Learned fear often relapses after extinction, suggesting that extinction training generates a new memory that coexists with the original fear memory; however, the mechanisms governing the expression of competing fear and extinction memories remain unclear. We used activity-dependent neural tagging to investigate representations of fear and extinction memories in the dentate gyrus. We demonstrate that extinction training suppresses reactivation of contextual fear engram cells while activating a second ensemble, a putative extinction engram. Optogenetic inhibition of neurons that were active during extinction training increased fear after extinction training, whereas silencing neurons that were active during fear training reduced spontaneous recovery of fear. Optogenetic stimulation of fear acquisition neurons increased fear, while stimulation of extinction neurons suppressed fear and prevented spontaneous recovery. Our results indicate that the hippocampus generates a fear extinction representation and that interactions between hippocampal fear and extinction representations govern the suppression and relapse of fear after extinction.
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All relevant data supporting the findings of this study are available from the corresponding author upon reasonable request.
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We thank J. Dunsmoor for comments on the manuscript. A.F.L. was supported by National Institutes of Health (NIH) grant nos. F31 MH111243 and T32 MH106454. S.L.S. was supported by grant no. PD/BD/128076/2016 from the Portuguese Foundation for Science and Technology. C.A.D. was supported by NIH grant no. DP5 OD017908 and New York Stem Cell Science grant no. C-029157. M.R.D. was supported by NIH grant nos. R01 MH102595, R01 MH117426 and R21 EY026446.
The authors declare no competing interests.
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Integrated supplementary information
(a, b) From Fig. 1. Extinction and alternative context exposure decreased the (a) percentage of Arc+ cells among eYFP+ cells and (b) percentage of eYFP+ cells among Arc+ cells compared to fear retrieval. No Ext: n = 8 mice; Ext: n = 8 mice; Alt Ctx: n = 6 mice. (c, d) From Fig. 2. During an extinction retrieval test, Ext-Tag mice displayed a (c) trend towards a greater percentage of Arc+ cells among eYFP+ cells and (d) a greater percentage of eYFP+ cells among Arc+ cells compared to Acq-Tag mice. The pattern reversed during the spontaneous recovery test. Acq-Tag/ExtTest: n = 8 mice; Ext-Tag/ExtTest: n = 8 mice; Acq-Tag/SpontRec: n = 8 mice; Ext-Tag/SpontRec: n = 7 mice. Data are means ± s.e.m. See Supplementary Table 1 for statistics. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Supplementary Figure 2 Silencing extinction- and fear acquisition-tagged neurons has no effect during second light ON and OFF epochs.
(a-c) From extinction-tagged neuron silencing experiment (Fig. 3). Silencing extinction-tagged neurons had no effect on freezing behavior during the second light ON and OFF epochs during (a) extinction retrieval test, (b) alternate context test, and (c) spontaneous recovery test. ArcCreERT2(-): n = 9 mice; ArcCreERT2(+): n = 9 mice. (d-f) From fear-tagged neuron silencing experiment (Fig. 4). Silencing fear-tagged neurons had no effect on freezing behavior during the second light ON and OFF epochs during (d) extinction retrieval test, (e) alternate context test, and (f) spontaneous recovery test. ArcCreERT2(-): n = 8 mice; ArcCreERT2(+): n = 7 mice. Data are means ± s.e.m. See Supplementary Table 1 for statistics.
Supplementary Figure 3 Silencing extinction-tagged neurons impairs extinction retrieval in second extinction retrieval test.
(a) Experimental design. Second extinction retrieval test occurred 1 d after the alternate context test. Same animals from Fig. 3. (b) Freezing behavior during the second extinction retrieval test. (c) Silencing extinction-tagged neurons increased freezing in ArcCreERT2(+) mice during the first light ON and light OFF epochs compared to ArcCreERT2(-) mice. ArcCreERT2(-): n = 9 mice; ArcCreERT2(+): n = 9 mice. Data are means ± s.e.m. See Supplementary Table 1 for statistics. * p < 0.05, ** p < 0.01.
Supplementary Figure 4 Stimulating fear acquisition- or extinction-tagged neurons induces c-Fos expression in eYFP+ cells in dorsal DG and CA3.
(a) Representative images of eYFP+ and c-Fos+ immunofluorescence in the DG of Acq-Tag(+) mice presented with light (top) or without light (bottom). White arrowheads denote co-labeled eYFP+/Arc+ cells. (b) Light increased the percentage of c-Fos+ cells among eYFP+ cells in Acq-Tag(+) and Ext-Tag(+) mice in the DG near the fiber optic location. (c) Same as (a), but in CA3. (d) Light increased the percentage of c-Fos+ cells among eYFP+ cells in Acq-Tag(+) and Ext-Tag(+) mice in dorsal CA3. Light: n = 5 mice; No Light: n = 3 mice. Data are means ± s.e.m. See Supplementary Table 1 for statistics. * p < 0.05, ** p < 0.01.
Supplementary Figure 5 Stimulating fear acquisition- or extinction-tagged neurons does not affect anxiety or exploratory behaviors.
(a) OF design. Light ON epoch occurred during minutes 5–10. Same animals from Fig. 5. (b-d) Stimulating fear acquisition- or extinction-tagged neurons did not affect (b) percentage of time in OF center, (c) center distance traveled, or (d) total distance traveled. Light OFF epochs averaged. Acq/Ext-Tag(-): n = 12 mice; Acq-Tag(+): n = 4 mice; Ext-Tag(+): n = 4 mice. Data are means ± s.e.m. See Supplementary Table 1 for statistics.
Supplementary Figure 6 Stimulating extinction-tagged neurons promotes extinction retrieval in extinction retrieval test following spontaneous recovery.
(a) Experimental design. Second extinction retrieval test occurred 1 d after spontaneous recovery test. Light was delivered during minutes 3–6. Same animals from Fig. 5. (b) Freezing behavior during the second extinction retrieval test. (c) Stimulating extinction-tagged neurons reduced freezing during the light ON epoch in Ext-Tag(+) mice compared to Acq/Ext-Tag(-) mice. Light OFF epochs averaged. Acq/Ext-Tag(-): n = 12 mice; Acq-Tag(+): n = 4 mice; Ext-Tag(+): n = 4 mice. Data are means ± s.e.m. See Supplementary Table 1 for statistics. * p < 0.05.
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Lacagnina, A.F., Brockway, E.T., Crovetti, C.R. et al. Distinct hippocampal engrams control extinction and relapse of fear memory. Nat Neurosci 22, 753–761 (2019). https://doi.org/10.1038/s41593-019-0361-z
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