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. 2022 Dec 23;26(2):259–273. doi: 10.1038/s41593-022-01223-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a, Experimental setup for b–e. Photostimulation activated mPFC engram inputs and induced EPSCs in tdT⁻ nonengram and tdT⁺ engram BLA neurons. b, Mice were tested for memory recall 28 d after CFC. c, Left: freezing behavior during memory recall (eight mice). Middle: image showing ChR2-eYFP⁺/tdT⁺ mPFC neurons (squares). Right: image showing ChR2-eYFP⁺ mPFC axons (green) and tdT⁺ BLA neurons (red). d, Traces of EPSCs induced by photostimulation of mPFC engram inputs and recorded in tdT⁻ and tdT⁺ BLA neurons. AMPAR and NMDAR EPSCs were recorded as in Fig. 2d. Scale bar, 10 μm. e, Left: comparison of EPSC_AMPAR in 20 pairs of tdT^– versus tdT⁺ BLA neurons. Repeated measures two-way ANOVA. Right: comparison of AMPA/NMDA ratios of EPSCs in tdT⁻ (19 cells) versus tdT⁺ BLA neurons (20 cells). Unpaired t-test. f, Experimental setup for g–j. mPFC engram neurons expressed ChR2-eYFP, whereas BLA engram neurons expressed TVA-G-GFP. mPFC relay neurons (R, red) were labeled with mCherry. Photostimulation activated ChR2⁺ mPFC engram inputs and induced EPSCs in mPFC relay neurons. g, After AAV injection, mice underwent CFC and received RV injection. h, Left: freezing behavior during memory recall (six mice). Middle: BLA engram neurons were labeled with TVA-G-GFP and/or mCherry. TVA-G-GFP⁺/mCherry⁺ neurons are circled. Right: image showing mCherry⁺ mPFC relay neurons. i, Left: EPSCs were induced by photostimulation of mPFC engram inputs and recorded in mCherry⁺ mPFC relay neurons (red). TTX blocked EPSCs (black), which were rescued by 4-AP (blue). Scale bar, 10 μm. Right: comparison of EPSC amplitude in mCherry⁺ versus mCherry⁻ mPFC neurons (11 pairs) in the presence of TTX, 4-AP and SR-95531. j, Photostimulation (blue) of mPFC engram inputs induced AP firings (red) in mCherry⁺ mPFC relay neurons in cell-attached mode in the presence of SR-95531. k, mPFC engram neurons project to BLA engram neurons monosynaptically (1) or are connected to BLA engram neurons through mPFC relay neurons (2). Data are presented as the mean ± s.e.m. Details of the statistical analyses are presented in Supplementary Tables 1 and 2.

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