Title: Recording Polysynaptic Circuit Architecture Across the Cerebellar-Cortical Axis with an Evolving Transsynaptic Virus

The cerebellum contains four times more neurons than the entire neocortex and maintains extensive reciprocal connections with cortical regions governing cognition, language, and social behavior. Disruption of this cerebellar-cortical dialogue underlies motor and neurological conditions ranging from ataxia to autism. Yet the synaptic organization of this axis remains largely unresolved, because no existing tool can follow signals through multiple connected neurons while maintaining the identity of individual cells or recording the order in which they were traversed.

I am developing a dual-barcode viral tracing system to solve this problem. The system is built on yellow fever 17D, which spreads from neuron to neuron in the forward direction, following the flow of information through a circuit. Each viral particle carries two molecular tags. The first is a fixed genetic barcode that permanently identifies the neuron where infection began, allowing individual cells to be tracked across multiple synaptic relays. The second is dynamic: it accumulates mutations at each successive neuron using the cell's own RNA editing machinery, writing a molecular record of transmission order directly into the viral genome. Sequencing both tags simultaneously across thousands of neurons reconstructs complete multi-step connectivity maps, revealing not just which neurons are wired together, but the precise sequence of connections between them.

I will deploy this system to map how the deep cerebellar nuclei – the primary output centers of the cerebellum – communicate with the neocortex through thalamic intermediates. This will reveal whether individual cerebellar neurons project with specificity or broadcast broadly, and whether distinct cerebellar subregions reach cortical targets through dedicated or shared pathways.

Beyond the cerebellum, this technology is circuit-agnostic and can be deployed to map any polysynaptic pathway in the brain, providing a generalizable platform for understanding how circuit organization breaks down in neurological and psychiatric disease, and how wiring principles vary across species.