Molecular diversity and specializations among the cells of the adult mouse brain

The paper grouped cells into 565 subclusters using network-based clustering on biological independent component cell loadings.

“We then grouped cells based on combinations of “biological IC” cell loadings into 565 subclusters using network-based clustering”

• cites Shekhar et al., 2016 sourcePartly supported

  The Methods section (page unavailable) supports the use of graph-based clustering, but says cells were clustered using scores on 37 significant principal components, not biological independent-component loadings. It reports 30 initial Louvain-Jaccard clusters, while the Results section (page unavailable) reports 26 putative cell-type clusters—not 565 subclusters. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Waltman and van Eck, 2013 sourcePartly supported

  The Conclusions (p. 19) say the source introduces an algorithm for modularity-based community detection in large networks, supporting its use as a network-clustering method. The source does not report grouping cells by biological IC loadings or obtaining 565 subclusters; in the citing sentence, “We” refers to the citing authors, not Waltman and van Eck. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: Transcriptional clusters derived from single-cell RNA-seq correspond to biologically meaningful cell types or states.

The third transcriptional pattern involved a constellation of genes underlying axonal and pre-synaptic function, called the Neurofilament IC, because three of the most strongly contributing genes encode neurofilament subunits.

“This transcriptional pattern involved a constellation of genes that underlie axonal and pre-synaptic function. We call this signal the “Neurofilament IC,” because three of the most strongly contributing genes encode the neurofilament subunits of the axonal cytoskeleton (Nefl, Nefm, and Nefh)”

• cites Yuan et al., 2012 sourcePartly supported

  The passage describing neurofilament composition identifies NEFL, NEFM and NEFH as genes for light, medium and heavy neurofilament subunits, supporting the reason for the label. It does not describe a transcriptional pattern, an independent component (IC), or which genes contributed most strongly to one; those are findings of the citing study. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Across cell populations, expression of nicotinic acetylcholine receptor genes exhibited two prominent correlation blocks, each containing genes that encode subunits of known heteromeric α/β channels.

“Across cell populations, expression of nAChR genes exhibited two prominent correlation blocks, each containing genes that encode subunits of known heteromeric α/β channels”

• cites Zoli et al., 2015 sourcePartly supported

  Section 2, “Structure of nicotinic receptor subtypes” (page unavailable), says nAChR genes encode α and β subunits that form heteromeric receptors. It does not report an analysis of gene-expression correlations across cell populations or identify two correlation blocks; searches for those terms found no such passage. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Subcluster 3-12 likely corresponds to the glutamatergic projection to thalamus.

“Of the remaining subclusters, 3–12 expressed Slc17a6±, likely corresponding to the glutamatergic projection to thalamus (Antal et al., 2014).”

• cites Antal et al., 2014 sourceSupported

Subclusters spanning borders might account for a neuron type synaptically integrated into the GPe yet with VP-like axonal projections.

“Border-spanning subclusters may explain a neuron type that is synaptically incorporated into the GPe, but exhibits VP-like axonal projections (Chen et al., 2015; Saunders et al., 2015).”

• cites Chen et al., 2015 sourcePartly supported

  The Results section, ‘Striatal projections to pallidocortical neurons’ (page unavailable), shows that cortically projecting GPe neurons receive striatal input, supporting their incorporation into GPe circuitry. The Discussion (page unavailable) describes GPe and nearby basal-forebrain projecting neurons as appearing continuous in retrograde tracing, but it does not identify border-spanning subclusters or a neuron type with VP-like axonal projections. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Saunders et al., 2015 sourcePartly supported

  The paper reports GP-to-frontal-cortex neurons clustered along GP borders and receiving synaptic input characteristic of GP circuitry (Abstract; page not specified). It does not describe their axonal projections as VP-like or propose border-spanning subclusters as an explanation for that combination. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

These subclusters point to neuron types shared between regions that differ in behavioral function and connectivity.

“Several subclusters (2–15, 2–21, and 2–18) expressed markers in the VP and bordering GPe (Figure 6D,E), suggesting neuron types shared across regions with different behavioral functions and connectivity (Gittis et al., 2014; Kita, 2007; Smith et al., 2009).”

• cites Gittis et al., 2014 sourceSupported
• cites Kita, 2007 sourceNot checked — no full text of the source
• cites Smith et al., 2009 sourceSupported

Subcluster 2-14 represents fast-spiking "prototypical" GPe neurons.

“Only Pvalb and Penk/Foxp2 were selectively expressed in GPe subclusters (Kita, 1994; Voorn et al., 1999), suggesting 2–14 represents fast-spiking “prototypical” neurons, while 2–19 represents the slow-firing “arkypallidal” population (Abdi et al., 2015; Mallet et al., 2012).”

• cites Abdi et al., 2015 sourcePartly supported

  In the section on firing rates and patterns in dopamine-intact rats (Fig. 6), the paper identifies PV-positive prototypic GPe neurons and reports that they fire at relatively high rates. It does not identify subcluster 2–14; assigning that subcluster to this cell type is the citing authors’ inference. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Mallet et al., 2012 sourcePartly supported

  The Discussion identifies predominantly parvalbumin-positive GP-TI neurons as prototypic, and the Introduction describes prototypic GPe neurons as fast-firing. The paper does not identify subcluster 2–14, so the assignment of that subcluster to this cell type is the citing authors’ inference, not a finding verified by this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Subcluster 2-19 represents the slow-firing "arkypallidal" GPe population.

“Only Pvalb and Penk/Foxp2 were selectively expressed in GPe subclusters (Kita, 1994; Voorn et al., 1999), suggesting 2–14 represents fast-spiking “prototypical” neurons, while 2–19 represents the slow-firing “arkypallidal” population (Abdi et al., 2015; Mallet et al., 2012).”

• cites Abdi et al., 2015 sourceSupported
• cites Mallet et al., 2012 sourcePartly supported

  The Results (Physiological Dichotomy; page unavailable) report that GP-TA neurons fired more slowly than prototypic GP-TI neurons during slow-wave activity, and the Discussion identifies GP-TA neurons as arkypallidal. The source does not identify subcluster 2–19, and the firing-rate distinction is condition-dependent: GP-TA neurons fired faster than GP-TI neurons during beta oscillations. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Some eSPN subsets share markers that earlier work used to distinguish SPN subtypes.

“Subsets of the eSPN class share markers previously reported to distinguish SPN subtypes (Gökce et al., 2016).”

• cites Gökce et al., 2016 sourceSupported

Canonical SPN marker expression indicates eSPNs may be molecularly camouflaged, including in mouse studies using Drd1- and Adora2a-driven transgenes to label or manipulate dSPNs/iSPNs.

“Expression of markers associated with canonical SPNs suggests eSPNs have been molecularly “camouflaged,” including in studies using mice that have employed Drd1 and Adora2a driven–transgenes to label and manipulate dSPNs or iSPNs (Heiman et al., 2008; Kozorovitskiy et al., 2012; Kravitz et al., 2012).”

• cites Heiman et al., 2008 sourcePartly supported

  In Results (“The BACarray approach and methodology”; page unavailable), Heiman et al. describe D1-driven BAC transgenic mice used to identify striatonigral neurons. Results (“BACarray profiling of striatonigral and striatopallidal MSNs”; page unavailable) identify Adora2a as a striatopallidal marker, but the study uses D2—not Adora2a—to drive its striatopallidal transgene. It does not establish the cited example of Adora2a-driven transgenes used to label or manipulate those neurons; the molecular-camouflage inference is the citing authors’ framing. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Kozorovitskiy et al., 2012 sourcePartly supported

  The paper’s indexed text (section labeled Abstract; page unavailable) describes using Drd1a-Cre mice to target direct-pathway MSNs and Adora2a-Cre mice as a driver line for indirect-pathway MSNs. It does not discuss eSPNs, canonical marker expression, or molecular camouflage, so it supports the cited example of transgenic mouse studies but not the sentence’s broader inference. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Kravitz et al., 2012 sourceSupported

Non-neuronal cells exhibited broadly consistent expression signals across brain regions.

“Non-neuronal cells exhibited broadly consistent expression signals across brain regions.”

Among fibroblast-like cells, two subclusters (4 and 5) selectively expressed many genes encoding membrane transporters and pumps.

“Among the fibroblast-like cells, two subclusters (4 and 5) selectively expressed many genes encoding membrane transporters (e.g., Slc38a2, Slc4a10, Slc26a2, and Slc47a1) and pumps (e.g., Fxyd5 and Atp1b1)”

Analysis of pairwise correlations across neuronal independent component gene loadings revealed three prominent correlation blocks.

“Analysis of pairwise correlations across neuronal IC gene loadings revealed three prominent correlation blocks (Figure 3A).”

Endothelial independent components had strong contributions from genes with arterial and veinous expression, suggesting heterogeneity related to vessel type.

“We identified endothelial ICs (Figure 2E) with strong contributions from genes with arterial (IC 5 and 20) and veinous (IC 3 and 12) expression, suggesting heterogeneity related to vessel type”

• cites Vanlandewijck et al., 2018 sourceNot checked — no full text of the source

Single-molecule fluorescent in situ hybridization experiments confirmed that expression of Pvalb, Syt2, and Nefm were highly correlated among Pvalb+ neurons in the frontal cortex.

“Single-molecule fluorescent in situ hybridization experiments confirmed that, as predicted, expression of Pvalb, Syt2, and Nefm were highly correlated among Pvalb+ neurons in the frontal cortex”

Among smooth muscle alpha actin cells, Acta2 expression correlates with a veinous versus arterial distribution.

“Among SMA cells, Acta2 expression correlates with a veinous versus arterial distribution”

• cites Vanlandewijck et al., 2018 sourceNot checked — no full text of the source

Cross-region analysis revealed a gene-expression module for synthesizing axonal and presynaptic components.

“including a gene-expression module for synthesizing axonal and presynaptic components”

A second correlation block arose from diverse thalamic neurons, suggesting a tissue-specific transcriptional program.

“A second block arose from diverse thalamic neurons, suggesting a tissue-specific transcriptional program.”

Neurofilament IC cell loading was visible among Pvalb+ interneurons following Drop-seq analysis of 28,194 single nuclei isolated from flash-frozen frontal cortex.

“Furthermore, Neurofilament IC cell loading was visible among Pvalb+ interneurons following Drop-seq analysis of 28,194 single nuclei isolated from flash-frozen frontal cortex”

The expression of genes with the strongest Neurofilament IC contributions tended to covary both within and across neuronal types.

“The expression of genes with the strongest Neurofilament IC contributions tended to covary both within and across neuronal types.”

Other co-regulated genes in the Neurofilament IC included Syt2, Vamp1, Cplx1, Pvalb, and Caln1.

“Other co-regulated genes included Syt2, Vamp1, and Cplx1 – which have roles in vesicle exocytosis – and Pvalb and Caln1, which bind presynaptic Ca2+ (Data S5).”

Distinct brain regions yielded cell classes in different proportions.

“Distinct brain regions yielded cell classes in different proportions (Figure S1E).”

The study identified 565 transcriptionally distinct cell groups using computational methods that separate biological from technical signals.

“We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals.”

• AssumptionRests on an assumption: Transcriptional clusters derived from single-cell RNA-seq correspond to biologically meaningful cell types or states.

One correlation block included 15 inter-correlated independent components representing immediate early gene expression signatures from different regions and neuronal types.

“One such block included 15 inter-correlated ICs representing IEG-expression signatures from different regions and neuronal types.”

Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons.

“Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons.”

These arterial/veinous genes showed continuous, reciprocal expression, consistent with smooth molecular transitions described for endothelial cells associated with arteries or veins.

“These genes (e.g. Tm4sf1 and Slc38a5) showed continuous, reciprocal expression (Data S4G), consistent with the smooth molecular transitions described for endothelial cells associated with arteries or veins”

• cites Vanlandewijck et al., 2018 sourceNot checked — no full text of the source

Cross-region analysis revealed specialization of glutamatergic neurons across cortical regions.

“specialization of glutamatergic neurons across cortical regions”

Subclusters 1, 2, and 3 of fibroblast-like cells expressed genes involved with extracellular matrix secretion, angiogenesis, and contraction.

“To varying extents, subclusters 1, 2, and 3 expressed genes involved with extracellular matrix (ECM) secretion, angiogenesis, and contraction”

The paper identified 7 mural subclusters from 7 biological independent components, comprising 4,713 cells.

“We identified 7 mural subclusters from 7 biological ICs (n=4,713 cells, Figure 2C and Data S4E).”

The first stage of analysis robustly grouped cells into 8-11 broad classes, including neurons, astrocytes, microglia/macrophages, oligodendrocytes, polydendrocytes, and vascular components.

“The first stage of analysis robustly grouped cells into 8–11 broad classes (Data S2), including neurons, astrocytes, microglia/macrophages, oligodendrocytes, polydendrocytes (oligodendrocyte progenitor cells), and components of the vasculature”

The paper created a draft mouse brain cell atlas by profiling 690,000 individual cells from nine major regions of the adult mouse brain using Drop-seq.

“Here, we describe a draft mouse brain cell atlas that we created by profiling (using Drop-seq) 690,000 individual cells from nine major regions of the adult mouse brain.”

• AssumptionRests on an assumption: The nine sampled brain regions are representative of the adult mouse brain.

In the cortex, glutamatergic neurons tend to be specialized by region.

“We found that, in the cortex, glutamatergic neurons tend to be specialized by region”

The paper discovered large-scale patterns in ion channel gene expression that may enact neurons' electrophysiological properties.

“discovered large-scale patterns in ion channel gene expression that may enact neurons’ electrophysiological properties”

In the striatum, the paper identifies a novel group of principal neurons that had been overlooked in decades of research.

“In the striatum, where neuronal diversity is well charted, we nonetheless identify a novel group of principal neurons that had been overlooked in decades of research.”

Cluster 3 expressed higher levels of different collagen genes, Col1a1 and Col3a1.

“Interestingly, cluster 3 expressed higher levels of different collagen genes (Col1a1 and Col3a1)”

Non-neuron cell classes, such as those making up the vasculature, can be variably specialized across cortical and subcortical areas.

“while non-neuron cell classes, such as those that make up the vasculature, can be variably specialized across cortical and subcortical areas”

A similar Neurofilament IC pattern was ascertained among cortical glutamatergic and subcortical neuromodulatory populations.

“We ascertained a similar pattern among cortical glutamatergic (Data S5) and subcortical neuromodulatory (Figure 7O) populations.”

Other endothelial independent components identified genes with more bimodal expression, indicating specializations within the artery/vein gradient.

“Other ICs identified genes with more bimodal expression, indicating specializations within this artery/vein gradient.”

Cross-region analysis revealed functional distinctions among the cells of the vasculature.

“functional distinctions among the cells of the vasculature”

Cross-region analysis revealed patterns in the co-deployment of voltage-gated ion channels.

“patterns in the co-deployment of voltage-gated ion channels”

Among interneurons, Neurofilament IC cell loading was most prominent in fast-firing Pvalb+ populations.

“Among interneurons, Neurofilament IC cell loading was most prominent in fast-firing Pvalb+ populations.”

The third correlation block consisted of independent components from many brain regions and neuronal types.

“We focused on the third correlation block, which consisted of ICs from many brain regions and neuronal types.”

IC 19 represented the veinous-arterial difference in a graded rather than categorical way, as Rgs5/Acta2 expression and IC cell scores were continuously distributed across these cells.

“IC 19 represented this difference in a graded rather than categorical way, as Rgs5/Acta2 expression and IC cell scores were continuously, rather than bimodally, distributed across these cells”

• cites Vanlandewijck et al., 2018 sourceNot checked — no full text of the source

Removing technical independent components reduced spurious distinctions among cells.

“Removing technical ICs reduced spurious distinctions among cells (Figure S2I).”

Across subclusters, patterns of similar RNA expression generally corresponded to cell class rather than brain region.

“Across subclusters, patterns of similar RNA expression generally corresponded to cell class rather than brain region (Figure S1F,G).”

The paper identified and validated a pervasive transcriptional program supporting axon and presynaptic function.

“we identified and validated a pervasive transcriptional program supporting axon and presynaptic function”

Several α-subunit genes, including known (Chrna7, Chrna9, Chrna10) and not yet known (Chrna1, Chrna2) homomeric channel genes, lacked positive pairwise correlations with other subunit genes.

“Several α-subunit genes had this property, including those known (Chrna7, Chrna9, and Chrna10) and others not yet known (Chrna1 and Chrna2) to form homomeric channels in brain”

• cites Gotti et al., 2006 sourceNot checked — no full text of the source

Cluster 1 contained cholinergic neurons (n=437 cells).

“Cluster 1 contained cholinergic neurons (n=437 cells) and cluster 3 contained neurons of the adjacent striatum and basolateral amygdala (n=9,847 cells).”

Subcluster 3-12 expressed Slc17a6±.

“Of the remaining subclusters, 3–12 expressed Slc17a6±, likely corresponding to the glutamatergic projection to thalamus (Antal et al., 2014).”

In cluster 2, eight of twenty-five subclusters seemed intrinsic to the GPe or neighboring ventral pallidum.

“Within cluster 2, 8 of 25 subclusters appeared intrinsic to the GPe or adjacent ventral pallidum (VP), of which 4 were GPe-exclusive (Figure 6B–E).”

• cites Figure 6B–ENot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Selective markers for these cell subclusters confirmed their asymmetric distributions across the cortical mantle.

“Selective markers for these cell subclusters confirmed their asymmetric distributions across the cortical mantle (Figure 5D and Figure S5).”

Expression of Chrna3 and Chrnb4 was positively correlated across a large range.

“For example, expression of Chrna3 and Chrnb4 (known to form functional receptors) was positively correlated across a large range (from 0.01 to 100 transcripts per 100K)”

The study identified 323 neuronal subclusters across nine regions.

“We identified 323 neuronal subclusters across 9 regions (Data S3).”

Four of those subclusters were GPe-exclusive.

“Within cluster 2, 8 of 25 subclusters appeared intrinsic to the GPe or adjacent ventral pallidum (VP), of which 4 were GPe-exclusive (Figure 6B–E).”

• cites Figure 6B–ENot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

One set of co-expressed genes encoded proteins that enable fast and persistent action potentials, including Kcnc1-3 (Kv3.1), Kcna1 (Kv1.1) and Scn8a (Nav1.6) channels.

“One set of co-expressed genes encoded proteins that enable fast and persistent action potentials, including the Kcnc1–3 (Kv3.1), Kcna1 (Kv1.1) and Scn8a (Nav1.6) channels”

• cites Chen et al., 2008 sourceNot checked — no full text of the source
• cites Goldberg et al., 2008 sourceNot checked — no full text of the source
• cites Rudy and McBain, 2001 sourceNot checked — no full text of the source

Of four global neuron clusters, cluster 3 (n=10,049 cells) contained SNr neurons.

“Of 4 global neuron clusters (Figure 6F), one (cluster 3, n=10,049 cells) contained SNr neurons (Figure 6G).”

• cites Figure 6FNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Figure 6GNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Gene expression correlations across cell types recapitulated known nicotinic acetylcholine receptor subunit combinations more accurately than correlations based on bulk expression profiles for the nine regions.

“Gene expression correlations across cell types recapitulated known nAChR receptor subunit combinations (Gotti et al., 2006) more accurately than correlations based on the bulk expression profiles for the nine regions (Figure S3B).”

• cites Gotti et al., 2006 sourceNot checked — no full text of the source

GPe neurons were present in cluster 2, which contained 11,103 cells.

“GPe neurons were present in cluster 2 (n=11,103 cells), one of three neuron clusters (Figure 6A).”

• cites Figure 6ANot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Expression of Slc6a8 and Hcn2 were strongly correlated and high in fast-firing cell types.

“Indeed, expression of Slc6a8 and Hcn2 were strongly correlated and high in fast-firing cell types (Figure 4E).”

Cluster 3 held neurons from the adjacent striatum and basolateral amygdala (n=9,847 cells).

“Cluster 1 contained cholinergic neurons (n=437 cells) and cluster 3 contained neurons of the adjacent striatum and basolateral amygdala (n=9,847 cells).”

The remaining seventeen subclusters mapped to the thalamic reticular nucleus, substantia innominata, and lateral olfactory tract.

“The other 17 subclusters mapped to the thalamic reticular nucleus, the substantia innominata, and the lateral olfactory tract (Figure S6A–D).”

• cites Figure S6A–DNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The paper observed strong correlation blocks involving specific subsets of voltage-gated potassium and sodium channel genes across neuronal populations.

“We observed strong correlation blocks involving specific subsets of VGK and VGNA genes (Figure 4C).”

Subclusters 2-15, 2-21, and 2-18 showed marker expression in the VP and GPe border.

“Several subclusters (2–15, 2–21, and 2–18) expressed markers in the VP and bordering GPe (Figure 6D,E), suggesting neuron types shared across regions with different behavioral functions and connectivity (Gittis et al., 2014; Kita, 2007; Smith et al., 2009).”

• cites Figure 6D,ENot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Pvalb and Penk/Foxp2 were the only markers selectively expressed in GPe subclusters.

“Only Pvalb and Penk/Foxp2 were selectively expressed in GPe subclusters (Kita, 1994; Voorn et al., 1999), suggesting 2–14 represents fast-spiking “prototypical” neurons, while 2–19 represents the slow-firing “arkypallidal” population (Abdi et al., 2015; Mallet et al., 2012).”

• cites Kita, 1994 sourceNot checked — no full text of the source
• cites Voorn et al., 1999 sourceNot checked — no full text of the source

Within cluster 3, 6 of 19 subclusters mapped to the SNr.

“Within cluster 3, 6 of the 19 subclusters mapped to the SNr (Figure 6H–J and Figure S6E–H).”

• cites Figure 6H–JNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Figure S6E–HNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Cluster 11 corresponded to iSPNs (n=25,305 cells).

“Two large clusters corresponded to dSPNs (Cluster 10, n=30,835 cells) and iSPNs (Cluster 11, n=25,305)(Striatum dataset, Figure 7A,B) and were distinguished by 68 differentially expressed genes, including known and undescribed pathway markers (Figure 7C).”

• cites Figure 7A,BNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Cluster 13 also expressed Adora2a and Drd1.

“Surprisingly, about 4% of SPNs (Ppp1r1b+) were observed in a third, smaller cluster that also expressed Adora2a and Drd1 (cluster 13: n=2,744 cells; 4.5% of Ppp1r1b+ neurons; Figure 7A,B).”

• cites Figure 7A,BNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Dopaminergic neurons were more heterogeneous than cholinergic neurons (DA: 6 biological ICs, 9 subclusters; ACh: 2 biological ICs, 2 subclusters).

“Subcluster analyses of the dopaminergic (SN/VTA, cluster 4, 919 cells) and cholinergic (GP/NB, cluster 1, n=218 cells) clusters revealed that dopaminergic neurons were indeed more heterogeneous than the cholinergic neurons (DA: 6 biological ICs, 9 subclusters; ACh: 2 biological ICs, 2 subclusters)(Figure 6A,F).”

• cites Figure 6A,FNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• AssumptionRests on an assumption: The GPe and SNr lie next to neuromodulatory populations releasing acetylcholine or dopamine, respectively.

Cluster 10 corresponded to dSPNs (n=30,835 cells).

“Two large clusters corresponded to dSPNs (Cluster 10, n=30,835 cells) and iSPNs (Cluster 11, n=25,305)(Striatum dataset, Figure 7A,B) and were distinguished by 68 differentially expressed genes, including known and undescribed pathway markers (Figure 7C).”

• cites Figure 7A,BNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The data account for all known striatal interneuron types (3.9% of total neurons).

“Our data account for all known striatal interneuron types (3.9% of total neurons)(Tepper and Bolam, 2004), suggesting by exclusion that eSPNs are not interneurons.”

• cites Tepper and Bolam, 2004 sourceNot checked — no full text of the source

The clustering analysis yielded between 11 and 22 global clusters for each of the nine brain regions, dividing cells into broad classes.

“This produced 11–22 Global clusters across the nine different tissues, partitioning cells into broad “classes.””

About 10% of iSPNs and dSPNs showed this patch signal.

“Approximately 10% of iSPNs and dSPNs exhibited this patch signal (Figure S7A,B).”

• cites Figure S7A,BNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

eSPN subclusters fell into two main groups, split by a gene set containing markers like Drd1 and Adora2a that distinguish canonical iSPNs from dSPNs.

“eSPN subclusters (Figure S7F–H) were divided into two major groups (Figure 7H), separated by a gene set that included markers used to distinguish canonical iSPNs from dSPNs, such as Drd1 and Adora2a (Figure 7I).”

• cites Figure 7HNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Figure 7INot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The study confirmed further eSPN diversity predicted by Drop-seq, including an ultra-rare Adora2a±/Th±/Npffr1+ subtype (13-5) making up only 0.3% of SPNs (n=88).

“We validated additional eSPN diversity predicted by Drop-seq, including an ultra-rare eSPN Adora2a±/Th±/Npffr1+ subtype (13–5) that accounts for just 0.3% of all SPNs (n=88 cells)(Figure 7J).”

• cites Figure 7JNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

These SPNs expressed many genes with little expression elsewhere in the striatum.

“These SPNs differed in expression from dSPNs+iSPNs by 110 genes (more than the 68 that distinguished dSPNs and iSPNs from each other) (Figure 7C), and expressed many genes that had little expression in the rest of the striatum (Figure 7D).”

• cites Figure 7DNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

eSPNs were mixed among other striatal SPNs without clear spatial organization.

“eSPNs were intermixed with other SPNs in the striatum with no obvious spatial organization (Figure 7E,F).”

• cites Figure 7E,FNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Although they share markers, Adora2a+ and Drd1+ eSPNs differ from canonical SPNs in expression levels of many genes (35 for Adora2a+; 96 for Drd1+).

“Despite sharing markers, Adora2a+ eSPNs and Drd1+ eSPNs are distinguished from their canonical SPN counterparts by expression levels of many genes (Adora2a+ SPNs: 35 genes; Drd1+ SPNs: 96 genes; Figure S7I).”

• cites Figure S7INot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Aspects of dopamine diversity related to spatial positioning, for example delineating dorsal (IC 10) from ventral (IC 12) VTA.

“While aspects of DA diversity related to spatial positioning – for example, delineating the dorsal (IC 10) from ventral (IC 12) VTA (Figure 6L–N) - cholinergic neurons exhibited only a Neurofilament-like signal (IC 4), with no spatial component (Figure 6O,P).”

• cites Figure 6L–NNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

A third, smaller cluster contained roughly 4% of Ppp1r1b+ SPNs (cluster 13: n=2,744 cells; 4.5% of Ppp1r1b+ neurons).

“Surprisingly, about 4% of SPNs (Ppp1r1b+) were observed in a third, smaller cluster that also expressed Adora2a and Drd1 (cluster 13: n=2,744 cells; 4.5% of Ppp1r1b+ neurons; Figure 7A,B).”

• cites Figure 7A,BNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

These SPNs showed expression differences from dSPNs and iSPNs at 110 genes.

“These SPNs differed in expression from dSPNs+iSPNs by 110 genes (more than the 68 that distinguished dSPNs and iSPNs from each other) (Figure 7C), and expressed many genes that had little expression in the rest of the striatum (Figure 7D).”

• cites Figure 7CNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The paper developed an analysis method based on independent components analysis.

“We therefore developed an analysis method based on independent components analysis (ICA)(Figure 1B–E).”

Diverse neuron types share a coordinated transcriptional program involving genes that facilitate maintenance, elaboration, or subcellular transport to the axon and presynaptic terminal.

“We conclude that diverse neuron types share a coordinated transcriptional program involving genes that facilitate maintenance, elaboration, or subcellular transport to the axon and presynaptic terminal.”

The cell suspensions had cells with morphologies characteristic of neurons, astrocytes, and oligodendrocytes.

“had cells with morphologies characteristic of neurons, astrocytes, and oligodendrocytes (Figure S1A).”

Neuronal types characterized by extensive axonal arbors, long-distance axonal projections, and/or faster firing rates tended to utilize this transcriptional program more than other neurons.

“Neuronal types characterized by extensive axonal arbors, long-distance axonal projections, and/or faster firing rates tended to utilize this transcriptional program more than other neurons.”

The paper developed analytical methods to separate biological and technical influences on single-cell data.

“We developed analytical methods to separate biological and technical influences on single-cell data, enabling a more transparent understanding of the relationships driving cellular classifications.”

The resulting cell suspensions recovered intact 40-50% of cells from most tissues.

“The resulting cell suspensions, which recovered intact 40–50% of cells from most tissues (cortex: 0.46 ± 19 mean ± sem; striatum: 0.39 ± 20, Figure S4A–C)”

Gene-expression profiles for the 565 cell populations are less influenced by biological and statistical noise than single-cell-level data.

“these data are less influenced by biological and statistical noise than single-cell-level data (Figure S3A).”

Both over-representation effects could be partially explained by cell-inclusion thresholds, in which small but real transcript libraries had been excluded from downstream analysis.

“Both effects could be partially explained by cell-inclusion thresholds, in which small but real transcript libraries had been excluded from downstream analysis”

The data exhibit modest skews in cellular representation driven by transcript abundance and viability.

“We conclude our data exhibit modest skews in cellular representation driven by transcript abundance and viability.”

These pairs suggest relatedness.

“Two subcluster pairs shared selective markers (3–18/3–19: Sema3a, Adarb2; 3–17/3–13: Pax5, Pou6f2), suggesting relatedness, but were distinguished by ion channels (Kcnc3, Kcna1), transmembrane proteins (Tmem132c), and transcription factors (Pou3f1) that could imply a state or subtype distinction (Figure 6I).”

• cites Figure 6INot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Subcluster 3-2 probably is a third GABAergic type, marked by Sox14 and Cplx3.

“Subcluster 3–2 was Gad2+/Pvalb− and expressed the developmental marker Zfpm2 (Lahti et al., 2016), likely representing a third GABAergic type marked by Sox14 and Cplx3 expression (Figure 6I).”

• cites Figure 6INot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

These correlations match prominent subunit combinations associated at a protein level, suggesting nicotinic acetylcholine receptor composition is achieved in part by cell-type-specific patterns of RNA expression.

“These correlations match prominent subunit combinations associated at a protein level, suggesting nAChR composition is achieved in part by cell-type-specific patterns of RNA expression.”

The method helped identify a transcriptional program thought to support axon and presynaptic function, varying within and across neuron types.

“This approach allowed us to identify a transcriptional program we believe is enacted to maintain axon and presynaptic function, to different degrees both within and across neuron types.”

An ICA-based computational method was created to separate technical effects from endogenous biological signals.

“We developed an ICA-based computational method that we used to disentangle technical effects from endogenous biological signals.”

Functional studies using markers believed to separate iSPNs from dSPNs have grouped eSPNs with canonical SPNs.

“We conclude that 1) eSPNs represent a third axis of SPN diversity, distinct from the dSPN/iSPN and patch/matrix distinctions; 2) eSPNs harbor rare, additional molecular diversity; and 3) by using markers thought to exclusively distinguish iSPNs from dSPNs, functional studies have lumped eSPNs in with canonical SPNs.”

Patch compartments do not confer identical transcriptional features on dSPNs and iSPNs.

“Our data suggest complex SPN specializations across pathways and compartments: the transcriptional features endowed by patch compartments are not identical for dSPNs and iSPNs, and some of these differences appear to eliminate pathway expression differences found in the matrix dSPNs and iSPNs (Figure S7E).”

• cites Figure S7ENot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

eSPNs are not interneurons.

“Our data account for all known striatal interneuron types (3.9% of total neurons)(Tepper and Bolam, 2004), suggesting by exclusion that eSPNs are not interneurons.”

eSPNs form a third dimension of SPN diversity, separate from dSPN/iSPN and patch/matrix divisions.

“We conclude that 1) eSPNs represent a third axis of SPN diversity, distinct from the dSPN/iSPN and patch/matrix distinctions; 2) eSPNs harbor rare, additional molecular diversity; and 3) by using markers thought to exclusively distinguish iSPNs from dSPNs, functional studies have lumped eSPNs in with canonical SPNs.”

Some differences seem to remove pathway expression distinctions seen in matrix dSPNs and iSPNs.

“Our data suggest complex SPN specializations across pathways and compartments: the transcriptional features endowed by patch compartments are not identical for dSPNs and iSPNs, and some of these differences appear to eliminate pathway expression differences found in the matrix dSPNs and iSPNs (Figure S7E).”

• cites Figure S7ENot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Dopamine neurons are more transcriptionally regionalized than cholinergic neurons.

“Thus, DA neurons are more transcriptionally regionalized than cholinergic neurons.”

eSPNs are striatal principal neurons.

“We conclude that eSPNs are striatal principal neurons.”

The computational method was essential to recognize and interpret these varied influences on RNA expression.

“Our computational approach was critical for recognizing and understanding these diverse effects on RNA expression, all of which can simultaneously affect a cell’s RNA expression profile.”

eSPNs harbor rare, additional molecular diversity.

“We conclude that 1) eSPNs represent a third axis of SPN diversity, distinct from the dSPN/iSPN and patch/matrix distinctions; 2) eSPNs harbor rare, additional molecular diversity; and 3) by using markers thought to exclusively distinguish iSPNs from dSPNs, functional studies have lumped eSPNs in with canonical SPNs.”

On average, roughly 66% of DAPI+ cells in frontal cortex and striatum were ZsGreen+.

“In both Frontal Cortex and Striatum, ~66% of DAPI+ cells were ZsGreen+ on average.”