Ascertaining cells’ synaptic connections and RNA expression simultaneously with barcoded rabies virus libraries

Abstract

Brain function depends on synaptic connections between specific neuron types, yet systematic descriptions of synaptic networks and their molecular properties are not readily available. Here, we introduce SBARRO (Synaptic Barcode Analysis by Retrograde Rabies ReadOut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its “starter” postsynaptic cell to that cell’s presynaptic partners. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNAs. We use SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, finding that different cell types have presynaptic networks with differences in average size and cell type composition. Patterns of RNA expression suggest that functioning synapses are critical for rabies virus uptake. By tracking individual rabies clones across cells, SBARRO offers new opportunities to map the synaptic organization of neural circuits.

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA andin situsequencing

“…Accurately identifying the network that each neuron belongs to requires sequencing all source cells. This requirement is difficult to achieve using single-cell RNA-seq-based approaches (Clark et al 2021; Saunders et al 2022). Because conventional single-cell approaches require tissue dissociation, some source cells are inevitably lost.…”

“…Nonetheless, we could infer which neuronal types were more likely to synapse together onto the same source cell, regardless of the identity of the source cell. This analysis goes beyond previous barcoded rabies-based approaches (Clark et al 2021;Saunders et al 2022), and represents a first proof-of-principle of inferring the statistics of synaptic connectivity of neuronal types using a barcoding-based approach. Further optimizing the technique to unambiguously determine connectivity between source cells and presynaptic neurons will not only generate insights into the connectivity of neuronal types, but also allow comparison to connectivity inferred from electron microscopy data(MICrONS Consortium et al 2021) and patch-seq data (Campagnola et al 2022) to validate the specificity of rabies monosynaptic tracing.…”

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

“…This type of barcode sharing can be minimized by employing sufficiently diverse and uniformly distributed barcodes (Kebschull et al, 2016). However, this is typically challenging to achieve with rabies virus, because the recombinant rabies virus production process usually leads to uneven amplification of barcodes, resulting in their highly skewed distribution (Clark et al, 2021;Saunders et al, 2022). Alternatively, two cells expressing the rabies glycoprotein can be interconnected.…”

Limitations and Optimizations of Cellular Lineages Tracking

“…As a result, the number of clones in each passaging decreases, while the mean clone size increases. In the simulation presented in this document we [28] 36, 000 0.05 − 0.1 5% − 10% cells fate [32] 5, 000 − 10, 000 0.5 • 10 6 synaptic networks [33] 1.29 e we calculated it using the absolute number of infected and uninfected cells given in [33] TABLE I. Cellular barcoding parameters in a variety of systems.…”