DNA methylation regulates associative reward learning


Reward-related memories are essential for adaptive behavior and evolutionary fitness, but they are also a core component of maladaptive brain diseases such as addiction. Reward learning requires dopamine neurons located in the ventral tegmental area (VTA), which encode relationships between predictive cues and future rewards. Recent evidence suggests that epigenetic mechanisms, including DNA methylation, are essential regulators of neuronal plasticity and experience-driven behavioral change. However, the role of epigenetic mechanisms in reward learning is poorly understood. Here we show that the formation of reward-related associative memories in rats upregulates key plasticity genes in the VTA, which are correlated with memory strength and associated with gene-specific changes in DNA methylation. Moreover, DNA methylation in the VTA is required for the formation of stimulus-reward associations. These results provide the first evidence that that activity-dependent methylation and demethylation of DNA is an essential substrate for the behavioral and neuronal plasticity driven by reward-related experiences.

Figure 1: Reward learning causes experience dependent changes in VTA immediate-early gene expression that are correlated with memory strength.
Figure 2: Reward learning increases EGR1 specifically in dopamine neurons.
Figure 3: Reward-related memory formation alters VTA DNA methylation profiles at Egr1 and Fos.
Figure 4: Neuronal activity alters immediate-early gene expression and gene body DNMT binding in vitro.
Figure 5: DNA methylation is required for normal activity-induced immediate-early gene expression in vitro.
Figure 6: Reward learning requires DNA methylation in the VTA.
Figure 7: Active DNA methylation in the VTA is not required for remote memory storage or retrieval.
Figure 8: Reward learning alters gene expression in the NAc but does not require NAc DNA methylation.


  1. 1

    Flagel, S.B. et al. An animal model of genetic vulnerability to behavioral disinhibition and responsiveness to reward-related cues: implications for addiction. Neuropsychopharmacology 35, 388–400 (2010).

  2. 2

    Hyman, S.E., Malenka, R.C. & Nestler, E.J. Neural mechanisms of addiction: the role of reward-related learning and memory. Annu. Rev. Neurosci. 29, 565–598 (2006).

  3. 3

    Saunders, B.T. & Robinson, T.E. Individual variation in resisting temptation: implications for addiction. Neurosci. Biobehav. Rev. doi:10.1016/j.neubiorev.2013.02.008 (21 February 2013).

  4. 4

    Saunders, B.T., Yager, L.M. & Robinson, T.E. Preclinical studies shed light on individual variation in addiction vulnerability. Neuropsychopharmacology 38, 249–250 (2013).

  5. 5

    Fields, H.L., Hjelmstad, G.O., Margolis, E.B. & Nicola, S.M. Ventral tegmental area neurons in learned appetitive behavior and positive reinforcement. Annu. Rev. Neurosci. 30, 289–316 (2007).

  6. 6

    Stuber, G.D. et al. Reward-predictive cues enhance excitatory synaptic strength onto midbrain dopamine neurons. Science 321, 1690–1692 (2008).

  7. 7

    Day, J.J., Roitman, M.F., Wightman, R.M. & Carelli, R.M. Associative learning mediates dynamic shifts in dopamine signaling in the nucleus accumbens. Nat. Neurosci. 10, 1020–1028 (2007).

  8. 8

    Flagel, S.B. et al. A selective role for dopamine in stimulus-reward learning. Nature 469, 53–57 (2011).

  9. 9

    Schultz, W., Dayan, P. & Montague, P.R. A neural substrate of prediction and reward. Science 275, 1593–1599 (1997).

  10. 10

    Di Ciano, P., Cardinal, R.N., Cowell, R.A., Little, S.J. & Everitt, B.J. Differential involvement of NMDA, AMPA/kainate, and dopamine receptors in the nucleus accumbens core in the acquisition and performance of pavlovian approach behavior. J. Neurosci. 21, 9471–9477 (2001).

  11. 11

    Tsai, H.C. et al. Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324, 1080–1084 (2009).

  12. 12

    Yun, I.A., Wakabayashi, K.T., Fields, H.L. & Nicola, S.M. The ventral tegmental area is required for the behavioral and nucleus accumbens neuronal firing responses to incentive cues. J. Neurosci. 24, 2923–2933 (2004).

  13. 13

    Feng, J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat. Neurosci. 13, 423–430 (2010).

  14. 14

    Miller, C.A. et al. Cortical DNA methylation maintains remote memory. Nat. Neurosci. 13, 664–666 (2010).

  15. 15

    Miller, C.A. & Sweatt, J.D. Covalent modification of DNA regulates memory formation. Neuron 53, 857–869 (2007).

  16. 16

    Roth, T.L., Lubin, F.D., Funk, A.J. & Sweatt, J.D. Lasting epigenetic influence of early-life adversity on the BDNF gene. Biol. Psychiatry 65, 760–769 (2009).

  17. 17

    Weaver, I.C. et al. Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847–854 (2004).

  18. 18

    Day, J.J. & Sweatt, J.D. DNA methylation and memory formation. Nat. Neurosci. 13, 1319–1323 (2010).

  19. 19

    Guo, J.U. et al. Neuronal activity modifies the DNA methylation landscape in the adult brain. Nat. Neurosci. 14, 1345–1351 (2011).

  20. 20

    Guo, J.U., Su, Y., Zhong, C., Ming, G.L. & Song, H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011).

  21. 21

    Borrelli, E., Nestler, E.J., Allis, C.D. & Sassone-Corsi, P. Decoding the epigenetic language of neuronal plasticity. Neuron 60, 961–974 (2008).

  22. 22

    Lubin, F.D., Roth, T.L. & Sweatt, J.D. Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. J. Neurosci. 28, 10576–10586 (2008).

  23. 23

    Levenson, J.M. & Sweatt, J.D. Epigenetic mechanisms in memory formation. Nat. Rev. Neurosci. 6, 108–118 (2005).

  24. 24

    Jones, M.W. et al. A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat. Neurosci. 4, 289–296 (2001).

  25. 25

    Bramham, C.R., Worley, P.F., Moore, M.J. & Guzowski, J.F. The immediate early gene arc/arg3.1: regulation, mechanisms, and function. J. Neurosci. 28, 11760–11767 (2008).

  26. 26

    Lamprecht, R. & Dudai, Y. Transient expression of c-Fos in rat amygdala during training is required for encoding conditioned taste aversion memory. Learn. Mem. 3, 31–41 (1996).

  27. 27

    Margolis, E.B., Coker, A.R., Driscoll, J.R., Lemaitre, A.I. & Fields, H.L. Reliability in the identification of midbrain dopamine neurons. PLoS ONE 5, e15222 (2010).

  28. 28

    Margolis, E.B., Toy, B., Himmels, P., Morales, M. & Fields, H.L. Identification of rat ventral tegmental area GABAergic neurons. PLoS ONE 7, e42365 (2012).

  29. 29

    van Zessen, R., Phillips, J.L., Budygin, E.A. & Stuber, G.D. Activation of VTA GABA neurons disrupts reward consumption. Neuron 73, 1184–1194 (2012).

  30. 30

    Levenson, J.M. et al. Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J. Biol. Chem. 281, 15763–15773 (2006).

  31. 31

    Greer, P.L. & Greenberg, M.E. From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function. Neuron 59, 846–860 (2008).

  32. 32

    Kim, T.K. et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010).

  33. 33

    LaPlant, Q. et al. Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens. Nat. Neurosci. 13, 1137–1143 (2010).

  34. 34

    Deng, J.V. et al. MeCP2 in the nucleus accumbens contributes to neural and behavioral responses to psychostimulants. Nat. Neurosci. 13, 1128–1136 (2010).

  35. 35

    Im, H.I., Hollander, J.A., Bali, P. & Kenny, P.J. MeCP2 controls BDNF expression and cocaine intake through homeostatic interactions with microRNA-212. Nat. Neurosci. 13, 1120–1127 (2010).

  36. 36

    Tye, K.M., Stuber, G.D., de Ridder, B., Bonci, A. & Janak, P.H. Rapid strengthening of thalamo-amygdala synapses mediates cue-reward learning. Nature 453, 1253–1257 (2008).

  37. 37

    Parkinson, J.A., Willoughby, P.J., Robbins, T.W. & Everitt, B.J. Disconnection of the anterior cingulate cortex and nucleus accumbens core impairs Pavlovian approach behavior: further evidence for limbic cortical-ventral striatopallidal systems. Behav. Neurosci. 114, 42–63 (2000).

  38. 38

    Deroche-Gamonet, V., Belin, D. & Piazza, P.V. Evidence for addiction-like behavior in the rat. Science 305, 1014–1017 (2004).

  39. 39

    Kreek, M.J., Nielsen, D.A., Butelman, E.R. & LaForge, K.S. Genetic influences on impulsivity, risk taking, stress responsivity and vulnerability to drug abuse and addiction. Nat. Neurosci. 8, 1450–1457 (2005).

  40. 40

    Suzuki, M.M. & Bird, A. DNA methylation landscapes: provocative insights from epigenomics. Nat. Rev. Genet. 9, 465–476 (2008).

  41. 41

    Day, J.J. & Sweatt, J.D. Epigenetic mechanisms in cognition. Neuron 70, 813–829 (2011).

  42. 42

    Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).

  43. 43

    Weber, M. et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat. Genet. 37, 853–862 (2005).

  44. 44

    Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).

  45. 45

    Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

  46. 46

    Landt, S.G. et al. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 22, 1813–1831 (2012).

  47. 47

    Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B. Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).

  48. 48

    Brueckner, B. et al. Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases. Cancer Res. 65, 6305–6311 (2005).

  49. 49

    Schirrmacher, E. et al. Synthesis and in vitro evaluation of biotinylated RG108: a high affinity compound for studying binding interactions with human DNA methyltransferases. Bioconjug. Chem. 17, 261–266 (2006).

  50. 50

    Métivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50 (2008).

  51. 51

    Rajasethupathy, P. et al. A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity. Cell 149, 693–707 (2012).

  52. 52

    Maddox, S.A. & Schafe, G.E. Epigenetic alterations in the lateral amygdala are required for reconsolidation of a Pavlovian fear memory. Learn. Mem. 18, 579–593 (2011).

  53. 53

    Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates (Elsevier, New York, 2005).

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We would like to thank all members of the Sweatt laboratory, particularly G. Kaas and I. Zovkic, for comments and suggestions during the completion of these studies. We would also like to thank the Intellectual and Developmental Disabilities Research Core at the University of Alabama at Birmingham for assistance with cell culture experiments. This work is supported by the US National Institute on Drug Abuse (DA029419 to J.J.D.), the US National Institutes of Mental Health (MH091122 and MH057014 to J.D.S.) and the Evelyn F. McKnight Brain Research Foundation.

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J.J.D. and J.D.S. designed the experiments and wrote the manuscript with help from all authors. J.J.D. carried out behavioral and biochemical experiments with assistance from D.C., M.C.G.-K., M.K., J.M., E.S. and A.T.

Correspondence to J David Sweatt.

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Day, J., Childs, D., Guzman-Karlsson, M. et al. DNA methylation regulates associative reward learning. Nat Neurosci 16, 1445–1452 (2013) doi:10.1038/nn.3504

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