Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain

Abstract

The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.

Copyright © 2016, American Association for the Advancement of Science.

Figures

Fig. 1

Single nucleus RNA sequencing (SNS) identified 16 neuronal subtypes over 6 neocortical regions. A. Overview of SNS pipeline. Post-mortem tissue from Brodmann Areas (BA) 8, 10, 17, 21, 22, and 41/42 were dissociated to single nuclei for NeuN+ and DAPI+ sorting and capture on C1 chips. Resultant libraries were sequenced, mapped to the reference genome (pie chart showing averaged proportions) and screened for doublet removal before clustering and classification. BA proportions are shown. FC = Frontal Cortex; TC = Temporal Cortex; VC = Visual Cortex. B. Neuronal subtypes (excitatory (Ex) and inhibitory (In)) shown by multidimensional plotting using 10-fold or greater differentially expressed genes (Table S3); NoN (no nomenclature), low expression outlier cluster. C. Heatmap showing unique marker gene expression (Table S5).

Fig. 2

SNS reveals distinct interneuron subtypes. A. Pie charts display relative proportions of subtypes amongst BAs, and fraction of positive (FOP) heatmaps for inhibitory (In) and excitatory (Ex) marker genes. B. Diagram of subpallial origins of interneurons from either the lateral or medial ganglionic eminence (LGE, MGE) with FOP heatmaps (see A for scale) for marker genes associated with cortical layer (L) (upper panel), subpallial origin (middle panel) and interneuron classification (bottom panel). Potential interneuron subtypes are indicated below. SOM, somatostatin or SST; NPY, neuropeptide Y; CB, calbindin-D-28k or CALB1; VIP, vasoactive intestinal peptide; RELN, reelin; nNOS, neuronal nitric oxide synthase or NOS1; PV, parvalbumin or PVALB; CCK, cholecystokinin; NDNF, neuron-derived neurotrophic factor; CRHBP, corticotropin releasing hormone binding protein. C. Violin plots showing select marker gene expression values by BA (colors indicated in A) for each inhibitory neuron subtype. nGenes, total number of genes identified.

Fig. 3

Excitatory neuronal subtypes show distinct spatial organization. A. Schematic of the prefrontal cortex showing projection neuron layers (L) and expected axonal projection destinations (layer 4 granule neurons typically receive outside inputs for distribution of signals locally). B. FOP heatmap (see Fig. 2A for scale) for layer specific marker genes showing expected cortical layer identity (L2–L6b) and excitatory neuron sub-classification. CPN = cortical projection neuron; GN = granule neuron; SCPN = subcortical projection neuron; CThPN = corticothalamic projection neuron. C. Violin plots showing selected marker gene expression values by Ex subtype and BA represented by colors (see Fig. 2A). nGenes = total number of genes identified. D. RNA ISH showing layer-specific expression of selected markers in the temporal cortex (Allen Human Brain Atlas, Table S11).

Fig. 4

Neuronal subtypes reveal heterogeneity amongst BAs. A. Multidimensional plot showing projection neuron subtypes distributed according to their predicted cortical layer (L) identity. Layer 4 Ex2 and Ex3 subtypes are indicated. B. Clusters shown in (A) colored by BA and with BA41/42 and BA17 subpopulations of Ex3 indicated. C. Violin plots showing differentially expressed genes between Ex2 and Ex3 subtypes (Table S8). D. Heatmap showing genes differentially expressed between BA17 and BA41/42 within the Ex3 subtype (Table S10).