Single-nuclei RNA-sequencing of activated neurons enables the examination of gene expression beyond immediate early genes

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 researchers from the Salk Institute for Biological Studies 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, the researchers observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone.

IEG RNA expression in single DGC nuclei is associated with animal experience

rna-seq

(a) log2(TPM+1) Normalized RNA-seq values from HC NEUN+PROX1+ (top, n=23), NE PROX1+FOS− (bottom, n=43), and NE PROX1+FOS+ (bottom, n=36) single-nuclei. NE FOS+ nuclei (red) exhibited higher levels of IEG expression than both NE FOS− (blue) and HC nuclei (white). Stars indicate statistically significant differences in expression using edgeR after multiple-testing correction (fdrtool; R). Pie charts indicate the proportion of nuclei with detectable gene expression (yellow=detected). (b) IEG expression in nuclei after exposure to NE. (c) Principal components analysis (PCA) of the full transcriptome for NE nuclei. pseudo-FOS+ cells in red with black outline. (d) Differential expression results for all genes between FOS+ and FOS− nuclei excluding pseudo-FOS+ nuclei. Genes expressed to a higher level in FOS+ nuclei are in red and genes expressed higher in FOS− nuclei are in blue.

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.

Lacar B, Linker SB, Jaeger BN, Krishnaswami S, Barron J, Kelder M, Parylak S, Paquola A, Venepally P, Novotny M, O’Connor C, Fitzpatrick C, Erwin J, Hsu JY, Husband D, McConnell MJ, Lasken R, Gage FH. (2016) Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat Commun 7:11022. [article]

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