We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large ‘rosehip’-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1–SST–CALB2–PVALB–) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.
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The authors thank the Allen Institute for Brain Science founders, Paul G. Allen and Jody Allen, for their vision, encouragement, and support. The authors thank L. Christiansen and F. Zhang from Illumina, Inc. for their assistance with RNA sequencing. This work was supported by the ERC Interimpact project (G.T.), the National Institute of Mental Health (USA) RFA MH 17 210 (E.S.L., G.T.), the Hungarian Academy of Sciences (G.T.), the National Research, Development and Innovation Office of Hungary (GINOP-2.3.2-15-2016-00018, VKSZ-14-1-2015-0155), and by the National Brain Research Program, Hungary (G.T.).
The authors declare no competing interests.
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Integrated supplementary information
A, Nuclei were first gated based on size (forward scatter area, FSC-A) and granularity (side scatter area, SSC-A). B, Nuclei were then gated on DAPI fluorescence, followed by gates to exclude doublets and aggregates (C – FSC-single cells [SC], D – SSC-SC). E, Lastly, nuclei were gated based on NeuN Alexa Fluor 594 signal (NeuN-AF594-A) to differentiate neuronal (NeuN+) and non-neuronal (NeuN-) nuclei.
A, Heatmap of log-normalized expression (CPM) in single nuclei grouped by clusters that have been ordered by transcriptomic similarity. Canonical gene markers (SNAP25, GAD1, SLC17A7) classify clusters into broad classes of excitatory and inhibitory neuron and non-neuronal cell types. Within these broad types, many genes discretely mark individual clusters. B, Colorimetric ISH in mouse cortex of marker genes shown for human temporal cortex in Figure 1. Red arrows highlight cells with expression in layer 1. ISH experiments were repeated on multiple tissue specimens as follows: Sst, Lhx6,Ndnf (n=2); Slc17a7, Vip, Sema3c (n=3), Cck (n=4); Reln, Cnr1 (n=5); Gad1, Pvalb (n=20).
Supplementary Figure 3 Transcriptomic matching of clusters to previously reported clusters in mouse and human cortex.
A, Heatmap of expression correlations between pairs of clusters from this study and human cortical clusters reported by Lake et al. 22 based on median expression of  genes with cluster-specific expression in both studies. Reciprocal best matching cluster pairs are labeled. B, Proportion of nuclei expressing (CPM > 1) 168 genes for each pair of matching clusters labeled in A. Genes that are cluster-specific in both studies are labeled. C, Heatmap of median log2-expression for select marker genes in both studies with matching clusters labeled. D, Heatmap of expression correlations between pairs of clusters from this study and mouse cortical clusters reported by 4. Note that the same color scale is used for correlations as in A. E, Proportion of nuclei or cells expressing (CPM > 1) 212 genes for each pair of best matching clusters labeled in D. Pearson’s correlations are reported at the top of each plot.
Supplementary Figure 4 Diverse expression patterns of voltage-gated ion channels and receptors among GABAergic clusters.
Violin plots show the distribution of expression levels (CPM) within clusters and are scaled independently for each gene by the maximum expression across clusters for that gene. Expression is on a linear scale and dots indicate median expression. Numbers denote the number of nuclei in each cluster.
Supplementary Figure 5 Additional molecular phenotype information of RCs in layer 1 of the human cerebral cortex.
A, Further immunolabeling showed that RCs were immunopositive for gamma-aminobutyric acid (GABA) (n=2), and for chicken ovalbumin upstream promoter transcription factor II (NR2F2) (n=2). In addition, all tested RCs were negative for many common interneuron markers including parvalbumin (n=3), neuronal nitric oxide synthase (n=4), neuropeptide Y (n=2), calbindin (n=2), and choline acetyltransferase (n=3). B, Expression distributions of marker genes in all cell types in human temporal cortex layer 1 (left) and mouse primary visual cortex (right; data from 4). Expression is on a linear scale and dots indicate median expression. Numbers denote the number of nuclei in each cluster. C, Multiplex FISH validation of rosehip marker co-expression. Arrowheads show examples of RCs based on marker gene expression identified from RNA-Seq data. D, Multiplex FISH of approximately 2 mm section of layer 1 and upper layer 2 with rosehip interneurons labeled based on marker gene expression. 300 µm diameter circles approximate the maximum extent of rosehip axonal arbors (see Figure 2E). Multiplex FISH experiments were conducted on n=2 tissue donors.
Supplementary Figure 6 Multiplex FISH validation of rosehip marker co-expression in parietal (Brodmann Area 40) and frontal (Brodmann Area 9) regions of cortex.
A-F, Three distinct gene combinations were run in each brain region. Arrowheads show examples of RCs located in layer 1 of each brain region based on marker gene expression identified from RNA-Seq data. RCs were double positive for the marker genes shown and were either negative for CNR1 or had very low levels of CNR1 (C, E) in comparison to adjacent CNR1-positive cells (C). Multiplex FISH experiments were conducted on n=2 tissue donors.
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Boldog, E., Bakken, T.E., Hodge, R.D. et al. Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type. Nat Neurosci 21, 1185–1195 (2018). https://doi.org/10.1038/s41593-018-0205-2
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