Phenotypic variation of transcriptomic cell types in mouse motor cortex

May combine two claims.

The resulting data set covered 77 out of the 90 neuronal t-types, with 73 t-types having at least one morphologically reconstructed neuron.

“The resulting data set covered 77 out of the 90 neuronal t-types (Fig. 1a), with 73 t-types having at least one morphologically reconstructed neuron.”

May combine two claims.

Coverage was good for interneurons derived from the caudal and medial ganglionic eminences (CGE and MGE) and for excitatory neurons.

“The coverage was good for interneurons derived from the caudal and medial ganglionic eminences (CGE and MGE) and for excitatory neurons.”

May combine two claims.

Three previously described morphological types of L5 Pvalb cells, as well as two morphological types of L5 Martinotti cells, corresponded to different t-types.

“We also showed that three previously described morphological types of L5 Pvalb cells5, as well as two morphological types of L5 Martinotti cells29,30, corresponded to different t-types.”

• cites [5] Jiang, X. et al. Principles of connectivity among morphologically defined cell types in adult neocortex. Science 350, aac9462 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [29] Muñoz, W., Tremblay, R., Levenstein, D. & Rudy, B. Layer-specific modulation of neocortical dendritic inhibition during active wakefulness. Science 355, 954–959 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [30] Nigro, M. J., Hashikawa-Yamasaki, Y. & Rudy, B. Diversity and connectivity of layer 5 somatostatin-expressing interneurons in the mouse barrel cortex. J. Neurosci. 38, 1622–1633 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Lamp5 Egln3_1 neurons had rather Vip- and Sst-like firing instead of the typical Lamp5 electrophysiology, and Vip Mybpc1 neurons often had Sst-like firing.

“Similarly, Lamp5 Egln3_1 neurons had rather Vip- and Sst-like firing instead of the typical Lamp5 electrophysiology, and Vip Mybpc1 neurons often had Sst-like firing.”

May combine two claims.

The selected sparse reduced-rank regression model used 25 genes with a 5-dimensional latent space and achieved a cross-validated R2 of 0.38.

“The selected model used 25 genes with a 5-dimensional latent space and achieved a cross-validated R2 of 0.38.”

• cites [32] Kobak, D. et al. Sparse reduced-rank regression for exploratory visualization of paired multivariate data sets. R. Stat. Soc. 70, 980–1000 (2021). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The observed phenotypes included most of the morphological and electrophysiological types of cortical neurons that have been described previously in mice and rats, allowing linking of transcriptomic and morpho-electric descriptions.

“The observed phenotypes included most of the morphological and electrophysiological types of cortical neurons that have been described previously in mice and rats5,6,24, allowing us to link transcriptomic and morpho-electric descriptions (Extended Data Fig. 3, Supplementary File 1).”

• cites [5] Jiang, X. et al. Principles of connectivity among morphologically defined cell types in adult neocortex. Science 350, aac9462 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [6] Markram, H. et al. Reconstruction and simulation of neocortical microcircuitry. Cell 163, 456–492 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [24] Gouwens, N. W. et al. Classification of electrophysiological and morphological neuron types in the mouse visual cortex. Nat. Neurosci. 22, 1182–1195 (2019). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Electrophysiological properties of the t-types within the Vip subclass varied continuously across the transcriptomic landscape; the membrane time constant had its largest values close to the Sncg subclass and gradually decreased towards Vip Gpc3.

“For example, electrophysiological properties of the t-types within the Vip subclass varied continuously across the transcriptomic landscape; the membrane time constant, for instance, had its largest values close to the Sncg subclass and gradually decreased towards Vip Gpc3 (Fig. 4a).”

May combine two claims.

Different transcriptomic families had largely distinct morpho-electric phenotypes, in agreement with them being well separated in the transcriptomic space.

“In summary, different transcriptomic families had largely distinct morpho-electric phenotypes, in agreement with them being well separated in the transcriptomic space4.”

• cites [4] Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Different Pvalb t-types had different preferred morphologies but showed substantial overlap.

“While we found that different t-types had different preferred morphologies (Extended Data Table 1), they showed substantial overlap, in agreement with the L5 Pvalb t-types themselves not having clear boundaries4 (Fig. 1d).”

• cites [4] Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

L4/5 and L5 IT t-types that were transcriptomically close to L2/3 IT t-types were located at the top of L5 close to the border between L2/3 and L5, whereas L5 IT t-types that were transcriptomically close to L6 IT t-types were located at the bottom of L5 close to the border with L6.

“For example, L4/5 and L5 IT t-types that were transcriptomically close to the L2/3 IT t-types were located at the top of L5 close to the border between L2/3 and L5, whereas L5 IT t-types that were transcriptomically close to L6 IT t-types were located at the bottom of L5 close to the border with L6.”

May combine two claims.

Between-family separability was strongest when electrophysiological and morphological features were combined into a joint representation, showing that these sets of properties are not redundant.

“The between-family separability was strongest when we had combined electrophysiological and morphological features into a joint representation (Fig. 3c, d), showing that these sets of properties are not redundant.”

May combine two claims.

These five major groups had distinct electrophysiological properties; for example, Pvalb neurons were characterized by high firing rates while Sst neurons had high values of the hyperpolarization sag and rebound.

“These groups had distinct electrophysiological properties: for example, as expected, Pvalb neurons were characterized by high firing rates while Sst neurons had high values of the hyperpolarization sag and rebound (Fig. 2, right).”

May combine two claims.

IT t-types did not form distinct groups for each cortical layer; instead, soma depth and RNA expression varied continuously along a one-dimensional manifold.

“However, we found that IT t-types did not form distinct groups for each cortical layer; instead, the soma depth and RNA expression varied continuously along a one-dimensional manifold (Fig. 4d), in agreement with parallel findings based on a spatial transcriptomics approach34.”

• cites [34] Zhang, M. et al. Spatially resolved cell atlas of the mouse primary motor cortex by MERFISH. Nature https://doi.org/10.1038/s41586-021-03705-x (2021). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

A t-type, L4/5 IT_1, was located on the boundary between L2/3 and L5 and probably corresponds to the quasi-L4 neurons described previously in motor cortex.

“We were also able to identify a t-type, L4/5 IT_1, that was located on the boundary between L2/3 and L5 and probably corresponds to the quasi-L4 neurons described previously in motor cortex31.”

• cites [31] Yamawaki, N., Borges, K., Suter, B. A., Harris, K. D. & Shepherd, G. M. A genuine layer 4 in motor cortex with prototypical synaptic circuit connectivity. eLife 3, e05422 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The correlation between transcriptomic and electrophysiological distances was also observed within multiple individual families and for many individual electrophysiological features.

“The correlation was also observed within multiple individual families and for many individual electrophysiological features (Extended Data Fig. 7).”

May combine two claims.

Our data sets are overall in good agreement and together offer an unprecedented view of cell type variability in the neocortex.

“Our data sets are overall in good agreement (Extended Data Fig. 9) and together offer an unprecedented view of cell type variability in the neocortex.”

May combine two claims.

The shape of the axonal arbor of Pvalb cells showed continuous changes across the transcriptomic landscape: small shrub-like basket cells, horizontally elongated basket cells, and vertically elongated classical basket cells were located in different corners of the t-SNE embedding, with intermediate morphologies in between.

“The shape of the axonal arbor showed continuous changes across the transcriptomic landscape (Fig. 4e): small shrub-like basket cells, horizontally elongated basket cells, and vertically elongated classical basket cells were located in different corners of the t-SNE embedding, with intermediate morphologies in between.”

May combine two claims.

Most t-types could be unambiguously placed into the correct transcriptomic family using electrophysiological features, but some t-types were in between two families.

“Most t-types could be unambiguously placed into the correct family (Fig. 5a), but some t-types were in between two families.”

May combine two claims.

Vip Mybpc1_2 was marked as having high electrophysiological variability and had high variance in input resistance, membrane time constant, and rebound.

“By contrast, Vip Mybpc1_2 was marked as having high electrophysiological variability and indeed had high variance in input resistance, membrane time constant, and rebound (Extended Data Fig. 4).”

May combine two claims.

Sst Pvalb Calb2 cells had high variability in maximum firing rate, but high-firing cells were mostly grouped in one part of the transcriptomic landscape.

“Similarly, Sst Pvalb Calb2 cells had high variability in terms of the maximum firing rate, but high-firing cells were mostly grouped in one part of the transcriptomic landscape (Fig. 5d).”

May combine two claims.

In the Sst subclass, morpho-electric properties varied continuously across the transcriptomic landscape, with neighbouring t-types consistently showing similar morphologies and similar rebound values.

“We observed the same in the Sst subclass, which is known to be transcriptomically4 and morpho-electrically29,30,33 diverse in L5. Here we also found that morpho-electric properties varied continuously across the transcriptomic landscape, with neighbouring t-types consistently showing similar morphologies and similar rebound values (Fig. 4b).”

• cites [4] Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [29] Muñoz, W., Tremblay, R., Levenstein, D. & Rudy, B. Layer-specific modulation of neocortical dendritic inhibition during active wakefulness. Science 355, 954–959 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [30] Nigro, M. J., Hashikawa-Yamasaki, Y. & Rudy, B. Diversity and connectivity of layer 5 somatostatin-expressing interneurons in the mouse barrel cortex. J. Neurosci. 38, 1622–1633 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [33] Naka, A. et al. Complementary networks of cortical somatostatin interneurons enforce layer specific control. eLife 8, e43696 (2019). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Within each class, cells were strongly segregated by soma depth, with excitatory cells forming mostly a one-dimensional manifold.

“Within each class, cells were strongly segregated by the soma depth, with excitatory cells forming mostly a one-dimensional manifold.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The resulting cDNA yield was not correlated with the hold time (Spearman correlation −0.01).

“The resulting cDNA yield was not correlated with the hold time (Spearman correlation −0.01).”

May combine two claims.

In Gnb4-Cre mice, no labelled cells were found in MOp; all labelled cells were far outside MOp and close to the claustrum.

“We were unable to find any labelled cells in MOp in the Gnb4-Cre mice: all labelled cells were far outside of MOp and close to the claustrum46.”

• cites [46] Peng, H. et al. Morphological diversity of single neurons in molecularly defined cell types. Nature https://doi.org/10.1038/s41586-021-03941-1 (2021). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The addition of the RNase inhibitor resulted in an increase in osmolarity to the desired value of 315-320 mOSM without a further dilution.

“The addition of the inhibitor resulted in an increase in osmolarity to the desired value of 315–320 mOSM without a further dilution17.”

• cites [17] Cadwell, C. R. et al. Multimodal profiling of single-cell morphology, electrophysiology, and gene expression using Patch-seq. Nat. Protocols 12, 2531 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

A substantial portion of the RNA extracted from the neurons was nascent and contained intronic sequences.

“A substantial portion of the RNA extracted from the neurons was nascent and contained intronic sequences.”

Probably not a substantial claim: may not pass S3 (load-bearing).

We detected 42,466 genes, including pseudogenes and annotated non-coding segments, in at least one cell.

“We detected 42,466 genes, including pseudogenes and annotated non-coding segments, in at least one cell.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

One hundred and ten cells mapped to the Sst subclass, 43 to IT, 12 to ET, 10 to Pvalb, and 2 to NP.

“One hundred and ten cells mapped to the Sst subclass, 43 to IT, 12 to ET, 10 to Pvalb, and 2 to NP.”

Probably not a substantial claim: may not pass S3 (load-bearing).

175 cells were assigned to L5 in the post hoc analysis.

“175 cells were assigned to L5 in the post hoc analysis (see below).”

May combine two claims.

Neurons form a hierarchy that consists of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families.

“Instead, neurons form a hierarchy that consists of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families.”

• Also stated in: § Discussion · plain

  “In this metaphor, neurons follow a hierarchy consisting of distinct, non-overlapping branches at the level of families (large leaves), but with a spectrum of cells forming continuous and correlated transcriptomic and morpho-electrical landscapes within each leaf.”

May combine two claims.

After excluding cells with poor mapping, 1,227 neurons remained for further analysis (817 inhibitory, 410 excitatory; 369 and 269 with morphological reconstructions, respectively).

“Cells that showed poor mapping (owing to a low read count or excessive RNA contamination) were excluded (Extended Data Fig. 2), leaving 1,227 neurons for further analysis (817 inhibitory, 410 excitatory; 369 and 269 with morphological reconstructions, respectively).”

Probably not a substantial claim: may not pass S2 (checkable).

To understand the roles of t-types in cortical computations, it is necessary to know their anatomy, connectivity, and electrophysiology.

“However, to understand the roles of t-types in cortical computations, it is necessary to know their anatomy, connectivity, and electrophysiology7.”

• cites [7] Zeng, H. & Sanes, J. R. Neuronal cell-type classification: challenges, opportunities and the path forward. Nat. Rev. Neurosci. 18, 530–546 (2017). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

The study performed whole-cell recordings from more than 2,000 cells, of which 1,329 passed initial quality control.

“In total, we performed whole-cell recordings from more than 2,000 cells, of which 1,329 cells (from 266 mice) passed initial quality control.”

May combine two claims.

The mRNA of the 1,329 cells was sequenced, yielding on average 1.3 million exonic and 0.7 million intronic reads and 9,100 ± 3,500 detected genes per cell.

“The mRNA of these 1,329 cells was sequenced, yielding on average 1.3 million exonic and 0.7 million intronic reads (medians; mean ± s.d. on a log10 scale: 6.0 ± 0.6 and 5.6 ± 0.8, respectively) and 9,100 ± 3,500 (mean ± s.d.) detected genes per cell (Extended Data Fig. 2).”

Classified as probably not a substantial claim.

Linking transcriptomically defined cell types to their phenotypes has remained a major challenge.

“High-throughput single-cell sequencing techniques have identified dozens of types of neuron on the basis of their transcriptional profiles3,4,14,15, but linking transcriptomically defined cell types (t-types) to their phenotypes has remained a major challenge16.”

• cites [16] Huang, Z. J. & Paul, A. The diversity of GABAergic neurons and neural communication elements. Nat. Rev. Neurosci. 20, 563–572 (2019). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S1 (asserted).

The current view is that a neuronal cell type is characterized by a common genetic profile that gives rise to distinct physiological and anatomical properties, including patterns of connectivity.

“The current view is that a neuronal cell type is characterized by a common genetic profile that gives rise to distinct physiological and anatomical properties, including patterns of connectivity7,9.”

• cites [7] Zeng, H. & Sanes, J. R. Neuronal cell-type classification: challenges, opportunities and the path forward. Nat. Rev. Neurosci. 18, 530–546 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [9] Masland, R. H. Neuronal cell types. Curr. Biol. 14, R497–R500 (2004). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Of the 1,329 neurons, 646 had sufficient staining for their morphologies to be reconstructed.

“Of these neurons, 646 had sufficient staining for their morphologies to be reconstructed.”

Classified as probably not a substantial claim. May combine two claims.

Cortical neurons exhibit extreme diversity in gene expression as well as in morphological and electrophysiological properties.

“Cortical neurons exhibit extreme diversity in gene expression as well as in morphological and electrophysiological properties1,2.”

• cites [1] Harris, K. D. & Shepherd, G. M. The neocortical circuit: themes and variations. Nat. Neurosci. 18, 170–181 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [2] Tremblay, R., Lee, S. & Rudy, B. GABAergic interneurons in the neocortex: from cellular properties to circuits. Neuron 91, 260–292 (2016). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

High-throughput single-cell sequencing techniques have identified dozens of types of neuron on the basis of their transcriptional profiles.

“High-throughput single-cell sequencing techniques have identified dozens of types of neuron on the basis of their transcriptional profiles3,4,14,15, but linking transcriptomically defined cell types (t-types) to their phenotypes has remained a major challenge16.”

• cites [3] Tasic, B. et al. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [4] Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [14] Zeisel, A. et al. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [15] Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014.e22 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Although all deep NGCs belong to the Lamp5 subclass, some are derived from the CGE and some from the MGE, as in the hippocampus.

“Our results suggest that although all deep NGCs belong to the Lamp5 subclass, some are derived from the CGE and some from the MGE, as in the hippocampus25–27.”

• cites [25] Tricoire, L. et al. Common origins of hippocampal Ivy and nitric oxide synthase expressing neurogliaform cells. J. Neurosci. 30, 2165–2176 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [26] Pelkey, K. A. et al. Hippocampal GABAergic inhibitory interneurons. Physiol. Rev. 97, 1619–1747 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [27] Harris, K. D. et al. Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics. PLoS Biol. 16, e2006387 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Recent transcriptomic and anatomical studies have argued that neurons in hippocampus, striatum, and cerebellum can be better described as forming partially continuous manifolds.

“Consistent with our interpretation, recent transcriptomic and anatomical studies have argued that neurons in hippocampus, striatum, and cerebellum can be better described as forming partially continuous manifolds27,37–39.”

• cites [27] Harris, K. D. et al. Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics. PLoS Biol. 16, e2006387 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [37] Muñoz-Manchado, A. B. et al. Diversity of interneurons in the dorsal striatum revealed by single-cell RNA sequencing and PatchSeq. Cell Rep. 24, 2179–2190.e7 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [38] Stanley, G. et al. Continuous and discrete neuron types of the adult murine striatum. Neuron 105, 688–699.e8 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [39] Wang, W. X. & Lefebvre, J. L. Morphological pseudotime ordering and fate mapping reveals diversification of cerebellar inhibitory interneurons. Preprint at https://doi.org/10.1101/2020.02.29.971366 (2020). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Although some within-type morpho-electric variability may be attributed to technical challenges of Patch-seq or to factors such as the exact spatial location of the cell within motor cortex, there are clear cases in the data that suggest within-type morpho-electric variability is related to within-type transcriptomic variability.

“Although we cannot fully exclude the possibility that some of this variability can be attributed to technical challenges of Patch-seq or to factors such as the exact spatial location of the cell within motor cortex36, there are clear cases in our data that suggest that within-type morpho-electric variability is related to within-type transcriptomic variability.”

• cites [36] Cembrowski, M. S. et al. Spatial gene-expression gradients underlie prominent heterogeneity of ca1 pyramidal neurons. Neuron 89, 351–368 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

While overall transcriptomic family was highly predictive of the cell phenotype, some t-types exhibited properties similar to those of another transcriptomic family.

“Thus, while overall transcriptomic family was highly predictive of the cell phenotype, some t-types exhibited properties similar to those of another transcriptomic family.”

Classified as probably not a substantial claim.

Previous studies using transgenic lines had shown that morpho-electric properties within these families can be highly variable.

“Previous studies using transgenic lines had shown that morpho-electric properties within these families can be highly variable5,24.”

• cites [5] Jiang, X. et al. Principles of connectivity among morphologically defined cell types in adult neocortex. Science 350, aac9462 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [24] Gouwens, N. W. et al. Classification of electrophysiological and morphological neuron types in the mouse visual cortex. Nat. Neurosci. 22, 1182–1195 (2019). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A parallel Patch-seq study of the inhibitory neurons in the mouse visual cortex focused on isolating multimodal neural types but also often observed continuous variation.

“A parallel Patch-seq study of the inhibitory neurons in the mouse visual cortex45 focused on isolating multimodal neural types (‘met-types’) but also often observed continuous variation.”

• cites [45] Gouwens, N. W. et al. Integrated morphoelectric and transcriptomic classification of cortical GABAergic cells. Cell 183, 1–19 (2020). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

No statistical methods were used to predetermine sample size.

“No statistical methods were used to predetermine sample size.”

May combine two claims.

Within-type distributions of soma depths agreed well with previous data and with the layer-specific nomenclature of excitatory t-types, confirming the validity of t-type assignment.

“Within-type distributions of soma depths (Fig. 1b) agreed well with previous data4 and with the layer-specific nomenclature of excitatory t-types, confirming the validity of our t-type assignment.”

• cites [4] Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Cortical studies have identified many intermediate cells with uncertain t-type assignments.

“Similarly, cortical studies have identified many intermediate cells with uncertain t-type assignments3,4.”

• cites [3] Tasic, B. et al. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [4] Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The RNA extraction process may have interfered with biocytin diffusion and MOp is quite thick, making it difficult to recover complete morphologies of some groups of neurons, such as deep L5 Martinotti cells.

“Second, as the RNA extraction process may have interfered with biocytin diffusion17 and as MOp is quite thick, it was difficult to recover complete morphologies of some groups of neurons, such as deep L5 Martinotti cells with thin long axons that reach all the way to L1.”

• cites [17] Cadwell, C. R. et al. Multimodal profiling of single-cell morphology, electrophysiology, and gene expression using Patch-seq. Nat. Protocols 12, 2531 (2017). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment, unless otherwise stated.

“The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment, unless otherwise stated.”

May combine two claims.

On a fine within-family scale, both transcriptomic and morpho-electric landscapes are continuous rather than discrete.

“This is in agreement with the idea that on a fine within-family scale, both transcriptomic and morpho-electric landscapes are continuous rather than discrete.”

Classified as probably not a substantial claim. May combine two claims.

We obtained high-quality electrophysiological recordings and extracted electrophysiological features of 184 cells.

“We obtained high-quality electrophysiological recordings and extracted electrophysiological features of 184 cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The investigators were blinded to the cell type of each sample during library construction and sequencing.

“The investigators were blinded to the cell type of each sample during library construction and sequencing.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Two inhibitory neurons were left out because some of the morphometric statistics could not be extracted owing to insufficient dendritic recovery; this left 367 inhibitory neurons (with 50 morphometric features) and 269 excitatory neurons (with 35 morphometric features).

“Two inhibitory neurons were left out because some of the morphometric statistics could not be extracted owing to insufficient dendritic recovery; this left 367 inhibitory neurons (with 50 morphometric features) and 269 excitatory neurons (with 35 morphometric features).”

Probably not a substantial claim: may not pass S3 (load-bearing).

We obtained soma depth information for 1,284 neurons out of 1,329.

“We obtained soma depth information for 1,284 neurons out of 1,329 (45 neurons were mistakenly recorded using a solution with insufficient biocytin concentration, and we could measure soma depths for only 2 of those; for 2 other neurons the measurements could not be carried out because the slices were lost).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Samples containing less than around 1 ng total cDNA (in the 15 μl final volume) or with an average size less than 1,500 bp were typically not sequenced (with some occasional exceptions).

“Samples containing less than around 1 ng total cDNA (in the 15 μl final volume) or with an average size less than 1,500 bp were typically not sequenced (with some occasional exceptions).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

We did not use any axonal features for the excitatory cells because only a small part of the axon could typically be reconstructed.

“We did not use any axonal features for the excitatory cells because only a small part of the axon could typically be reconstructed.”

May combine two claims.

We sequenced in total 185 neurons, obtained from 8 adult mice (7 Sst-Cre/Ai9 and 1 Pvalb-Cre/Ai9), of which 177 neurons passed the transcriptomic quality control and got a t-type assignment.

“We sequenced in total 185 neurons, obtained from 8 adult mice (7 Sst-Cre/Ai9 and 1 Pvalb-Cre/Ai9), of which 177 neurons passed the transcriptomic quality control and got a t-type assignment (see below).”

Probably not a substantial claim: may not pass S3 (load-bearing).

We took special care to exclude reconstructions of all neurons that showed any signs of damage, lack of contrast, or poor overall staining.

“We took special care to exclude reconstructions of all neurons that showed any signs of damage, lack of contrast, or poor overall staining.”

Probably not a substantial claim: may not pass S3 (load-bearing).

One cell was probably located outside MOp, based on the slice anatomy, and was excluded.

“In addition, one cell was probably located outside MOp, based on the slice anatomy, and was excluded as well.”

Classified as probably not a substantial claim.

For all statistical analysis, we used 17 of the 29 extracted electrophysiological features, excluding features that were zero for many cells, highly correlated with other features, highly skewed, or undefined for some cells.

“For all statistical analysis we used 17 features out of the extracted 29, excluding features that were equal to zero for many cells (afterdepolarization, burstiness, rebound number of APs, sag area, sag time, wildness), two Fano factor features that were highly correlated with the corresponding coefficient of variation features (AP Fano factor, ISI Fano factor) and another measure of latency that”

Probably not a substantial claim: may not pass S3 (load-bearing).

This is effectively a leave-one-out cross-validation procedure.

“This is effectively a leave-one-out cross-validation procedure.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

To prevent overlap between these two populations, we added a small constant value of 0.25 to the excitatory block-diagonal block, leading to the strong excitatory-inhibitory separation in Fig. 3b.

“To prevent overlap between these two populations, we added a small constant value of 0.25 to the excitatory block-diagonal block, leading to the strong excitatory–inhibitory separation in Fig. 3b.”

Probably not a substantial claim: may not pass S3 (load-bearing).

We used the entropies of a Leiden clustering35 as an alternative way to approach the same question.

“We used the entropies of a Leiden clustering35 as an alternative way to approach the same question.”

• cites [35] Traag, V. A., Waltman, L. & van Eck, N. J. From Louvain to Leiden: guaranteeing well-connected communities. Sci. Rep. 9, 5233 (2019). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

We did not use any features measuring morphological properties in the slice depth (y) direction because of possible slice shrinkage artefacts.

“We did not use any features measuring morphological properties in the slice depth (y) direction because of possible slice shrinkage artefacts.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Forty-five sequenced cells were mistakenly recorded using a solution with a much smaller concentration of biocytin, and their morphologies could not be recovered.

“Forty-five sequenced cells were mistakenly recorded using a solution with a much smaller concentration of biocytin, and their morphologies could not be recovered.”

Classified as probably not a substantial claim.

We used bootstrapping over genes to assess the confidence of each t-type assignment.

“We used bootstrapping over genes to assess the confidence of each t-type assignment.”

Classified as probably not a substantial claim.

To provide a sensible baseline for the range of possible normalized total variances in a population of morpho-electrically homogeneous types, we used a clustering analysis.

“To provide a sensible baseline for the range of possible normalized total variances in a population of morpho-electrically homogeneous types, we used a clustering analysis.”

• AssumptionThe authors state this as an assumption: The k-means clusters represent morpho-electrically homogeneous types.

Probably not a substantial claim: may not pass S3 (load-bearing).

The remaining 1,232 cells passed quality control and entered the second round.

“The remaining 1,232 cells passed quality control and entered the second round.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The distributions of the remaining features were visually checked for outliers and for meaningful variation between transcriptomic types, leading to a further exclusion of three features for the inhibitory cells.

“The distributions of the remaining features were visually checked for outliers and for meaningful variation between transcriptomic types, leading to a further exclusion of three features for the inhibitory cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Special care was taken to ensure that the seal between the pipette and the cell membrane remained intact to reduce contamination from the extracellular environment.

“Special care was taken to ensure that the seal between the pipette and the cell membrane remained intact to reduce contamination from the extracellular environment.”

Probably not a substantial claim: may not pass S3 (load-bearing).

We did not use the Sncg and NP families owing to insufficient coverage in our data set (Fig. 1).

“We did not use the Sncg and NP families owing to insufficient coverage in our data set (Fig. 1).”

Classified as probably not a substantial claim. May combine two claims.

We used the following quality control criteria: (1) seal resistance value >1 GΩ before achieving whole-cell configuration; (2) access resistance <30 MΩ.

“We used the following quality control criteria: (1) seal resistance value >1 GΩ before achieving whole-cell configuration; (2) access resistance <30 MΩ.”

Classified as probably not a substantial claim.

In each case, we excluded features with coefficient of variation below 0.25 (among the features with only positive values).

“In each case, we excluded features with coefficient of variation below 0.25 (among the features with only positive values).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The final number of cells with t-type assignment was 1,227.

“The final number of cells with t-type assignment was 1,227.”

Classified as probably not a substantial claim. May combine two claims.

We reduced each set to five PCs, discarded PC1 (it was strongly correlated with the normalized soma depth and made the resulting embedding strongly influenced by the soma depth), and scaled the PCs by the standard deviation of the respective PC2.

“We reduced each set to five PCs, discarded PC1 (it was strongly correlated with the normalized soma depth and made the resulting embedding strongly influenced by the soma depth), and scaled the PCs by the standard deviation of the respective PC2.”

May combine two claims.

The three large transcriptomic groups (CGE-derived interneurons, MGE-derived interneurons, and excitatory neurons) are perfectly separated from each other with no transcriptomically intermediate cells.

“the three large transcriptomic groups (CGE-derived interneurons, MGE-derived interneurons, and excitatory neurons) that are perfectly separated from each other with no transcriptomically intermediate cells4.”

• cites [4] Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

This shrinkage was not compensated for in our analysis.

“This shrinkage was not compensated for in our analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Tissue shrinkage due to the fixation and staining procedures was about 10-20%.

“Consistently with previous studies, tissue shrinkage due to the fixation and staining procedures was about 10–20%5,28,49.”

• cites [5] Jiang, X. et al. Principles of connectivity among morphologically defined cell types in adult neocortex. Science 350, aac9462 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [28] Scala, F. et al. Layer 4 of mouse neocortex differs in cell types and circuit organization between sensory areas. Nat. Commun. 10, 4174 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [49] Markram, H., Lübke, J., Frotscher, M., Roth, A. & Sakmann, B. Physiology and anatomy of synaptic connections between thick tufted pyramidal neurones in the developing rat neocortex. J. Physiol. (Lond.) 500, 409–440 (1997). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The measured electrophysiological properties of the 45 cells mistakenly recorded with a smaller biocytin concentration were not systematically different from those of the other sequenced cells.

“We made sure that the measured electrophysiological properties of these cells were not systematically different from those of the the other sequenced cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Four features were log-transformed to make their distribution more Gaussian-like: AP coefficient of variation, ISI coefficient of variation, ISI adaptation index, and latency.

“Four features were log-transformed to make their distribution more Gaussian-like: AP coefficient of variation, ISI coefficient of variation, ISI adaptation index, and latency.”

Classified as probably not a substantial claim.

Cells meeting any of the following exclusion criteria were declared low quality and did not get a t-type assignment.

“Cells meeting any of the following exclusion criteria were declared low quality and did not get a t-type assignment (Extended Data Fig. 2e): cells with the highest correlation below 0.4 (78 cells); cells that would be assigned to non-neural t-types, presumably owing to RNA contamination51 (14 cells; see also Extended Data Fig. 2j–n); cells with the highest correlation less than 0.02 above the max”

Classified as probably not a substantial claim.

These four cells were excluded from all analyses and visualizations.

“These four cells were excluded from all analyses and visualizations (as if they did not pass the transcriptomic quality control).”

May combine two claims.

The confidences obtained with seven reference data sets agreed well with each other and were averaged to obtain the consensus confidence.

“The confidences obtained with seven reference data sets agreed well with each other (Extended Data Fig. 2i) and were averaged to obtain the consensus confidence.”

Classified as probably not a substantial claim.

Subsampling was done to eliminate a possible bias due to the t-type abundance.

“Subsampling was done to eliminate a possible bias due to the t-type abundance.”