![]() ![]() MAPseq can be combined with in situ sequencing of starter cells to identify their Sindbis-encoded barcodes and fluorescence in situ hybridization (FISH) to assign these starter cells to particular cell types ( Chen et al., 2019). ![]() A structure of interest is infected with Sindbis virus encoding RNA barcodes that are trafficked into axons and detected by RNA-seq of target structures. A recently described sequencing-based method, multiplexed analysis of projections by sequencing (MAPSeq), traces connectivity anterogradely ( Kebschull et al., 2016). Because this method does not resolve single cells, it obscures heterogeneity within projection populations moreover, it must be performed on just one projection target at a time. A third sequencing-based approach, retroTRAP (translating ribosome affinity purification), relies on retrograde viruses to express tagged ribosomal subunits in projection neurons followed by immunoprecipitation of tagged mRNA ( Ekstrand et al., 2014). Both approaches are laborious, requiring separate rounds for each projection target and population of interest and additional steps for selective isolation of labeled cells, and costly, because each projection target is sequenced separately. In another, Patch-seq, cells are targeted for intracellular recording before the cellular contents are aspirated and sequenced ( Cadwell et al., 2016 Fuzik et al., 2016). In one approach, retrogradely labeled cells are isolated and sequenced ( Tasic et al., 2018). Many exploit dyes or viruses that are injected into the target structure, internalized at axon terminals, and trafficked retrogradely to label cell bodies in the source structure ( Wickersham and Feinberg, 2012). Therefore, in recent years, several approaches have been developed to focus transcriptional profiling on projection populations of interest. This is a powerful, systematic approach, but entails obtaining or generating Cre transgenic mice for each population and is mouse and labor intensive. One approach to this correspondence problem is to sequence a tissue, identify markers for many different cell types, and perform viral anterograde tracing in a panel of transgenic mice expressing Cre recombinase ( Ding et al., 2020). However, a challenge in interpreting single-cell RNA-seq datasets is to link populations identified through RNA expression to anatomy, connectivity, and circuit properties, a conundrum known as a “correspondence problem” ( Lein et al., 2017). Single-cell RNA sequencing (RNA-seq) technologies offer insights into the physiology of each identified neuronal population and molecular markers that could be used for targeted monitoring and manipulations during behavior. A central goal of neuroscience is deciphering the properties and behavioral functions of the myriad projection neuron subtypes in the brain. For example, primary visual cortex contains functionally distinct populations that project to higher visual cortical areas, contralateral cortex, and subcortical targets such as striatum, thalamus, and superior colliculus and substantia nigra pars reticulata in midbrain harbors subtypes that project to 39 target structures and differ in neurotransmitters released, response tuning, and intrinsic excitability ( Antal et al., 2014 Jiang et al., 2003 Kim et al., 2015 Lur et al., 2016 McElvain et al., 2021 Poulin et al., 2016). Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.įunctionally and molecularly diverse projection neurons with distinct targets are intermingled in most brain areas. ![]() 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. 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. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). 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. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Behavior arises from concerted activity throughout the brain. ![]()
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