Fig. 2

In vivo Ca²⁺ imaging of engram cells identified with the photoconvertible protein, Kikume Green Red (KikGR). a Schematic diagram showing labelling and visualization of engram and non-engram cells in Thy1-G-CaMP7 × c-fos-tTA double transgenic mice injected with TRE-KikGR lentivirus (LV). b Experimental design for calcium imaging. Top: snapshot of KikGR (scale bar, 200 µm), before (1) and after (2) contextual exploration, and after photoconversion (3), and representative photographs of behaviour during contextual learning, sleep and retrieval. Bottom: Ca²⁺ imaging paradigm. Photoconversion by 365 nm light (dark purple arrow) and snapshot (pink arrow) before OFF doxycycline (Dox), then calcium imaging during contextual learning and sleep with OFF Dox, followed by a second snapshot and photoconversion after reinstatement of ON Dox, then a calcium imaging session during retrieval. c Schematic diagram showing the processing of the Ca²⁺ imaging for the entire movie. Step 1: binning, motion correction, and background removal for each session independently; Step 2: concatenation of all movies into one long movie; motion correction run on the entire movie using a single reference frame; Step 3: separation of the entire movie into single sessions; ∆F/F performed on each movie; Step 4: re-concatenation of the single movies and procession to auto-detection of Ca²⁺ signals. Mot Corr, motion correction. d Field of view (FOV) from a representative animal; white enclosed area is the area that showed KikGR expression and was incorporated in the analysis, with some representative engram and non-engram cells indicated in red and blue, respectively, together with their ID labels. e Regions of interest (ROI) of the labelled cells and their representative traces (extracted between the frames indicated) on days 1 and 2 for both engram cells (left) and non-engram cells (right). Arrowheads point to the time selected for evaluating the cell location. The number of frames is indicated under each ROI