Classification of electrophysiological and morphological neuron types in the mouse visual cortex

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

The majority of cells (1,599) came from primary visual cortex (VISp), with the rest from nearby higher visual areas.

“Most cells (n=1,599) were from primary visual cortex (VISp); the remainder were in nearby higher visual areas.”

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

The authors released the experimental data and analysis tools publicly through the Allen Cell Types Database and the Allen Software Development Kit.

“The experimental data and analysis tools are made available as a public resource as part of the Allen Cell Types Database and the Allen Software Development Kit.”

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

The authors created a public online resource, the Allen Cell Types Database, for displaying the data sets.

“We built a publicly-accessible online database, the Allen Cell Types Database, to display these data sets.”

May combine two claims.

The authors produced a taxonomy for mouse visual cortex containing 17 electrophysiological types, 38 morphological types, and 46 joint types.

“We established a taxonomy of morphologically- and electrophysiologically-defined cell types for this region of cortex with 17 e-types, 38 m-types, and 46 me-types”

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

For each data subset, sparse principal components analysis usually yielded 1 to 8 components above an adjusted explained-variance cutoff.

“We applied a sparse principal components analysis20,21 (sPCA) procedure to each collected subset of the data, which typically identified 1-8 components that exceeded an adjusted explained variance threshold per subset”

• cites [20] Zou, H., Hastie, T. & Tibshirani, R. Sparse principal component analysis. J. Comput. Graph. Stat. 15, 265–286 (2006). sourceNot checked yet

  Not checked in the free run.

• cites [21] Baden, T. et al. The functional diversity of retinal ganglion cells in the mouse. Nature 529, 345–350 (2016). sourceNot checked yet

  Not checked in the free run.

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

At the time of writing, the website held electrophysiological data for 1,861 of 1,938 cells and 431 of 461 reconstructions.

“As of this writing, 1,861 out of 1,938 cells had electrophysiological data on the website, and 431 out of 461 reconstructions were on the website.”

May combine two claims.

Applying their approach to juvenile rat somatosensory cortex morphologies yielded spiny clusters that either split BBP qualitative m-types (the single L2/3 PC type became six) or merged them (L5 thick tufted 1 and 2).

“we identified spiny clusters that subdivided the BBP qualitative m-types (e.g., the single L2/3 PC m-type was split into 6 m-types), and those that merged m-types (e.g., L5 thick tufted 1 and 2”

May combine two claims.

When excitatory and inhibitory cells were clustered together, the result was a similar but not identical count of 14 clusters, including one mixed cluster.

“clustering them together resulted in a similar, though not identical, number of clusters (14 total), but it also produced one cluster that contained both excitatory and inhibitory neurons”

May combine two claims.

Excitatory e-types resembled one another, having broad action potentials and comparable membrane capacitance and input resistance distributions.

“The excitatory e-types were relatively similar to each other, with wide APs and similar distributions of membrane capacitance and input resistance”

May combine two claims.

Inhibitory me-types aligned with the various inhibitory e-types, with some mixing among fast-spiking e-types Inh_8 through Inh_13.

“Inhibitory me-types were consistent with the different inhibitory e-types, although there was some mixing between, for example, the different fast-spiking e-types (Inh_8 through Inh_13”

May combine two claims.

Clustering produced 17 e-types overall: four for the 928 excitatory neurons and thirteen for the 1,010 inhibitory neurons.

“This procedure yielded 17 total clusters, or e-types: four e-types for the 928 excitatory neurons and thirteen for the 1,010 inhibitory neurons.”

• AssumptionThe authors state this as an assumption: Spiny neurons are excitatory.
• AssumptionThe authors state this as an assumption: Aspiny or sparsely spiny neurons are inhibitory.

May combine two claims.

For inhibitory cells, e-types were relatively consistent, m-types scored lower, and most me-types were highly consistent with transcriptomic subclasses; often one subclass scored high and the others low.

“On the inhibitory side, e-types showed relatively high consistency, m-types had lower scores, and most me-types were again highly consistent with transcriptomic subclasses—for many me-types, a single subclass had a high score while the rest were low”

May combine two claims.

Spiny m-types were mostly confined to one layer but not exclusively so, except for Spiny_1, 3, 10, 14, and 19.

“Spiny m-types were predominantly found in a single layer, but were not exclusive to that layer, although see Spiny_1, 3, 10, 14, and 19”

May combine two claims.

The 19 inhibitory interneuron m-types spanned all cortical layers L1-L6b; most occurred in multiple layers, except for a few L2/3-specific types.

“The 19 m-types for inhibitory interneurons were distributed across all cortical layers (L1-L6b), with most m-types found in multiple layers with the exception of several L2/3-specific m-types”

May combine two claims.

These me-types usually matched an excitatory m-type, although sometimes several m-types were collapsed into fewer me-types.

“Each of these me-types was usually consistent with an excitatory m-type (Fig. 5c), though in some cases multiple m-types were collapsed into fewer me-types.”

May combine two claims.

Almost all inhibitory e-types had lower membrane capacitance than excitatory e-types, with Inh_4 as a clear exception.

“membrane capacitance values were nearly all lower than those of the excitatory e-types (though Inh_4 was a notable exception)”

May combine two claims.

M-type distributions differed across transgenic lines, and many m-types occupied fairly consistent positions on the electrophysiology-based t-SNE projection, though not all did.

“The distribution of m-types varied across transgenic lines (Supplementary Fig. 22), and many, but not all, m-types were found in relatively consistent locations on the electrophysiology-based t-SNE projection”

May combine two claims.

Soma depth, dendrite and axon size, and their distributions were all important for separating inhibitory m-types.

“Relative soma depth, dendrite and axon size and distribution were all important for distinguishing among inhibitory m-types”

May combine two claims.

The analysis identified 38 m-types in total: 19 spiny and 19 aspiny.

“In total, 38 clusters or m-types were identified, 19 for spiny neurons (Fig. 3) and 19 for aspiny neurons (Fig. 4).”

May combine two claims.

Of the 461 reconstructed cells, 452 underwent electrophysiological classification; the nine excluded mainly failed to fire an action potential during the 25 pA/s ramp.

“All but 9 of those 461 cells underwent electrophysiological classification; the primary reason for not including these nine was failure to elicit an action potential with the 25 pA/s ramp stimulus.”

May combine two claims.

Within a layer, aspiny m-types from the same subclass (such as nest and small BCs) often merged, yet Martinotti, double-bouquet, and chandelier cells formed fairly distinct groups.

“Aspiny m-types within a subclass (e.g., nest and small BCs) were frequently merged within a layer, but we also identified fairly distinct groupings of Martinotti cells, double-bouquet cells, and chandelier cells”

May combine two claims.

Some e-types contained more neurons with bursting or delayed firing (Exc_4 and Inh_1, respectively), yet these patterns did not strictly define the types.

“here certain e-types had higher proportions of neurons that exhibited these patterns (e.g., Exc_4 for bursting, Inh_1 for delayed firing) but were not strictly demarcated by these patterns.”

May combine two claims.

For excitatory cells, most e-types had low consistency scores except Exc_4; m-types scored higher, and me-types higher still.

“Among excitatory cells, we found that most excitatory e-types had low consistency scores (except Exc_4), while more m-types had higher scores, and even more me-types had high scores”

May combine two claims.

The 1,938 cells included 928 spiny neurons and 1,010 aspiny or sparsely spiny neurons.

“Of these, 928 were spiny neurons, which we assume to be excitatory, and 1,010 were aspiny or sparsely spiny, which we assume to be inhibitory”

• AssumptionThe authors state this as an assumption: Spiny neurons are excitatory.
• AssumptionThe authors state this as an assumption: Aspiny or sparsely spiny neurons are inhibitory.

May combine two claims.

Pvalb-labeled cells sat next to Sst cells, while Vip cells occupied the opposite side of the Sst cells.

“Pvalb-labeled cells were adjacent to Sst cells, and Vip cells were found on the other side of Sst cells.”

May combine two claims.

The study identified 17 e-types, 38 m-types, and 46 me-types.

“This led to the identification of 17 e-, 38 m-, and 46 me-types”

May combine two claims.

The study identified 46 me-types by joint clustering, in contrast to 120 observed combinations by intersecting e- and m-types.

“For example, we identify 46 me-types by joint clustering, in contrast to the 120 observed combinations by just intersecting e- and m-types (Supplementary Fig. 29).”

May combine two claims.

Penk-IRES2-Cre-neo in L5-L6 and Oxtr-T2A-Cre in L6, which labeled overlapping sets of t-types, both identified the same me-type ME_Exc_6.

“Interestingly, Penk-IRES2-Cre-neo in L5-L6 and Oxtr-T2A-Cre in L6, which labeled overlapping sets of t-types, both identified the same me-type (ME_Exc_6)”

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

Sparse PCA yielded 56 components for inhibitory neurons, 54 for excitatory neurons, and 54 for all neurons combined.

“Analysis of inhibitory neurons yielded 56 total components, excitatory neurons yielded 54 components, and all neurons combined yielded 54 components.”

May combine two claims.

L5-L6 neurons labeled by a Nos1/Sst intersectional strategy were quite coherent in the electrophysiological t-SNE projection and had mostly one of two similar me-types.

“The L5-L6 neurons labeled by a Nos1/Sst intersectional strategy were quite coherent in the electrophysiological t-SNE projection and had mostly one of two similar me-types”

May combine two claims.

Other transgenic line/layer combinations labeling between one and three t-types predominantly had a single me-type, and those cells were tightly clustered in the electrophysiological t-SNE projection.

“However, other combinations labeling between one and three t-types predominantly had a single me-type (Fig. 7b-d), which were also tightly clustered in the electrophysiological t-SNE projection.”

May combine two claims.

These L1-L4 Nkx2-1-labeled neurons had me-types that contained fast-spiking cells and had basket-cell and chandelier-cell morphologies.

“Accordingly, we found that these neurons had me-types that contained fast-spiking cells and had basket-cell and chandelier-cell morphologies (see Figs. 5d and 6b).”

May combine two claims.

The mouse morpho-electric visual cortical dataset is large and diverse.

“This mouse morpho-electric visual cortical dataset is large and diverse”

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

Some cells exhibited a sag in membrane potential during hyperpolarizing step responses, attributable to Ih activation.

“This emphasized the “sag” in the membrane potential due to the activation of Ih observed in some cells.”

May combine two claims.

Random forest achieved 79/70% out-of-bag prediction accuracy and SVM achieved 89/79% leave-one-out cross-validation accuracy for spiny/aspiny neurons.

“Random forest classifier gave 79/70% out-of-bag prediction accuracy and support vector machine showed 89/79% prediction accuracy in leave-one-out cross validation for spiny/aspiny neurons.”

Classified as probably not a substantial claim.

The dendritic morphology of 1938 neurons was classified as aspiny, sparsely spiny, or spiny based on qualitative microscopic assessment.

“The dendritic morphology of each neuron (N=1938) was identified as either aspiny, sparsely spiny or spiny49 based on a qualitative assessment of the neuron’s dendrites by viewing the slides under the microscope and/or in the 63X image.”

• cites [49] Tripathy, S. J. et al. Transcriptomic correlates of neuron electrophysiological diversity. PLoS Comput. Biol. 13, e1005814 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Some cell types have been quantified, but those studies often relied on extra labels such as individual gene markers to define the types beforehand.

“Specific cell types have been characterized quantitatively9–11, but these studies frequently have used additional labels (individual gene markers) to pre-define cell types.”

• cites [9] Bortone, D. S., Olsen, S. R. & Scanziani, M. Translaminar inhibitory cells recruited by layer 6 corticothalamic neurons suppress visual cortex. Neuron 82, 474–485 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [10] Kim, E. J., Juavinett, A. L., Kyubwa, E. M., Jacobs, M. W. & Callaway, E. M. Three types of cortical layer 5 neurons that differ in brain-wide connectivity and function. Neuron 88, 1253–1267 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [11] Dehorter, N. et al. Tuning of fast-spiking interneuron properties by an activity-dependent transcriptional switch. Science 349, 1216–1220 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The authors used many transgenic mouse lines to cover excitatory and inhibitory classes across cortical layers, target rare populations, and connect with other approaches like transcriptomic characterization.

“A diverse set of transgenic mouse lines14–18 were used to ensure broad coverage of excitatory and inhibitory classes across all cortical layers, to enable selective targeting of rare cell populations, and to link our study with other experimental approaches, such as transcriptomic characterization19.”

• cites [14] 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 [15] Peng, H., Ruan, Z., Long, F., Simpson, J. H. & Myers, E. W. V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets. Nat. Biotechnol. 28, 348–353 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [16] Harris, J. A. et al. Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation. Front. Neural Circuits 8, 76 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [17] Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [18] Daigle, T. L. et al. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174, 465–480.e22 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [19] Bernard, A., Sorensen, S. A. & Lein, E. S. Shifting the paradigm: new approaches for characterizing and classifying neurons. Curr. Opin. Neurobiol. 19, 530–536 (2009). sourceNot checked yet

  Not checked in the free run.

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

Earlier work described excitatory neuron groups whose properties are often tied to cortical layer and axonal projection targets.

“excitatory neuron populations with features often linked to their laminar locations as well as the locations to which they project their axons2–4”

• 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.

• cites [3] Harris, K. D. & Shepherd, G. M. G. The neocortical circuit: themes and variations. Nat. Neurosci. 18, 170–181 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [4] Lodato, S. & Arlotta, P. Generating neuronal diversity in the mammalian cerebral cortex. Annu. Rev. Cell Dev. Biol. 31, 699–720 (2015). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

The entire pipeline-including slice preparation, recording, stimulation, staining, imaging, reconstruction, and atlas mapping-used highly standardized and quality-controlled methods.

“Every aspect of the pipeline, from slice preparation, recording, and stimulation, to staining, imaging, reconstruction, and mapping of cells to a reference atlas, employed highly standardized and quality-controlled methodology.”

May combine two claims.

Methods that use extra labels can reveal heterogeneity between chosen populations, yet those differences might not co-vary with other features or show discontinuities across a wider set of cortical cells.

“these approaches may successfully identify heterogeneity in features between specific populations under investigation, but these differences may not necessarily co-vary with other features or display discontinuities when examined in the context of a broader set of cortical cells—significant criteria for cell-type classification1.”

• cites [1] 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.

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

The features that separate types may be particular to the studied population and may not generalize to all cortical neurons.

“features used to distinguish types can be specific to the population being studied and may not be broadly applicable (or distinguishing) across all cortical neurons.”

May combine two claims.

The authors used a uniform protocol to characterize mouse visual cortex neurons and built an unsupervised classification of cell types from electrophysiology and morphology.

“We characterized mouse visual cortex neurons with a uniform experimental protocol and developed an unsupervised classification scheme of cell types based on electrophysiology and morphology.”

May combine two claims.

Qualitative descriptions may agree with statistical feature analyses, but they are constrained by how many criteria can be used at once to separate types.

“Such qualitative descriptions are valuable and can be consistent with statistical analyses of features8, but they can be limited by the number of criteria that can be simultaneously used to differentiate types.”

• cites [8] Druckmann, S., Hill, S., Schürmann, F., Markram, H. & Segev, I. A hierarchical structure of cortical interneuron electrical diversity revealed by automated statistical analysis. Cereb. Cortex 23, 2994–3006 (2013). sourceNot checked yet

  Not checked in the free run.

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

Recent transcriptomic work with single-cell RNA sequencing used unsupervised classification to build a taxonomy of transcriptomic types in mouse neocortex.

“Recent studies using transcriptomic characterization based on single-cell RNA-sequencing techniques performed unsupervised classification to generate a taxonomy of transcriptomic types in the mouse neocortex12–14.”

• cites [12] 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 [13] 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 [14] 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).

Large-scale efforts, such as the Blue Brain Project's work on juvenile rat somatosensory cortex, addressed these limitations by using large coherent data sets to uncover principles of local connectivity and computation in a cortical column.

“Recent studies performed at larger scales5,6, including the Blue Brain Project’s thorough classification of morpho-electric diversity in juvenile rat somatosensory cortex, have overcome many of these issues, using large, coherent data sets to identify principles of local connectivity and computation within a cortical column.”

• 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.

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

Such transcriptomic methods depend on data from thousands of neurons produced under standardized conditions.

“These approaches rely on data from thousands of neurons generated in a standardized manner.”

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

Earlier studies identified three main interneuron groups in neocortex-Pvalb+, Sst+, and Htr3a+-each containing distinct types.

“leading to the characterization of three major populations (i.e., Pvalb+, Sst+, and Htr3a+) of neocortical interneurons, each with distinct types”

• 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.

• cites [3] Harris, K. D. & Shepherd, G. M. G. The neocortical circuit: themes and variations. Nat. Neurosci. 18, 170–181 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [4] Lodato, S. & Arlotta, P. Generating neuronal diversity in the mammalian cerebral cortex. Annu. Rev. Cell Dev. Biol. 31, 699–720 (2015). sourceNot checked yet

  Not checked in the free run.

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

This finding matches earlier descriptions of excitatory m-type distribution in other brain regions.

“This agrees with previous descriptions of excitatory m-type distribution in other brain regions8,21.”

• cites [8] Druckmann, S., Hill, S., Schürmann, F., Markram, H. & Segev, I. A hierarchical structure of cortical interneuron electrical diversity revealed by automated statistical analysis. Cereb. Cortex 23, 2994–3006 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [21] Baden, T. et al. The functional diversity of retinal ganglion cells in the mouse. Nature 529, 345–350 (2016). sourceNot checked yet

  Not checked in the free run.

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

Every recorded cell followed the same workflow, and only those meeting predefined quality-control criteria entered the final set of 1,938 cells that passed electrophysiology QC.

“All recorded cells underwent the same workflow (Fig. 1a), and only cells that met pre-defined quality control (QC) standards were included in the final data set, which consisted of 1,938 cells passing electrophysiology QC.”

Classified as probably not a substantial claim.

Intrinsic electrophysiological properties were measured using a standardized current-clamp protocol with diverse stimuli such as square pulses, ramps, and noisy current injections.

“We characterized the intrinsic electrophysiological properties with a standardized current-clamp protocol that contained a variety of stimuli, including square pulses, ramps, and noisy current injections”

May combine two claims.

Within a type, properties can vary widely if the variation is gradual; such intermediates leave no clear boundary for splitting the cluster.

“Properties may vary considerably within a type as long as gradual variation is present; the existence of these intermediates means that there is no identifiable place to subdivide the cluster.”

May combine two claims.

The coherence of transgenic groups implies that electrophysiological features are similar within genetically defined cortical populations and distinct between non-overlapping ones.

“This cohesiveness suggests that there is similarity in e-features within genetically-defined populations of cortical cells, as well as distinctiveness between non-overlapping populations.”

Classified as probably not a substantial claim.

Prior single-cell RNA-seq studies have defined taxonomies of mouse neocortical cell types from isolated neurons and glia.

“Recent studies have defined taxonomies of mouse neocortical cell types by single-cell RNA-seq transcriptomic profiling of isolated neurons and glia12–14.”

• cites [12] 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 [13] 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 [14] 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). May combine two claims.

Image quality control for 63X Z-stacks included a z-stack plane count, a visual check for proper stitching alignment and even illumination, and evaluation based on quality metrics that would impact cell reconstruction.

“Image quality control included a z-stack plane count, a visual check for proper stitching alignment and even illumination throughout the images. 63X Z-stacks were evaluated based on quality metrics that would impact cell reconstruction, as opposed to aesthetic quality.”

May combine two claims.

The study's approach is quantitative, reproducible, transferable to new data, and robust across the varied characteristics of the entire population of neurons studied.

“Our approach, while certainly not free of biases (e.g., choice of stimuli delivered to the cells, morphological features quantified), has the advantage of being quantitative, reproducible, transferable to new data (e.g., Supplementary Fig. 24-26), and robust across the varied characteristics of the entire population of neurons studied.”

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

The study's methods and data are publicly accessible as part of the Allen Cell Types Database.

“Our methods and data are publicly accessible as part of the Allen Cell Types Database at celltypes.brain-map.org”

Classified as probably not a substantial claim.

The study used both electrophysiological and morphological features measured from the same cell together when possible.

“we used both electrophysiological and morphological features measured from the same cell together when possible.”

May combine two claims.

Electrophysiologically, truncated neurons were distributed in each type and found throughout the t-SNE representation of electrophysiology space.

“Electrophysiologically, truncated neurons were distributed in each type and found throughout the t-SNE representation of electrophysiology space.”

Classified as probably not a substantial claim.

Repeating the entire procedure, including robustness evaluation, on 90% subsets generated a cellwise co-clustering matrix that supported cluster stability assessment.

“The entire procedure (including the robustness evaluation) was repeated on 90% subsets of the dataset to generate a cellwise co-clustering matrix, which supported the assessment of cluster stability.”

May combine two claims.

Exc_4 has the lowest fraction of neurons with a truncated apical dendrite, and the region of the t-SNE projection where Exc_4 is primarily represented appears to have fewer truncated neurons.

“However, Exc_4 has the lowest fraction of neurons with a truncated apical dendrite, and the region of the t-SNE projection where Exc_4 is primarily represented appears to have fewer truncated neurons (Supplementary Fig. 32).”

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

Recent work found shared t-types among inhibitory cells but different t-types among excitatory cells from separate cortical areas.

“The recent finding of shared t-types among inhibitory cells but different t-types among excitatory cells from separate cortical areas14”

• cites [14] 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. May combine two claims.

Only spiny neurons with an intact apical dendrite were included in morphological analysis, while all neurons were included in electrophysiology analysis, with apical dendrite truncation annotated.

“Although only spiny neurons with an intact apical dendrite were included in morphological analysis, all neurons were included in electrophysiology analysis, though the truncation of the apical dendrite was annotated.”

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

Image quality evaluation included qualitative evaluation of signal to noise, in-focus cell body, absent or negligible tessellation, and overall evenness of section illumination and bounding box region.

“Image quality evaluation included a qualitative evaluation of signal to noise for the imaged object (with high signal apparent in the cell body and dendrites, as opposed to background stain in the surrounding tissue, which can occur when cell filling leaks), in-focus cell body, and absent or negligible tessellation (tiling and stitching edge artifact). Overall evenness of section illumination and”

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

The study's electrophysiological results reflect the specific experimental protocols used here.

“we note that our electrophysiological results reflect the specific experimental protocols used here”

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

All procedures were carried out in accordance with the Institutional Animal Care and Use Committee at the Allen Institute for Brain Science.

“All procedures were carried out in accordance with Institutional Animal Care and Use Committee at the Allen Institute for Brain Science.”

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

Individual t-types can exhibit substantial continuous variation within a type.

“It is known that individual t-types can exhibit substantial continuous variation within a type14”

• cites [14] 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. May combine two claims. The quote only partly states this claim.

Cluster stability was assessed by generating 100 random 90% subsamples, fitting them with the best GMM, merging as before, and dissolving clusters whose average Jaccard similarity was below 0.5.

“Cluster stability was then evaluated by subsampling analysis23.”

• Also stated in: § Electrophysiological classification. · plain

  “clusters that had average Jaccard similarities below 0.5 were dissolved by merging into other clusters (using the entropy criterion above), resulting in a final set of clusters, or e-types.”

• cites [23] Hennig, C. Cluster-wise assessment of cluster stability. Comput. Stat. Data Anal. 52, 258–271 (2007). sourceNot checked yet

  Not checked in the free run.

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

To be included in analysis, a >1 GΩ seal was recorded prior to break-in and the initial access resistance was <20 MΩ and <15% of the Rinput.

“To be included in analysis, a > 1 GΩ seal was recorded prior to break-in and the initial access resistance < 20 MΩ and < 15% of the Rinput.”

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

The composition of bath and internal solution and preparation methods were designed to maximize tissue quality of slices from adult mice and to align with solution compositions typically used in the field.

“The composition of bath and internal solution as well as preparation methods were made to a) maximize the tissue quality of slices from adult mice, and b) align with solution compositions typically used in the field (to maximize the chance of comparison to previous studies).”

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

For an individual sweep to be included, the bridge balance was <20 MΩ and <15% of the Rinput, bias current was 0 +/- 100 pA, RMS noise in short and long windows was <0.07 mV and 0.5 mV respectively, and the voltage difference between the end of the sweep and immediately prior to stimulus onset was <1 mV.

“For an individual sweep to be included: (1) the bridge balance was < 20 MΩ and < 15% of the Rinput, (2) bias (leak) current 0 +/- 100 pA”

• Also stated in: § Patch clamp recording. · plain

  “(3) root mean square (RMS) noise measurements in a short window (1.5 ms, to gauge high frequency noise) and longer window (500 ms, to measure patch instability) < 0.07 mV and 0.5 mV, respectively and (4) the difference in the voltage at the end of the data sweep (measured over 500 ms of rest) and the voltage measured immediately prior to the stimulus onset < 1 mV.”

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

Analyzing putatively excitatory and inhibitory cells separately produced results similar to analyzing all cells together.

“Data from putatively excitatory cells and inhibitory cells (determined by the presence and type of dendritic spines) were analyzed separately, though similar results were observed when all cells were analyzed together (see Supplementary Fig. 6).”

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

Applying the methods of this study to a different cortical area may identify the same inhibitory me-types.

“suggests that applying the methods of this study to a different cortical area may identify the same inhibitory me-types.”

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

The procedure was also run on 20%, 40%, 60%, and 80% random subsamples (10 draws each) to evaluate how the number of clusters varied with sample size.

“In addition, the procedure was used on 20%, 40%, 60%, and 80% random subsamples of the data (each drawn 10 times) to evaluate how the number of clusters identified varied with the number of samples used in the analysis.”

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

The code for electrophysiological feature analysis is publicly available as part of the Allen SDK.

“The code for feature analysis is publicly available as part of the Allen SDK.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Direct comparisons with previous studies should take into account that specific protocols and solution composition vary within the literature.

“direct comparisons with previous studies should take into account the fact that specific protocols and solution composition vary within the literature.”

Classified as probably not a substantial claim.

Large-scale transcriptomic studies have provided informative taxonomies of cortical cell types.

“Large-scale transcriptomic studies have provided informative taxonomies of cortical cell types12–14.”

• cites [12] 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 [13] 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 [14] 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. May combine two claims.

Clusters with average Jaccard similarity below 0.5 were deemed unstable and merged with another cluster having the highest between-cluster rate if that rate exceeded 35%, a threshold chosen to match the 5th percentile of within-cluster rates in stable clusters.

“Clusters with average Jaccard similarities of below 0.5 were considered unstable and were merged with another cluster with the highest between-cluster rate if it exceeded 35% (chosen because it was similar to the 5th percentile of the within-cluster rates of all cells in stable clusters).”

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

An alternate analyst checked for missed branches or inappropriate connections as a final step in manual correction and curation.

“As a final step in the manual correction and curation process, an alternate analyst checked for missed branches or inappropriate connections.”

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

The different stimulus protocols were not presented in randomized order.

“The different stimulus protocols were not presented in a randomized order.”

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

Data collection and analysis were not blinded to experimental conditions because there was a single experimental condition for all acquired data.

“Data collection and analysis were not performed blind to the conditions of the experiments as there was a single experimental condition for all acquired data.”

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

No randomization was used during data collection because all acquired data came from a single experimental condition.

“No randomization was used during data collection as there was a single experimental condition for all acquired data.”

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

Automated reconstructions were extensively manually corrected and curated using tools in the Mozak extension of Terafly tools in Vaa3D.

“Automated reconstructions were then extensively manually corrected and curated using a range of tools (e.g., virtual finger, polyline) in the Mozak extension (Zoran Popovic, Center for Game Science, University of Washington) of Terafly tools50,53 in Vaa3D.”

• cites [50] Neher, E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 207, 123–131 (1992). sourceNot checked yet

  Not checked in the free run.

• cites [53] Peng, H., Bria, A., Zhou, Z., Iannello, G. & Long, F. Extensible visualization and analysis for multidimensional images using Vaa3D. Nat. Protoc. 9, 193–208 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Restoring reconstructions to original volumes, aligning them to the perpendicular cortical axis, and re-running clustering showed good agreement with the original clustering results.

“However, to test the effects of z-shrinkage and tilt angle on the classification results, we restored reconstructions to their original volumes and aligned them to the perpendicular cortical axis (based on registration to the common mouse reference atlas), and re-ran our clustering analysis (Supplementary Fig. 33). We found good agreement between the clustering results before and after adjustment.”

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

No statistical methods pre-determined sample sizes, but the sample sizes were similar to those in previous publications.

“No statistical methods were used to pre-determine sample sizes, but the sample sizes here are similar to those reported in previous publications5,6,12.”

• 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 [12] 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.

Classified as probably not a substantial claim.

p-values were adjusted for multiple testing using the two-stage Benjamini-Hochberg step-up procedure to control the false discovery rate.

“The p-values were adjusted for multiple testing using the two-stage Benjamini and Hochberge step-up procedure to control the false discovery rate.”

• AssumptionThe authors state this as an assumption: Morphological feature distributions were assumed to be normal for the t-test, but this was not formally tested.