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, 484 (7394), 381-5

Optogenetic Stimulation of a Hippocampal Engram Activates Fear Memory Recall


Optogenetic Stimulation of a Hippocampal Engram Activates Fear Memory Recall

Xu Liu et al. Nature.


A specific memory is thought to be encoded by a sparse population of neurons. These neurons can be tagged during learning for subsequent identification and manipulation. 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) 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.


Figure 1
Figure 1. Basic experimental protocols and selective labeling of the DG cells by ChR2-EYFP
a, The c-fos-tTA mouse was injected with AAV9-TRE-ChR2-EYFP and implanted with an optical fiber targeting DG. b, When off Dox, training induces the expression of c-fos-tTA, which binds to TRE and drives the expression of ChR2-EYFP, labeling a subpopulation of activated cells (yellow) in DG. c, Basic experimental scheme. Mice were habituated in context A with light stimulation while on Dox for five days, then taken off Dox for two days and fear conditioned (FC) in context B. Mice were put back on Dox and tested for five days in context A with light stimulation. d, Representative image showing the expression of ChR2-EYFP in a mouse that was taken off Dox for two days and underwent FC training. An image of each rectangular area in (d) is magnified showing DG (e), CA1 (f), and CA3 (g). The green signal from ChR2-EYFP in the DG spreads throughout entire granule cells, including dendrites (e), while the green signal confined to the nuclei in CA1 and CA3 is due to shEGFP expression from the c-fosshEGFP construct of the transgenic mouse (f, g). Blue is nuclear marker DAPI.
Figure 2
Figure 2. Activity dependent expression and stimulation of ChR2-EYFP
a–f, Representative images of DG from c-fos-tTA mice injected with AAV9-TRE-ChR2-EYFP and sacrificed after the following treatments: a, On Dox. b, Off Dox for two days in home cage. c, Same as (b) followed by fear conditioning (FC). d, Same as (c) except no shock was delivered (NS). e, Same as (c), five days post-training. f, Same as (c), 30 days post-training. g, Same as (b) followed by kainic acid injection to induce seizure. Residual green signal in (a) and (f) are from nuclear-localized c-fos-shEGFP (see Fig. 1 legend). h, Percentage of ChR2-EYFP–positive cells after various treatments represented by (a) to (g) (n = 5 subjects each; F6,28 = 94.43, *P < 0.05; ***P < 0.001). i, j, Representative DG cells after light stimulation in c-fos-tTA mice injected with AAV9-TRE-ChR2-EYFP (i) or AAV9-TRE-EYFP (j). k, Percentage of c-fos–positive cells among ChR2-EYFP–positive cells or EYFP-positive cells after light stimulation (n = 3 subjects each; ***P < 0.001). l, Light-evoked single unit activity of a DG neuron from a c-fos-tTA mouse injected with AAV9-TRE-ChR2-EYFP. Peri-event histogram (top) and raster plot (bottom) show reliable and precisely time-locked spiking relative to the onset of 15 ms light pulses (blue bar). Inset shows an overlay of waveforms for all the spikes during light stimulation. m, Spike probability and peak latency for all the light-responsive cells (n = 10) recorded as in (l). n, Multi-unit activity in the DG from a c-fos-tTA mouse injected with AAV9-TRE-ChR2-EYFP in response to trains of 10 light pulses (15 ms; blue bars) at 20 Hz. Scale bar in (a) 250 μm.
Figure 3
Figure 3. Optical stimulation of engram-bearing cells induces post-training freezing
a, c-fos-tTA mice injected with AAV9-TRE-ChR2-EYFP and trained with FC (Exp group) show elevated freezing during three min light-on epochs. Freezing for each epoch represents 5-day average (Supplementary Fig. 5a, g). Freezing levels for the two light-off and light-on epochs are further averaged in the inset. (n = 12, F1,22 = 37.98, ***P < 0.001). b, Mice trained similar to (a) but without foot shock (NS group) do not show increased light-induced freezing (n = 12). c, Mice injected with AAV9-TRE-EYFP and trained with FC (EYFP group) do not show increased light-induced freezing (n = 12). d, Mice trained similar to (a) but kept off Dox for one day before FC training (Exp-1day group) showed greater freezing during test light-on epochs compared to Exp group (n = 5, F1,8 = 38.26, ***P <0.001). e, Mice trained similar to (a) but bilaterally injected with AAV9-TRE-ChR2-EYFP and implanted with optical fibers (Exp-Bi group) showed even higher levels of freezing during test light-on epochs (n = 6, F1,10 = 85.14, ***P < 0.001). f, Summary of freezing levels of the five groups during test light-on epochs (F4,42 = 37.62, *P < 0.05; ***P < 0.001).
Figure 4
Figure 4. Labeling and stimulation of independent DG cell populations
a, c-fos-tTA mice injected with AAV9-TRE-ChR2-EYFP were taken off Dox and exposed to context C to label activated cells with ChR2-EYFP (yellow), then put back on Dox and trained with FC in context B to activate endogenous c-fos (red). Representative images of DG from these mice are shown in (b) to (e). b, ChR2-EYFP–labeled cells activated in context C. c, c-fos–labeled cells activated in context B. d, Nuclear marker DAPI. e, Merge. The white and red circles show examples of ChR2-EYFP–positive and c-fos–positive cells, respectively. The c-fos–positive cells in (e) appear yellow because they express both endogenous c-fos (red) and the nuclear-localized c-fos-shEGFP (green) (see Figure 1 legend). f, Percentage of ChR2-EYFP–positive, endogenous c-fos–positive, and double-positive cells among total cells (DAPI+) (n = 5). g, Observed percentage of double-positive cells is the same as what would be expected if the two cell populations were independent (i.e. a product of the observed percentage of ChR2-EYFP–single-positive and c-fos–single-positive cells.) h, Behavior setup for mice exposed to an open field in context C while off Dox and subsequently fear conditioned in context B while on Dox (OF-FC). i, OF-FC mice (n = 5) do not show increased light-induced freezing. Scale bar in (b) 10 μm.

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