Optogenetic stimulation of a hippocampal engram activates fear memory recall

Abstract

A specific memory is thought to be encoded by a sparse population of neurons1,2. These neurons can be tagged during learning for subsequent identification3 and manipulation4,5,6. Moreover, their ablation or inactivation results in reduced memory expression, suggesting their necessity in mnemonic processes. However, the question of sufficiency remains: it is unclear whether it is possible to elicit the behavioural output of a specific memory by directly activating a population of neurons that was active during learning. Here we show in mice that optogenetic reactivation of hippocampal neurons activated during fear conditioning is sufficient to induce freezing behaviour. We labelled a population of hippocampal dentate gyrus neurons activated during fear learning with channelrhodopsin-2 (ChR2)7,8 and later optically reactivated these neurons in a different context. The mice showed increased freezing only upon light stimulation, indicating light-induced fear memory recall. This freezing was not detected in non-fear-conditioned mice expressing ChR2 in a similar proportion of cells, nor in fear-conditioned mice with cells labelled by enhanced yellow fluorescent protein instead of ChR2. Finally, activation of cells labelled in a context not associated with fear did not evoke freezing in mice that were previously fear conditioned in a different context, suggesting that light-induced fear memory recall is context specific. Together, our findings indicate that activating a sparse but specific ensemble of hippocampal neurons that contribute to a memory engram is sufficient for the recall of that memory. Moreover, our experimental approach offers a general method of mapping cellular populations bearing memory engrams.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Basic experimental protocols and selective labelling of DG cells by ChR2–EYFP.
Figure 2: Activity-dependent expression and stimulation of ChR2–EYFP.
Figure 3: Optical stimulation of engram-bearing cells induces post-training freezing.
Figure 4: Labelling and stimulation of independent DG cell populations.

References

  1. 1

    Josselyn, S. A. Continuing the search for the engram: examining the mechanism of fear memories. J. Psychiatry Neurosci. 35, 221–228 (2010)

    Article  Google Scholar 

  2. 2

    Silva, A. J. et al. Molecular and cellular approaches to memory allocation in neural circuits. Science 326, 391–395 (2009)

    CAS  Article  Google Scholar 

  3. 3

    Reijmers, L. G., Perkins, B. L., Matsuo, N. & Mayford, M. Localization of a stable neural correlate of associative memory. Science 317, 1230–1233 (2007)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Han, J. H. et al. Neuronal competition and selection during memory formation. Science 316, 457–460 (2007)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Han, J. H. et al. Selective erasure of a fear memory. Science 323, 1492–1496 (2009)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Zhou, Y. et al. CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala. Nature Neurosci. 12, 1438–1443 (2009)

    CAS  Article  Google Scholar 

  7. 7

    Boyden, E. S. et al. Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neurosci. 8, 1263–1268 (2005)

    CAS  Article  Google Scholar 

  8. 8

    Tye, K. M. et al. Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature 471, 358–362 (2011)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Martin, S. J. & Morris, R. G. New life in an old idea: the synaptic plasticity and memory hypothesis revisited. Hippocampus 12, 609–636 (2002)

    CAS  Article  Google Scholar 

  10. 10

    Gerber, B., Tanimoto, H. & Heisenberg, M. An engram found? Evaluating the evidence from fruit flies. Curr. Opin. Neurobiol. 14, 737–744 (2004)

    CAS  Article  Google Scholar 

  11. 11

    Lever, C. et al. Long-term plasticity in hippocampal place-cell representation of environmental geometry. Nature 416, 90–94 (2002)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Phillips, R. G. & LeDoux, J. E. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav. Neurosci. 106, 274–285 (1992)

    CAS  Article  Google Scholar 

  13. 13

    Kim, J. J. & Fanselow, M. S. Modality-specific retrograde amnesia of fear. Science 256, 675–677 (1992)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Ramamoorthi, K. et al. Npas4 regulates a transcriptional program in CA3 required for contextual memory formation. Science 334, 1669–1675 (2011)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Treves, A. & Rolls, E. T. Computational analysis of the role of the hippocampus in memory. Hippocampus 4, 374–391 (1994)

    CAS  Article  Google Scholar 

  16. 16

    McHugh, T. J. et al. Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317, 94–99 (2007)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Nakashiba, T. et al. Young dentate granule cells mediate pattern separation whereas old granule cells facilitate to pattern completion. Cell (2012)

  18. 18

    Schmidt, B., Marrone, D. F. & Markus, E. J. Disambiguating the similar: The dentate gyrus and pattern separation. Behav. Brain Res. 226, 56–65 (2012)

    Article  Google Scholar 

  19. 19

    Chawla, M. K. et al. Sparse, environmentally selective expression of Arc RNA in the upper blade of the rodent fascia dentata by brief spatial experience. Hippocampus 15, 579–586 (2005)

    MathSciNet  CAS  Article  Google Scholar 

  20. 20

    Satvat, E. et al. Changes in task demands alter the pattern of zif268 expression in the dentate gyrus. J. Neurosci. 31, 7163–7167 (2011)

    CAS  Article  Google Scholar 

  21. 21

    Kubik, S., Miyashita, T. & Guzowski, J. F. Using immediate-early genes to map hippocampal subregional functions. Learn. Mem. 14, 758–770 (2007)

    Article  Google Scholar 

  22. 22

    Shockett, P. E. & Schatz, D. G. Diverse strategies for tetracycline-regulated inducible gene expression. Proc. Natl Acad. Sci. USA 93, 5173–5176 (1996)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Leutgeb, J. K., Leutgeb, S., Moser, M. B. & Moser, E. I. Pattern separation in the dentate gyrus and CA3 of the hippocampus. Science 315, 961–966 (2007)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Lee, I. & Kesner, R. P. Differential contributions of dorsal hippocampal subregions to memory acquisition and retrieval in contextual fear-conditioning. Hippocampus 14, 301–310 (2004)

    Article  Google Scholar 

  25. 25

    Goshen, I. et al. Dynamics of retrieval strategies for remote memories. Cell 147, 678–689 (2011)

    CAS  Article  Google Scholar 

  26. 26

    Seidenbecher, T., Laxmi, T. R., Stork, O. & Pape, H. C. Amygdalar and hippocampal theta rhythm synchronization during fear memory retrieval. Science 301, 846–850 (2003)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Schafe, G. E., Doyere, V. & LeDoux, J. E. Tracking the fear engram: the lateral amygdala is an essential locus of fear memory storage. J. Neurosci. 25, 10010–10014 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Rogan, M. T., Staubli, U. V. & LeDoux, J. E. Fear conditioning induces associative long-term potentiation in the amygdala. Nature 390, 604–607 (1997)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Paxinos, G. & Franklin, K. The Mouse Brain in Stereotaxic Coordinates (Academic, 2001)

    Google Scholar 

  30. 30

    Rasband, W. S. Image J. http://imagej.nih.gov/ij/ (National Institutes of Health, 1997–, 2011)

    Google Scholar 

Download references

Acknowledgements

We thank S. Huang, G. Lin, M. Ragion and X. Zhou for help with the experiments, T. Ryan, A. Rivest, J. Young, R. Redondo and G. Dragoi for comments and discussions on the manuscript, and all the members of the Tonegawa laboratory for their support. This work is supported by National Institutes of Health grants R01-MH078821, P50-MH58880 to S.T. and RIKEN Brain Science Institute.

Author information

Affiliations

Authors

Contributions

X.L., S.R., A.G. and S.T. contributed to the study design. X.L., S.R. and P.T.P. contributed to the data collection and interpretation. X.L. cloned all constructs. X.L. and S.R. conducted the surgeries and the behaviour experiments. S.R. conducted the expression timeline experiments. P.T.P. conducted the electrophysiology experiments. C.B.P. contributed to the setup of the electrophysiology apparatus and wrote the Matlab software to analyse the data. K.D. provided the original ChR2 construct. X.L., S.R. and S.T. wrote the paper. All authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Susumu Tonegawa.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1–11. (PDF 8878 kb)

Supplementary Movie 1

This file contains a movie showing one representative mouse from the Exp-Bi group during a test session post-training. The first minute of the movie shows the light-off epoch and the subsequent three minutes show the light-on epoch. The movie is played at 4× normal speed. Note that freezing levels increase dramatically only during the light-on epoch. (MOV 20221 kb)

Supplementary Movie 2

This file contains a movie showing one representative mouse from the Exp-Bi group pre-training (on Dox, habituation session). The first minute of the movie shows the light-off epoch and the subsequent three minutes show the light-on epoch. The movie is played at 4× normal speed. Note that freezing levels do not increase throughout the session. (MOV 15272 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liu, X., Ramirez, S., Pang, P. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012). https://doi.org/10.1038/nature11028

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

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