Abstract
Recent advances in single-cell technologies are paving the way to a comprehensive understanding of the cellular complexity in the brain. Protocols for single-cell transcriptomics combine a variety of sophisticated methods for the purpose of isolating the heavily interconnected and heterogeneous neuronal cell types in a relatively intact and healthy state. The emphasis of single-cell transcriptome studies has thus far been on comparing library generation and sequencing techniques that enable measurement of the minute amounts of starting material from a single cell. However, in order for data to be comparable, standardized cell isolation techniques are essential. Here, we analyzed and simplified methods for the different steps critically involved in single-cell isolation from brain. These include enzymatic digestion, tissue trituration, improved methods for efficient fluorescence-activated cell sorting in samples containing high degree of debris from the neuropil, and finally, highly region-specific cellular labeling compatible with use of stereotaxic coordinates. The methods are exemplified using medium spiny neurons (MSN) from dorsomedial striatum, a cell type that is clinically relevant for disorders of the basal ganglia, including psychiatric and neurodegenerative diseases. We present single-cell RNA sequencing (scRNA-Seq) data from D1 and D2 dopamine receptor expressing MSN subtypes. We illustrate the need for single-cell resolution by comparing to available population-based gene expression data of striatal MSN subtypes. Our findings contribute toward standardizing important steps of single-cell isolation from adult brain tissue to increase comparability of data. Furthermore, our data redefine the transcriptome of MSNs at unprecedented resolution by confirming established marker genes, resolving inconsistencies from previous gene expression studies, and identifying novel subtype-specific marker genes in this important cell type.
Highlights
The structural complexity of the human brain presents challenges to a mechanistic understanding of its development and function
As a starting point for developing a reproducible and efficient single-cell isolation protocol from adult rodent brain we took two previous studies that aimed at extraction of major, postmitotic central nervous system (CNS) cell types including hippocampal pyramidal neurons and striatal medium spiny neurons (MSN) (Brewer and Torricelli, 2007; Ena et al, 2013)
The study of cell type-specific gene expression is of great interest for heterogeneous tissues such as the brain, both to increase our scientific understanding and the discovery of novel therapeutic targets
Summary
The structural complexity of the human brain presents challenges to a mechanistic understanding of its development and function. With the exception of a recent report by Gokce et al (2016) using scRNA-Seq across striatal cell types (Gokce et al, 2016), these studies provide limited transcriptome coverage due to their reliance on multiplexed qPCR technology (Portmann et al, 2014; Fuccillo et al, 2015) This level of understanding is imperative as many subcortical neuronal subtypes including striatal MSN support critical information processing and have been implicated in various neurological disorders. The indirect pathway is thought to provide an inhibitory signal referred to as avoidance or punishment that reduces the likelihood of action initiation Both MSN subtypes are strongly modulated by dopamine, the striatal release of which shifts the balance between the direct and indirect pathway through activation of Gαs-coupled D1 receptors and Gαi-coupled D2 receptors. MSN function and imbalance is highly relevant to disorders of the basal ganglia and the dopamine system including Huntington’s disease, Parkinson’s disease, drug addiction, schizophrenia, ADHD and other psychiatric and neurodegenerative conditions
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