Optimizing stereotaxic injection strategy for AAV-mediated corticospinal tract tracing in mice

The corticospinal tract (CST) is a major descending motor pathway essential for voluntary motor control. While adeno-associated virus-mediated anterograde tracing is widely used to label CST projections in mice, what the best stereotaxic injection coordinates and post-injection intervals remain unclear.

Here, we systematically evaluated eight cortical injection strategies, differing in anterior-posterior (AP) and medial-lateral (ML) coordinates, the number of injection sites, and post-injection intervals. CST labeling was quantitatively assessed at cervical 2, cervical 5, thoracic 2, thoracic 6, and lumbar 2 spinal levels using transduced axon count (TAC), mean fluorescence intensity (MFI) and transduced area within the dorsal columns, normalized to the C2 segment.

Across the tested AP/ML coordinates and single- versus multi-site injections, TAC and MFI were broadly comparable across cervical and thoracic levels, with the exception of reduced L2 labeling in Group IV (AP +0.70 mm). Furthermore, reducing the post-injection interval from four weeks to two weeks did not compromise labeling efficiency.

Conventional CST tracing typically requires multiple injections and ≥ 4-week intervals, increasing complexity and duration. Our optimized single-injection, 2-week protocol achieves comparable labeling fidelity while reducing procedural burden and improving reproducibility.

We suggest that effective CST labeling from L2 and rostral segments can be achieved with a single-point injection at AP coordinates between 0.0 and + 0.7 mm (ML fixed at 1.2 mm) or ML coordinates from + 0.7 to + 1.5 mm (AP fixed at 0.0 mm). These results establish a simplified, reproducible strategy for CST tracing.