Brain-wide immunolabeling and tissue clearing applications for engram research

In recent years, there has been significant progress in memory research, driven by genetic and imaging technological advances that have given unprecedented access to individual memory traces or engrams. Although Karl Lashley argued since the 1930s that an engram is not confined to a particular area but rather distributed across the entire brain, most current studies have focused exclusively on a single or few brain regions. However, this compartmentalized approach overlooks the interactions between multiple brain regions, limiting our understanding of engram mechanisms. More recently, several studies have begun to investigate engrams across the brain, but research is still limited by a lack of standardized techniques capable of reconstructing multiple ensembles at single-cell resolution across the entire brain. In this review, we guide researchers through the latest technological advancements and discoveries in immediate early gene (IEG) techniques, tissue clearing methods, microscope modalities, and automated large-scale analysis. These innovations could propel the field forward in building brain-wide engram maps of normal and disease states, thus, providing unprecedented new insights. Ultimately, this review aims to bridge the gap between research focused on single brain regions and the need for a comprehensive understanding of whole-brain engrams, revealing new approaches for exploring the neuronal mechanisms underlying engrams.