Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration

Cell Rep. 2021 Dec 21;37(12):110131. doi: 10.1016/j.celrep.2021.110131.

Abstract

Behavior 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 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.

Keywords: connectivity; lateral posterior thalamus; methodology; neuronal cell types; single-cell sequencing; superior colliculus; viral barcoding; zona incerta.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Expression Profiling / methods
  • Genetic Techniques
  • High-Throughput Screening Assays / methods*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Neural Pathways / physiology
  • Neurons / classification*
  • Neurons / physiology*
  • Optogenetics
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • Transgenes