Non-viral traditional tracers are effective tools for visualizing a large number of neuronal connections. These tracers are often injected and can be used in any animal of interest, eliminating the necessity for genetically modified mice with a specific genetic background.

Neuroanatomical tracing was first discovered by Waller, who found that damaged neuronal axons gradually degenerate from the lesion site to the distal end. Subsequently, "Waller-like" degeneration of neurons was found in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). In the 1960s, chemical tracers were developed and became the mainstream solution for neurotracing for a long time.

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