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Optogenetic stimulation of a hippocampal engram activates fear memory recall


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.

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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.


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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.

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Authors and Affiliations



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.

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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)

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

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