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. 2021 Jan 15;22(2):810. doi: 10.3390/ijms22020810

Figure 1.

Figure 1

Experimental procedures and behavioral results of in vivo optogenetics of the Prelimbic (PrL) pyramidal neurons during Contextual Fear Conditioning (CFC). (A) On day 1 (Conditioning phase) of CFC, each Thy1-COP4 mouse was allowed to explore the conditioning chamber for 3 min (Baseline). Afterward, only a part of the entire sample received three foot-shocks. On days 2, 3, 4, 7, and 14 (Extinction phase), the fear-conditioned (n = 20) and not fear-conditioned (n = 20) mice were placed again in the conditioning chamber for 6 min. During the Extinction phase, no shock was delivered and the mice received three optogenetic (OPTO FEAR and OPTO NOT FEAR groups, n = 10/group) or sham (SHAM FEAR and SHAM NOT FEAR groups, n = 10/group) stimulations of PrL pyramidal neurons. To control for the effects of learned but not yet extinguished fear, on day 1 (Conditioning phase) of CFC other Thy1-COP4 mice (No-EX group, n = 6) were allowed to explore the conditioning chamber for 3 min (Baseline) and then they received three foot-shocks. The day after the Conditioning phase (day 2) these animals were placed again in the conditioning chamber for 6 min (without receiving any optogenetic stimulation) and then sacrificed for electrophysiological and morphological analyses. A1) Representative image of the expression of the transgenic ChR2-YFP fusion protein detected in pyramidal cortical layer 5 neurons of PrL cortex. Scale bar 50 μm. (B) Freezing behavior measured during 0–3 min of CFC. The animals belonging to OPTO FEAR, OPTO NOT FEAR, SHAM FEAR, and SHAM NOT FEAR groups showed similar responses in the Conditioning phase and only the fear-conditioned animals (OPTO FEAR and SHAM FEAR groups) showed increased freezing times on day 2. While SHAM FEAR group progressively extinguished fear memories over time, impaired extinction of fear memories was observed in OPTO FEAR group. (C) Freezing times measured during 0–6 min of day 14. OPTO FEAR group showed the highest freezing times in comparison to the remaining groups (*** p = 0.0005). (D) Freezing behavior measured during 0–3 min of day 1 and 2 of CFC. Only the fear-conditioned animals (No-EX, OPTO FEAR, and SHAM FEAR groups) increased their freezing times between day 1 and day 2 (**** p < 0.0001), showing similar consolidation of fear memory. Data are reported as mean ± SEM.