AAV-delivered TurboRFP enables streamlined tracing and analysis of corticospinal tract sprouting.

Quantitative analysis of corticospinal tract (CST) sprouting after injury requires reliable labeling of long-range axons and fine collateral branches. Conventional biotinylated dextran amine (BDA) tracing has limited sensitivity and requires additional surgeries, while some viral-based approaches, although robust, rely on extensive tissue processing and signal amplification. Here, we describe a streamlined adeno-associated virus (AAV)-based workflow for CST sprouting analysis using TurboRFP that enables robust labeling of descending CST axons, including sprouting fibers after unilateral pyramidotomy. This approach allows direct visualization of fine CST axons without immunostaining or signal amplification, simplifying tissue processing and reducing experimental variability. Using a standardized workflow, we enable consistent CST labeling and reproducible quantification of CST remodeling. In addition, compatibility with co-delivery of other AAVs enables simultaneous circuit tracing and genetic manipulation within the same neuronal population. This workflow provides a practical platform that lowers technical barriers to axon repair research and potentially improves reproducibility across laboratories.