Abstract
Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.
Highlights
Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions
To determine whether the common whole-cell dissociation method using papain was suitable for studying activity-induced expression, whole dentate granule cells (DGCs) were dissociated and examined by single-cell RNA-seq
The mice used in this experiment expressed cytoplasmic green fluorescent protein linked to the Prox[1] promoter[27], which enabled sorting for DGCs without permeabilizing the cell
Summary
Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr[1]. The response of individual dentate granule cells (DGCs) to an experience, such as exposure to a novel environment (NE), is highly variable and relatively unpredictable based on current staining approaches[10]. Such exposure has a well-documented rapid effect on the neural transcriptome, namely through activation of immediate early genes (IEGs)[11]. Despite this understanding of immediate events, much remains unknown with respect to the heterogeneity of gene expression that is both upstream and downstream of these IEGs
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