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. 2023 Jul 4;12:e82697. doi: 10.7554/eLife.82697

Figure 2. Behavioral experiment design.

Figure 2.

(A) Schematic of the behavioral experiment. Animals were habituated in context A for 4 days prior to the tagging of engram neurons in context B. During tagging, some animals underwent a foot shock (FS+) while others were placed in context B but without any stimulus (FS-). Following recall, animals were re-exposed to context A and underwent reactivation of the engram neurons with four different stimulation strategies on distinct days (trials). The experiment concluded with a final recall in context B. (B) FS+ (Bi) and FS- (Bii) animals had a similar number of dentate gyrus (DG) cells tagged as shown by the number of granule cells expressing EYFP-ChR2. FS+ animals received a foot shock in context A, while FS- animals did not. (C) Trends of mouse baseline freezing during habituation. (Ci) Habituation of animals over 4 days in context A resulted in decreased in freezing, indicating comfort with the setup. Black line shows the trend for the mean (n=26 animals). (Cii) Average of the percent time freezing over four epochs of trials on the last day of habituation (day 4) indicates 5% increase in percent time freezing due to fatigue later in the trial. This value serves as a baseline for subsequent analysis. ‘x’ indicates No Stim epochs. Shaded area indicates 95% confidence interval (n=26 animals). (D) Both FS+ and FS- animals showed minimal freezing in context B prior to the foot shocks (FS+ n = 17 animals, FS- n=9 animals; independent t-test with Bonferroni correction, p=0.7). However, FS+ animals showed significantly higher freezing post foot shock that persisted on the following day, indicating recall (FS+ n = 17, FS- n=9; independent t-test with Bonferroni correction, ****p<0.00001). (E) On the last day of habituation, both FS+ and FS- groups exhibited minimal baseline freezing (FS+ n = 17, FS- n=9; independent t-test with Bonferroni correction, p=0.6). However, post fear conditioning (FC), the FS+ group showed significantly higher baseline freezing (FS+ n = 17 animals, FS- n=9 animals; independent t-test with Bonferroni correction, ***p<0.0001). The elevated baseline freezing for the FS+ animals is sustained throughout all 4 days of the experiment. The black line shows the trend for the mean freezing. (For all figures, box shows the quartiles of the dataset, while whiskers show the rest of the distribution. Outliers are shown using diamonds.)