Virally Encoded Connectivity Transgenic Overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration

Abstract

SummaryBehavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing of defined projection populations are low-throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, Virally Encoded Connectivity Transgenic Overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes, e.g., recombinases and fluorescent proteins, by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.

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

“…SBARRO complements and extends other virus-based systems for studying cell-type-specific axonal projections and cell populationbased synaptic connectivity. For example, MAPseq 40 and VECTORseq 59 enable anterograde and retrograde labeling of axonal projections, respectively, in combination with transcriptional analyses of individual cells. Herpes virus-based Connect-seq 60 enables putative presynaptic networks (which are not strictly monosynaptic) to be identified upstream of individual Cre-expression postsynaptic cell populations (but not individual cells).…”

Single-Nucleus RNA Sequencing of Developing and Mature Superior Colliculus Identifies Neuronal Diversity and Candidate Mediators of Circuit Assembly

“…Tsai et al developed the Transseq method which used modified adeno-associated virus (AAV)-wheat germ agglutinin (WGA) to identify and profile SC neurons that are post-synaptic to RGCs (Tsai et al, 2022). Cheung et al developed Vector-seq using retrograde viruses to profile SC projection neurons that send axons to different brain regions (Cheung et al, 2021). In both studies, the authors provided userdefined subtypes of SC neurons, but how these subtypes compare across studies is not known, and whether these populations are detected in our development time-course dataset is unclear.…”

“…Trans-seq count matrix data were accessed from GEO with the accession GSE202257. Processed data from the Vectorseq study (Cheung et al 2021) were pulled from links from the Github repository associated with the publication (https://github.com/vic-cheung/vectorseq). Vector-seq count matrix data were accessed from GEO with the accession GSE189907.…”

Cell-type specific transcriptomic signatures of neocortical circuit organization and their relevance to autism

“…Finally, each major neuronal class contains unique subtypes that may form specialized microcircuit motifs (Tasic et al, 2018;Chen et al, 2019;Kim et al, 2020;Cheung et al, 2021;Que et al, 2021). In future studies, it will be interesting to use the additional metadata provided by the Allen Institute to further refine our analysis to consider these subtypes.…”