UC San Diego

Neural circuit tracing is a method utilized to map brain connectivity and function of the mammalian nervous system. Tracing is often utilized to illustrate axonal projections and examine the regenerative response to disease and neurotrauma. Current methodology involves delivering axonally transported organic compounds, fluorescent molecules and dyes, and viral vectors to label the axonal connections directly. Delivery of fluorescent reporters can be achieved through AAV-based neural circuit tracing to create digital atlases of brain connectivity that comprehensively map the axonal connections among brain regions. The non-viral tracer biotinylated dextran amine (BDA) is a common tracer utilized in visualization of neuronal structures, but newer viral tracers and improved detection methods could improve the sensitivity of axon tracing. Testing of new AAV-based tracers in cortical motor neurons resulted in strong, widespread labeling of corticospinal axon fibers in the spinal cord, including fine fibers extending beyond the ventral neuronal cell body layer. Hence, improved methods for axon tracing could uncover connectivity among brain regions, advance our understanding of the healthy and diseased mammalian brain, and support more comprehensive mapping of the brain connectome. In this experiment, we compared BDA and a control virus against three candidate AAV axon tracers with or without tyramide signal amplification (TSA). This study provides evidence that tyramide signal amplification improves the sensitivity of axon tracing by AAV vectors and biotinylated dextran amine (BDA) and that AAV vectors label more corticospinal axons than BDA in the mouse cervical spinal cord. Additionally, targeting tracer proteins to the inner cell membrane using a Ras farnesylation tag improves the subjective clarity of tracing, with more continuous brightness of labeling along the length of the axon. Lastly, of the candidate axon tracers examined in this study, AAV carrying Ruby2sm_Flag with a Ras farnesylation tag performed the best overall when detected using TSA.