Multiplexed mapping of long-range projections with FluoCode, a combinatorial fluorescent barcoding system

Abstract

Comprehensive mapping of long-range projections remains a major challenge in neuroscience. Conventional virus tracing labels only one or a few pathways per animal, and inter-individual comparison requires registering whole-brain images to a standard atlas. However, inter-individual variability in morphology and wiring limits fine-scale mapping. Here we report "FluoCode", a combinatorial fluorescent barcoding system for multiplexed projection mapping within single animals. We developed adeno-associated virus (AAV) vectors that express two of seven different fluorescent proteins, yielding 21 distinct color codes, and a "FluoCode Reader" that automatically classifies neurite signals by color combinations and morphology. Using FluoCode, we mapped projections from up to 21 cortical areas to subcortical regions in individual mice, revealing fine-scale topographic maps in small subcortical nuclei. In the marmoset, mapping from the primary visual cortex demonstrated topographic projections in higher visual areas and thalamic nuclei. FluoCode thus enables fine-scale, within-animal comparison of long-range projections in rodents and primates.