Mapping brain-wide connectivity of neuronal types using barcoded connectomics

Mapping the brain-wide connections of neurons provides a foundation for understanding the structure and functions of a brain. Neuroanatomical techniques based on light-microscopy or electron microscopy have advanced tremendously in throughput and cost in recent years, but it remains challenging to scale them up to systematically interrogate large non-human primate (NHP) brains. Here we propose to develop sequencing-based neuroanatomical approaches to achieve high throughput and highly multiplexed brain- wide mapping of neuronal projections and synaptic connectivity in NHPs at cellular resolution. Unlike microscopy-based techniques, which rely on visually tracing individual axons from the somas to axonal termini, sequencing-based approaches label neurons with unique virally encoded RNA sequences, or “barcodes.” Sequencing and matching barcodes thus reveals the projections and/or synaptic connectivity of neurons. Thus, by transforming projection and connectivity mapping into sequencing problems, sequencing-based neuroanatomical approaches are not constrained by the same tradeoffs that plague microscopy-based techniques.

Specifically, we will develop and optimize three techniques for brain-wide mapping. First, we will optimize BRICseq (brain-wide individual animal connectome sequencing), which can currently map the projections of tens to hundreds of thousands of neurons in a single mouse brain at cellular resolution. We aim to adapt BRICseq for NHP brains, further reduce cost and increase throughput, to achieve the ability to map a million neurons per brain at cellular resolution at extremely low cost per neuron. Second, we will optimize BARseq (barcoded anatomy resolve by sequencing) for NHP brains. BARseq uses in situ sequencing of the same viral barcodes used in BRICseq to achieve higher resolution in projection mapping and to also read out gene expression in the same neurons. Thus, BARseq can associate neuronal projections with cell types defined by gene expression in individual neurons. We will automate in situ sequencing, reduce probe cost, and scale up BARseq to achieve the ability to map brain- wide projections in NHP brains. Finally, we will develop barcoded rabies virus-based monosynaptic tracing to achieve highly multiplexed mapping of synaptic connectivity of neuronal types at cellular resolution. 

Determining the synaptic connectivity of neuronal types will powerfully constrain and test computational models of circuit function beyond what knowing the axonal projections allows. We will apply all three techniques to generate a multi-resolution projection and synaptic connectivity map of the macaque visual cortex. With the ability to generate massive single-neuron datasets and the ability to link projections and synaptic connectivity to neuronal types, our proposed techniques complement mature techniques deployed at BRAIN CONNECTS centers to achieve an unprecedented view of NHP brains.