during late withdrawal but, unlike cocaine, with no detectable change in surface or total AMPAR subunits, mGlu1, or mGlu1-Homer coupling, leaving the translation finding correlational. The open problem is to establish causally whether and how elevated GluA1 translation during METH withdrawal produces synaptic CP-AMPAR accumulation and incubated seeking; to identify the upstream withdrawal-dependent mechanism, in particular whether it is the retinoic-acid-dependent homeostatic cascade shown in 2024-2025 to drive CP-AMPAR accumulation in cocaine incubation or a distinct pathway; to localize the relevant receptor pool at subcellular resolution; and to determine how accumbens-core plasticity cooperates with central amygdala and dorsal striatum in the expression of incubated METH craving. As of August 2026 the METH side of this question remains untested.

Background

Relapse to methamphetamine (METH) use is preceded by cue-induced craving that paradoxically grows stronger the longer abstinence lasts. This phenomenon, termed incubation of craving, is modeled in rats by extended-access intravenous drug self-administration followed by forced abstinence: cue-induced drug seeking is low on withdrawal day 1 (WD1) and progressively intensifies over subsequent weeks, remaining elevated for months. Incubation of cue-induced craving is also observed in humans, which makes the rat model a leading framework for the persistence of relapse vulnerability.

Much of the mechanistic work has located the critical plasticity in the nucleus accumbens (NAc), a ventral-striatal structure that integrates dopaminergic and glutamatergic input to govern motivated behavior; its core subregion (NAc core) is required for cue-controlled drug seeking. The principal NAc neurons are medium spiny neurons (MSNs), GABAergic projection neurons that can be classified by whether they express dopamine D1 or D2 receptors. Fast excitatory transmission onto MSNs is mediated by AMPA receptors (AMPARs). Most AMPARs contain the GluA2 subunit and are Ca2+-impermeable; AMPARs lacking GluA2 (largely homomeric GluA1 receptors), termed Ca2+-permeable AMPARs (CP-AMPARs), pass calcium, have high single-channel conductance, and are scarce at NAc synapses in drug-naive adult rats.

For cocaine, incubated seeking ultimately depends on accumulation of CP-AMPARs at NAc core synapses (Conrad et al. 2008): by about one month of abstinence CP-AMPAR levels are stably elevated, and removing or blocking them (with Naspm, a CP-AMPAR-selective blocker, or via activation of the metabotropic glutamate receptor mGlu1, a G-protein-coupled receptor whose stimulation triggers CP-AMPAR internalization) abolishes the expression of incubated cocaine seeking. Maintaining this potentiated state requires ongoing protein synthesis in the NAc: inhibiting translation in the NAc core normalizes CP-AMPAR transmission and attenuates incubated cocaine seeking (Scheyer et al. 2014; Werner et al. 2018), and translation of GluA1, but not GluA2, is elevated after incubation of cocaine craving (Stefanik et al. 2018). Two early-withdrawal cocaine adaptations are thought to permit CP-AMPAR accumulation: a withdrawal-dependent decrease in mGlu1 surface expression (mGlu1 normally limits synaptic CP-AMPAR levels) and the emergence of NMDA receptors containing the atypical GluN3 subunit (Christian et al. 2021). In cultured NAc neurons, lowering mGlu1 tone raises surface CP-AMPAR levels through retinoic-acid signaling and protein translation (Loweth et al. 2019). For METH, the same synaptic endpoint is reached faster: CP-AMPARs are elevated in NAc core by WD7-8, and their removal blocks expression of incubated METH seeking (Scheyer et al. 2016).

The source of this problem, Murray et al. (2019), asked which cocaine-associated biochemical adaptations accompany METH incubation, and the answer sharpened rather than settled the mechanism. On WD30, when CP-AMPARs are stably elevated, newly synthesized GluA1, labeled by puromycin incorporation into nascent polypeptide chains followed by GluA1 immunoprecipitation, was elevated in METH rats, exactly as found for cocaine. But, in contrast to cocaine, no change was detected in surface or total GluA1, GluA2, or GluA3 (assessed by surface biotinylation, which labels plasma-membrane proteins, and immunoblotting), nor in mGlu1 surface or total levels, in coupling of mGlu1 to Homer scaffolding proteins (postsynaptic proteins that link group-I mGluRs to intracellular effectors), or in mGlu5 (the other group-I metabotropic glutamate receptor) function (Murray et al. 2021). Repeated injections of a positive allosteric modulator of mGlu1 during the rising phase of incubation failed to delay METH incubation. Thus elevated GluA1 translation is the one shared biochemical adaptation identified so far, yet it is a correlation without demonstrated necessity, and the newly translated GluA1 pool is biochemically invisible at the whole-tissue level.

For cocaine this mechanistic gap has since been largely filled. In retinoic-acid-mediated homeostatic synaptic scaling, a form of plasticity in which prolonged reduction of neuronal activity disinhibits retinoic-acid (RA) synthesis in dendrites, RA acting on its dendritic receptor RARalpha rapidly increases GluA1 translation and synaptic insertion of homomeric GluA1 CP-AMPARs. Wunsch et al. (2024), using viral vectors expressing short hairpin RNA (shRNA, a gene-silencing method) to manipulate the RA-synthetic enzyme Aldh1a1 and the RA-degradative enzyme Cyp26b1 in NAc core, showed that RA signaling bidirectionally controls CP-AMPAR accumulation and incubated cocaine seeking, and Hwang et al. (2025) showed that interrupting RA signaling in the NAc core of incubated rats normalizes both the elevated CP-AMPAR levels and incubated cocaine seeking, with the effect confined to D1-type MSNs. Kawa et al. (2025) went on to map cell-type-specific changes in actively translating mRNAs during cocaine incubation. Whether this cascade drives CP-AMPAR accumulation during METH withdrawal is unknown, and the question is non-trivial: METH incubation lacks the early-withdrawal mGlu1 decrease, and GluN3-containing NMDARs have not been examined in the METH incubation model, so a shared mechanism would require a different trigger (or an as-yet-untested trigger). CP-AMPAR accumulation in NAc also accompanies incubation of oxycodone craving (Wong et al. 2022), so identifying which upstream pathways are shared across drugs is a general question about how addictive drugs converge on this synaptic endpoint. At the circuit level, expression of incubated METH seeking depends on the central amygdala (CeA) and dorsal striatum (DS), whereas cocaine incubation additionally requires ventromedial prefrontal cortex; how NAc core plasticity relates to these other withdrawal-sensitive regions during expression of METH incubation remains unknown (Murray et al. 2019; Chow et al. 2025).

Problem Statement

Establish, with causal evidence in the rat incubation model, the mechanistic chain in nucleus accumbens core that links withdrawal from methamphetamine self-administration to the synaptic accumulation of homomeric GluA1 CP-AMPARs that mediates incubated METH craving, and determine how this pathway relates to the corresponding cocaine mechanism. Concretely, the problem comprises four linked questions; resolving the problem requires addressing all four, and an answer to a proper subset is a partial result rather than a resolution:

  1. Causality of translation: test whether elevated GluA1 translation during METH withdrawal is necessary, sufficient, or both for synaptic CP-AMPAR accumulation in NAc core and for expression of incubated METH seeking. At present the link is only correlational.

  2. Upstream mechanism and drug comparison: identify the withdrawal-dependent signal that elevates GluA1 translation in METH incubation, and determine whether it is the retinoic-acid/RARalpha-dependent homeostatic cascade established causally for cocaine (Wunsch et al. 2024; Hwang et al. 2025) or a distinct pathway. Note that METH incubation lacks the early-withdrawal decrease in mGlu1 surface expression, and GluN3-containing NMDARs have not been examined in the METH incubation model, so a shared mechanism would require a different trigger (or an as-yet-untested trigger), making this a substantive comparative question rather than an extrapolation.

  3. Subcellular and cellular locus: determine where the relevant plasticity resides, that is, whether newly translated GluA1 is preferentially inserted at synapses and in which compartment and MSN subtype, given that bulk surface biotinylation and immunoblotting detect no change in GluA1 protein and the postsynaptic-density-associated pool has not been directly measured.

  4. Circuit embedding: establish how this NAc core plasticity interacts with the other withdrawal-sensitive regions required for METH incubation, notably the central amygdala and dorsal striatum, to produce the expression of incubated craving.

References

  1. Murray CH, Loweth JA, Milovanovic M, Stefanik MT, Caccamise AJ, Dolubizno H, Funke JR, Foster Olive M, Wolf ME (2019). AMPA receptor and metabotropic glutamate receptor 1 adaptations in the nucleus accumbens core during incubation of methamphetamine craving. Neuropsychopharmacology 44(9):1534-1541. doi:10.1038/s41386-019-0425-5. https://doi.org/10.1038/s41386-019-0425-5
  2. Scheyer AF, Loweth JA, Christian DT, Uejima J, Rabei R, Le T, Dolubizno H, Stefanik MT, Murray CH, Sakas C, Wolf ME (2016). AMPA receptor plasticity in accumbens core contributes to incubation of methamphetamine craving. Biological Psychiatry 80(9):661-670. doi:10.1016/j.biopsych.2016.04.003. https://doi.org/10.1016/j.biopsych.2016.04.003
  3. Conrad KL, Tseng KY, Uejima JL, Reimers JM, Heng LJ, Shaham Y, Marinelli M, Wolf ME (2008). Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving. Nature 454(7200):118-121. doi:10.1038/nature06995. https://doi.org/10.1038/nature06995
  4. Scheyer AF, Wolf ME, Tseng KY (2014). A protein synthesis-dependent mechanism sustains calcium-permeable AMPA receptor transmission in nucleus accumbens synapses during withdrawal from cocaine self-administration. The Journal of Neuroscience 34(8):3095-3100. doi:10.1523/JNEUROSCI.4940-13.2014. https://doi.org/10.1523/JNEUROSCI.4940-13.2014
  5. Stefanik MT, Milovanovic M, Werner CT, Spainhour JCG, Wolf ME (2018). Withdrawal from cocaine self-administration alters the regulation of protein translation in the nucleus accumbens. Biological Psychiatry 84(3):223-232. doi:10.1016/j.biopsych.2018.02.012. https://doi.org/10.1016/j.biopsych.2018.02.012
  6. Werner CT, Stefanik MT, Milovanovic M, Caccamise A, Wolf ME (2018). Protein translation in the nucleus accumbens is dysregulated during cocaine withdrawal and required for expression of incubation of cocaine craving. The Journal of Neuroscience 38(11):2683-2697. doi:10.1523/JNEUROSCI.2412-17.2018. https://doi.org/10.1523/JNEUROSCI.2412-17.2018
  7. Loweth JA, Reimers JM, Caccamise A, Stefanik MT, Woo KKY, Chauhan NM, Werner CT, Wolf ME (2019). mGlu1 tonically regulates levels of calcium-permeable AMPA receptors in cultured nucleus accumbens neurons through retinoic acid signaling and protein translation. European Journal of Neuroscience 50(3):2590-2601. doi:10.1111/ejn.14151. https://doi.org/10.1111/ejn.14151
  8. Christian DT, Stefanik MT, Bean LA, Loweth JA, Wunsch AM, Funke JR, Briggs CA, Lyons J, Neal D, Milovanovic M, D'Souza GX, Stutzmann GE, Nicholson DA, Tseng KY, Wolf ME (2021). GluN3-containing NMDA receptors in the rat nucleus accumbens core contribute to incubation of cocaine craving. The Journal of Neuroscience 41(39):8262-8277. doi:10.1523/JNEUROSCI.0406-21.2021. https://doi.org/10.1523/JNEUROSCI.0406-21.2021
  9. Murray CH, Christian DT, Milovanovic M, Loweth JA, Hwang EK, Caccamise AJ, Funke JR, Wolf ME (2021). mGlu5 function in the nucleus accumbens core during the incubation of methamphetamine craving. Neuropharmacology 186:108452. doi:10.1016/j.neuropharm.2021.108452. https://doi.org/10.1016/j.neuropharm.2021.108452
  10. Wunsch AM, Hwang EK, Funke JR, Baker R, Moutier A, Milovanovic M, Green TA, Wolf ME (2024). Retinoic acid-mediated homeostatic plasticity in the nucleus accumbens core contributes to incubation of cocaine craving. Psychopharmacology 241(10):1983-2001. doi:10.1007/s00213-024-06612-x. https://doi.org/10.1007/s00213-024-06612-x
  11. Hwang EK, Wunsch AM, Wolf ME (2025). Retinoic acid-mediated homeostatic plasticity drives cell type-specific CP-AMPAR accumulation in nucleus accumbens core and incubation of cocaine craving. Molecular Psychiatry 30(7):3175-3187. doi:10.1038/s41380-025-03026-9. https://doi.org/10.1038/s41380-025-03026-9
  12. Funke JR, Hwang EK, Wunsch AM, Baker R, Engeln KA, Murray CH, Milovanovic M, Caccamise AJ, Wolf ME (2023). Persistent neuroadaptations in the nucleus accumbens core accompany incubation of methamphetamine craving in male and female rats. eNeuro 10(3):ENEURO.0480-22.2023. doi:10.1523/ENEURO.0480-22.2023. https://doi.org/10.1523/ENEURO.0480-22.2023
  13. Chow JJ, Pitts KM, Negishi K, Madangopal R, Dong Y, Wolf ME, Shaham Y (2025). Neurobiology of the incubation of drug craving: An update. Pharmacological Reviews 77(2):100022. doi:10.1016/j.pharmr.2024.100022. https://doi.org/10.1016/j.pharmr.2024.100022
  14. Wolf ME (2025). Targeting neuroplasticity in substance use disorders: Implications for therapeutics. Annual Review of Pharmacology and Toxicology 65:259-280. doi:10.1146/annurev-pharmtox-061724-080548. https://doi.org/10.1146/annurev-pharmtox-061724-080548
  15. Kawa AB, Hashimoto JG, Beutler MM, Guizzetti M, Wolf ME (2025). Changes in nucleus accumbens core translatome accompanying incubation of cocaine craving. Neuropsychopharmacology 50(8):1305-1316. doi:10.1038/s41386-025-02112-4. https://doi.org/10.1038/s41386-025-02112-4
  16. Wong B, Zimbelman AR, Milovanovic M, Wolf ME, Stefanik MT (2022). GluA2-lacking AMPA receptors in the nucleus accumbens core and shell contribute to the incubation of oxycodone craving in male rats. Addiction Biology 27(6):e13237. doi:10.1111/adb.13237. https://doi.org/10.1111/adb.13237
  17. Daiwile AP, McCoy MT, Ladenheim B, Subramaniam J, Cadet JL (2024). Incubation of methamphetamine craving in punishment-resistant individuals is associated with activation of specific gene networks in the rat dorsal striatum. Molecular Psychiatry 29(7):1990-2000. doi:10.1038/s41380-024-02455-2. https://doi.org/10.1038/s41380-024-02455-2
  18. Daiwile AP, Ladenheim B, Jayanthi S, Cadet JL (2025). Punishment-induced suppression of methamphetamine self-administration is accompanied by activation of the CPEB4/GLD2 polyadenylation complex of the translational machinery. International Journal of Molecular Sciences 26(6):2734. doi:10.3390/ijms26062734. https://doi.org/10.3390/ijms26062734
  19. Altshuler RD, Burke MAM, Garcia KT, Class K, Cimbro R, Li X (2025). Profiling gene alterations in striatonigral neurons associated with incubation of methamphetamine craving by cholera toxin subunit B-based fluorescence-activated cell sorting. Frontiers in Cellular Neuroscience 19:1542508. doi:10.3389/fncel.2025.1542508. https://doi.org/10.3389/fncel.2025.1542508
  20. Shi S, Sun YW, Ding JJ, Cheng YJ, Wu MQ, Su H, Gong WK, Liu F, Yuan TF, Zhao M (2026). Circuit and molecular mechanisms underlying incubation of methamphetamine craving in the prelimbic cortex. Neuron 114(13):2456-2472.e7. doi:10.1016/j.neuron.2026.02.017. https://doi.org/10.1016/j.neuron.2026.02.017