Fig 1. Pipeline schematic.

(A) The ArcCreER^T2 x eYFP tagging strategy allows for labelling of Arc-expressing cells with eYFP following injection of 4-OHT. (B) Indelible labelling of Arc⁺ ensembles with eYFP followed by immunolabelling for c-Fos⁺ allows for identification of a co-labeled ensembles active during two distinct timepoints. (C) eYFP⁺ and c-Fos⁺ cells are immunolabelled and imaged across brain-wide coronal sections. (D) eYFP⁺ and c-Fos⁺ populations are automatically segmented and co-labeled cells are identified in ImageJ/Fiji. Images are automatically pre-processed and flattened for registration alignment downstream. (E) The object-oriented infrastructure of SMARTR package in R allows for importation of segmentation data, registration, and mapping, and statistical analysis and visualization using a user-friendly API.