Single-Cell RNA-Seq of Neurons in the Human Nervous System

Abstract

The human nervous system comprises highly heterogeneous neuronal populations. Our group has applied patch clamp electrophysiology, fluorescence microscopy, and next-generation single-cell RNA sequencing to correlate function, morphology and gene expression profiles at the single-cell level in both developing and mature human central nervous system. We performed whole cell patch clamp followed by cytoplasm extraction and single-cell RNA sequencing of neurons in acute slices and organotypic brain slice cultures from human adult cerebral cortex and cerebellum; and fetal brain and spinal cord of gestational age 10-20 weeks.We sequenced the mRNA with a modified aRNA method alongside with Truseq stranded RNA kit (Vn gelder et al., 1990). On average, we can detect ∼6,000 genes for each sample. Heterogeneity in gene expression and function was evident between neurons from different types of tissue when data were analyzed for Principle Components. Cajal-Retzius neurons are of special interest, as they are morphologically distinct and large neurons, located in the outermost layer of the developing cortex, where they are believed to contribute to defining the layered structure of the cortex. We showed that Cajal-Retzius neurons separate from subplate neurons with PC analysis and identified sets of genes that are significantly differentially expressed in Cajal-Retzius neurons compared to subplate neurons. Electrophysiological recordings revealed for the first time, spontaneous synaptic activity and action potential firing in human Cajal-Retzius neurons. Our data are significant for demonstrating the synergistic potential of combining functional and transcriptome analysis at the single-cell level.