Function of the Drosophila CPEB protein Orb2 in long-term courtship memory


Both long-term behavioral memory and synaptic plasticity require protein synthesis, some of which may occur locally at specific synapses. Cytoplasmic polyadenylation element–binding (CPEB) proteins are thought to contribute to the local protein synthesis that underlies long-term changes in synaptic efficacy, but a role has not been established for them in the formation of long-term behavioral memory. We found that the Drosophila melanogaster CPEB protein Orb2 is acutely required for long-term conditioning of male courtship behavior. Deletion of the N-terminal glutamine-rich region of Orb2 resulted in flies that were impaired in their ability to form long-term, but not short-term, memory. Memory was restored by expressing Orb2 selectively in fruitless (fru)-positive γ neurons of the mushroom bodies and by providing Orb2 function in mushroom bodies only during and shortly after training. Our data thus demonstrate that a CPEB protein is important in long-term memory and map the molecular, spatial and temporal requirements for its function in memory formation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Structure and expression of Drosophila orb2.
Figure 2: Targeted mutations in Drosophila orb2 disrupt long-term memory.
Figure 3: Memory does not persist beyond 9 h in orb2ΔQ mutants.
Figure 4: Rescue of long-term memory.
Figure 5: orb2 function is required during or shortly after training.
Figure 6: orb2 is required in mushroom body γ neurons.

Accession codes




  1. 1

    Klann, E. & Dever, T.E. Biochemical mechanisms for translational regulation in synaptic plasticity. Nat. Rev. Neurosci. 5, 931–942 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Sutton, M.A. & Schuman, E.M. Dendritic protein synthesis, synaptic plasticity and memory. Cell 127, 49–58 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Martin, K.C. et al. Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 91, 927–938 (1997).

    CAS  Article  Google Scholar 

  4. 4

    Frey, U. & Morris, R.G. Synaptic tagging and long-term potentiation. Nature 385, 533–536 (1997).

    CAS  Article  Google Scholar 

  5. 5

    Hake, L.E. & Richter, J.D. CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation. Cell 79, 617–627 (1994).

    CAS  Article  Google Scholar 

  6. 6

    Lantz, V., Ambrosio, L. & Schedl, P. The Drosophila orb gene is predicted to encode sex-specific germline RNA-binding proteins and has localized transcripts in ovaries and early embryos. Development 115, 75–88 (1992).

    CAS  PubMed  Google Scholar 

  7. 7

    Stebbins-Boaz, B., Hake, L.E. & Richter, J.D. CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs, and is necessary for oocyte maturation in Xenopus. EMBO J. 15, 2582–2592 (1996).

    CAS  Article  Google Scholar 

  8. 8

    Lantz, V., Chang, J.S., Horabin, J.I., Bopp, D. & Schedl, P. The Drosophila orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity. Genes Dev. 8, 598–613 (1994).

    CAS  Article  Google Scholar 

  9. 9

    Christerson, L.B. & McKearin, D.M. orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis. Genes Dev. 8, 614–628 (1994).

    CAS  Article  Google Scholar 

  10. 10

    Chang, J.S., Tan, L. & Schedl, P. The Drosophila CPEB homolog orb is required for oskar protein expression in oocytes. Dev. Biol. 215, 91–106 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Chang, J.S., Tan, L., Wolf, M.R. & Schedl, P. Functioning of the Drosophila orb gene in gurken mRNA localization and translation. Development 128, 3169–3177 (2001).

    CAS  PubMed  Google Scholar 

  12. 12

    Wu, L. et al. CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 21, 1129–1139 (1998).

    CAS  Article  Google Scholar 

  13. 13

    Si, K. et al. A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in Aplysia. Cell 115, 893–904 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Alarcon, J.M. et al. Selective modulation of some forms of Schaffer collateral-CA1 synaptic plasticity in mice with a disruption of the CPEB-1 gene. Learn. Mem. 11, 318–327 (2004).

    Article  Google Scholar 

  15. 15

    Berger-Sweeney, J., Zearfoss, N.R. & Richter, J.D. Reduced extinction of hippocampal-dependent memories in CPEB knockout mice. Learn. Mem. 13, 4–7 (2006).

    Article  Google Scholar 

  16. 16

    Huang, Y.S., Kan, M.C., Lin, C.L. & Richter, J.D. CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA. EMBO J. 25, 4865–4876 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Si, K., Lindquist, S. & Kandel, E.R. A neuronal isoform of the Aplysia CPEB has prion-like properties. Cell 115, 879–891 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Heisenberg, M. Mushroom body memoir: from maps to models. Nat. Rev. Neurosci. 4, 266–275 (2003).

    CAS  Article  Google Scholar 

  19. 19

    Manoli, D.S. et al. Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour. Nature 436, 395–400 (2005).

    CAS  Article  Google Scholar 

  20. 20

    Stockinger, P., Kvitsiani, D., Rotkopf, S., Tirian, L. & Dickson, B.J. Neural circuitry that governs Drosophila male courtship behavior. Cell 121, 795–807 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Siegel, R.W. & Hall, J.C. Conditioned responses in courtship behavior of normal and mutant Drosophila. Proc. Natl. Acad. Sci. USA 76, 3430–3434 (1979).

    CAS  Article  Google Scholar 

  22. 22

    Gailey, D.A., Jackson, F.R. & Siegel, R.W. Conditioning mutations in Drosophila melanogaster affect an experience-dependent behavioral modification in courting males. Genetics 106, 613–623 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    McBride, S.M. et al. Mushroom body ablation impairs short-term memory and long-term memory of courtship conditioning in Drosophila melanogaster. Neuron 24, 967–977 (1999).

    CAS  Article  Google Scholar 

  24. 24

    Kamyshev, N.G., Iliadi, K.G. & Bragina, J.V. Drosophila conditioned courtship: two ways of testing memory. Learn. Mem. 6, 1–20 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Dukas, R. Experience improves courtship in male fruit flies. Anim. Behav. 69, 1203–1209 (2005).

    Article  Google Scholar 

  26. 26

    Dukas, R. Learning in the context of sexual behaviour in insects. Anim. Biol. 56, 125–141 (2006).

    Article  Google Scholar 

  27. 27

    Reif, M., Linsenmair, K.E. & Heisenberg, M. Evolutionary significance of courtship conditioning in Drosophila melanogaster. Anim. Behav. 63, 143–155 (2002).

    Article  Google Scholar 

  28. 28

    Tully, T., Preat, T., Boynton, S.C. & Del Vecchio, M. Genetic dissection of consolidated memory in Drosophila. Cell 79, 35–47 (1994).

    CAS  Article  Google Scholar 

  29. 29

    Mery, F. & Kawecki, T.J. A cost of long-term memory in Drosophila. Science 308, 1148 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Sitnik, N.A., Tokmacheva, E.V. & Savvateeva-Popova, E.V. The ability of Drosophila mutants with defects in the central complex and mushroom bodies to learn and form memories. Neurosci. Behav. Physiol. 33, 67–71 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Heisenberg, M., Borst, A., Wagner, S. & Byers, D. Drosophila mushroom body mutants are deficient in olfactory learning. J. Neurogenet. 2, 1–30 (1985).

    CAS  Article  Google Scholar 

  32. 32

    de Belle, J.S. & Heisenberg, M. Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. Science 263, 692–695 (1994).

    CAS  Article  Google Scholar 

  33. 33

    Zars, T., Fischer, M., Schulz, R. & Heisenberg, M. Localization of a short-term memory in Drosophila. Science 288, 672–675 (2000).

    CAS  Article  Google Scholar 

  34. 34

    Isabel, G., Pascual, A. & Preat, T. Exclusive consolidated memory phases in Drosophila. Science 304, 1024–1027 (2004).

    CAS  Article  Google Scholar 

  35. 35

    McGuire, S.E., Le, P.T., Osborn, A.J., Matsumoto, K. & Davis, R.L. Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 1765–1768 (2003).

    CAS  Article  Google Scholar 

  36. 36

    McGuire, S.E., Le, P.T. & Davis, R.L. The role of Drosophila mushroom body signaling in olfactory memory. Science 293, 1330–1333 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Krashes, M.J., Keene, A.C., Leung, B., Armstrong, J.D. & Waddell, S. Sequential use of mushroom body neuron subsets during Drosophila odor memory processing. Neuron 53, 103–115 (2007).

    CAS  Article  Google Scholar 

  38. 38

    Heimbeck, G., Bugnon, V., Gendre, N., Keller, A. & Stocker, R.F. A central neural circuit for experience-independent olfactory and courtship behavior in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 98, 15336–15341 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Ejima, A., Smith, B.P., Lucas, C., Levine, J.D. & Griffith, L.C. Sequential learning of pheromonal cues modulates memory consolidation in trainer-specific associative courtship conditioning. Curr. Biol. 15, 194–206 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Mehren, J.E., Ejima, A. & Griffith, L.C. Unconventional sex: fresh approaches to courtship learning. Curr. Opin. Neurobiol. 14, 745–750 (2004).

    CAS  Article  Google Scholar 

  41. 41

    Manoli, D.S., Meissner, G.W. & Baker, B.S. Blueprints for behavior: genetic specification of neural circuitry for innate behaviors. Trends Neurosci. 29, 444–451 (2006).

    CAS  Article  Google Scholar 

  42. 42

    Kurtovic, A., Widmer, A. & Dickson, B.J. A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446, 542–546 (2007).

    CAS  Article  Google Scholar 

  43. 43

    van der Goes van Naters, W. & Carlson, J.R. Receptors and neurons for fly odors in Drosophila. Curr. Biol. 17, 606–612 (2007).

    CAS  Article  Google Scholar 

  44. 44

    Lin, H.H., Lai, J.S., Chin, A.L., Chen, Y.C. & Chiang, A.S. A map of olfactory representation in the Drosophila mushroom body. Cell 128, 1205–1217 (2007).

    CAS  Article  Google Scholar 

  45. 45

    Tuite, M.F. Yeast prions and their prion-forming domain. Cell 100, 289–292 (2000).

    CAS  Article  Google Scholar 

  46. 46

    Mendez, R. et al. Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 404, 302–307 (2000).

    CAS  Article  Google Scholar 

  47. 47

    Schaeren-Wiemers, N. & Gerfin-Moser, A. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100, 431–440 (1993).

    CAS  Article  Google Scholar 

  48. 48

    Rong, Y.S. & Golic, K.G. Gene targeting by homologous recombination in Drosophila. Science 288, 2013–2018 (2000).

    CAS  Article  Google Scholar 

  49. 49

    Backhaus, B., Sulkowski, E. & Schlote, F.W. A semi-synthetic, general-purpose medium for Drosophila melanogaster. Drosoph. Inf. Serv. 60, 210–212 (1984).

    Google Scholar 

  50. 50

    Gailey, D.A., Hall, J.C. & Siegel, R.W. Reduced reproductive success for a conditioning mutant in experimental populations of Drosophila melanogaster. Genetics 111, 795–804 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank M. Novatchkova for bioinformatic analysis of CPEB proteins, R. Fuchs and E. Viragh for technical assistance, R. Davis for the TARGET stocks, K. Golic and E. Demir for stocks and vectors for homologous recombination, F. Bertolucci for advice on learning assays, R. Neumüller for help in examining mushroom body morphology, and M. Heisenberg, A. Keene and S. Rumpel for comments on the manuscript. We also thank M. Heisenberg for generously hosting K.K. during the initial behavioral assays and for his constant support and advice. This work was supported by funding from Boehringer Ingelheim and the Austrian Academy of Sciences.

Author information




B.J.D. and K.K. designed the experiments. K.K. generated the orb2 alleles and carried out all anatomical and behavioral studies together with S.K. M.A. performed the in situ hybridization experiments. B.J.D. wrote the manuscript.

Corresponding authors

Correspondence to Krystyna Keleman or Barry J Dickson.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1,2 and Tables 1–9 (PDF 860 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Keleman, K., Krüttner, S., Alenius, M. et al. Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nat Neurosci 10, 1587–1593 (2007).

Download citation

Further reading


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing