Fig. 5: Stimulation of cholinergic inputs to ACx at the encoding stage influences the reactivation of BLA engram cells. | Translational Psychiatry

Fig. 5: Stimulation of cholinergic inputs to ACx at the encoding stage influences the reactivation of BLA engram cells.

From: ACx-projecting cholinergic neurons in the NB influence the BLA ensembles to modulate the discrimination of auditory fear memory

Fig. 5

A Schematic of virus injection. In ChAT-cre mice, AAV9-DIO-NpHR3.0-mCherry was infused into the NB. AAV9-c-fos-tTA and AAV9-TRE-Gcamp6s or AAV9-TRE-EGFP were infused into the BLA. B Behavioral design. C, E The mean and SEM of the corresponding Ca2+ signal of BLA engram cells in mice without (C) or with (E) inhibition of ACx-projecting cholinergic neurons of the NB at the encoding stage and their corresponding heatmaps. The under red line indicated bins between 10 kHz or 1 kHz tone retrieval, with two-tailed unpaired t-test significance (P < 0.05). D, F the correlation between freezing and the amplitudes of Ca2+ signal without (related to C) or with (related to E) inhibition of ACx-projecting cholinergic neurons of the NB. G, H Quantification of the averaged amplitude of increased signal against baseline between non-inhibition trials (related to C) and inhibition trials (related to E) during the 1 kHz tone retrieval (F) or 10 kHz tone retrieval (H). I–L Representative confocal images (I and J) and the statistical numbers (K and L) of the reactivated BLA engram cells during 1 kHz retrieval stage (I and K) or 10 kHz retrieval stage (J and L) in mice under inhibition of ACx-projecting cholinergic neurons of NB at the encoding stage. (Blue, DAPI; green, the activated BLA engram cells at day 1 that were marked by EGFP; red: the activated BLA neurons marked by c-fos in mice that were given 1 kHz tone (I) or 10 kHz tone (J) at day 2. Scale bar: 50 μm. In the middle graph of K, the NpHR3.0 group displayed higher percentages of c-fos+/DAPI+ and EGFP+c-fos+/EGFP+ cells than mCherry group during the 1 kHz retrieval test (n = 9 images from three mice). In middle graph of L, the NpHR3.0 group displayed lower percentages of c-fos+/DAPI+ and EGFP+c-fos+/EGFP+ cells than mCherry group during the 10 kHz retrieval test (n = 9 images from three mice). Statistical tests: D, F, correlation: two-tailed Pearson (R) correlation. G, H signal variation: unpaired two-tailed Mann–Whitney-U-test. K, L unpaired two-tailed t-test. ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM.

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