Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type

May combine two claims.

There appear to be ten inhibitory cell types within layer 1, although it is not clear whether any are completely restricted to layer 1.

“Therefore, there appear to be ten inhibitory cell types within layer 1, although it is not clear whether any of these types are completely restricted to layer 1.”

May combine two claims.

Voltage-gated ion channels and GABA and glutamate receptor subunits show diverse expression patterns among interneurons, including cell type-specific expression of CACNA2D1, GABRG1, KCNH5, and SCN5A.

“voltage-gated ion channels and GABA and glutamate receptor subunits show diverse expression patterns among interneurons, including highly cell type-specific expression of CACNA2D1, GABRG1, KCNH5, and SCN5A”

May combine two claims.

Light microscopic examination identified neurogliaform cells (n=16, 21%) and a novel group of rosehip cells (n=10, 13%) among recovered layer 1 interneurons.

“Light microscopic examination of these cells identified neurons with previously described morphological features, e.g. neurogliaform cells (NGFCs, n=16, 21%; Fig.2C,D)1,21,24 as well as a novel group of interneurons having large, rosehip-shaped axonal boutons forming very compact, bushy arborizations (rosehip cells, RCs, n=10, 13%; Fig.2A,D).”

May combine two claims.

Bouton density decreased with distance from soma; it was lower in basket cells at 30-50 μm and higher in neurogliaform cells at 70-220 μm and basket cells at 130-220 μm.

“The bouton density of rosehip, neurogliaform and BCs almost monotonously decreased with increasing distances from the soma; however, bouton densities were lower in BCs 30-50 µm (p<0.04 for 30-50, MW U-test) and higher in NGFCs 70-220 µm (p<0.02, MW U-test) and in BCs 130-220 µm (p<0.03) from the soma.”

May combine two claims.

Rosehip cells have significantly different total dendritic length and dendritic node frequency from basket cells and similar values to neurogliaform cells.

“Total dendritic length (1.96±0.90 mm) and dendritic node frequency per 100 µm (0.66±0.21) of RCs were significantly different from those of BCs (3.41±0.58 mm, p<0.031; 0.29±0.10, p<0.009, respectively, MW U-test) and were similar to those of NGFCs (2.62±1.08 mm, 1.50±1.47).”

May combine two claims.

Rosehip cells have significantly smaller maximal radial axon extent than basket cells but not different from neurogliaform cells.

“Maximal radial extent of axon of RCs (263.42±69.09 µm) was significantly smaller than that of BCs (713.22±124.87 µm, p<0.005, MW U-test), but were not different from those of NGFCs (323.18±49.60 µm).”

May combine two claims.

Rosehip cells have fewer primary dendrites than neurogliaform cells but similar numbers to basket cells.

“The number of primary dendrites of RCs (5.50±1.87) was similar to that of BCs (6.2±2.17, n=5) and was significantly fewer compared to NGFCs (8.6±2.19, n=5, p<0.04, Mann-Whitney (MW) U-test).”

May combine two claims.

Rosehip cells have an immunohistochemical profile that is GAD1/CCK-positive and CNR1/SST/CALB2/PVALB-negative, matching a single transcriptomically defined cell type.

“These rosehip cells show an immunohistochemical profile (GAD1/CCK-positive, CNR1/SST/CALB2/PVALB-negative) matching a single transcriptomically-defined cell type”

May combine two claims.

Rosehip cells have longer interbouton intervals than neurogliaform cells and shorter intervals than basket cells.

“RCs had longer interbouton intervals compared to NGFCs (3.97±0.49 and 3.10±0.32 µm, respectively, p<0.038, MW U-test) and shorter compared to BCs (5.63±0.51 µm, p<0.024) measured as linear distances between neighboring boutons.”

May combine two claims.

In human MTG layer 1, SST and VIP but not PVALB are seen by in situ hybridization.

“Interestingly, both SST and VIP (but not PVALB) are seen in human MTG layer 1 by ISH”

May combine two claims.

A novel group of human layer 1 interneurons has large rosehip-like axonal boutons and compact arborization, anatomical features not described in rodents.

“characterize a novel group of human interneurons with anatomical features never described in rodents having large, “rosehip”-like axonal boutons and compact arborization”

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

Several layer 1 interneuron markers appear related to interaction with endothelia, including EDNRA and EGF.

“several of these markers appear to be related to interaction with endothelia, including endothelin receptor type A (EDNRA) and epidermal growth factor (EGF).”

May combine two claims.

The axon of rosehip cells usually emerges from the basal part of the soma and forms compact, dense axonal trees predominantly in layer 1, with tortuous collaterals and spindle-shaped boutons whose diameters are not seen in other human layer 1 interneurons in the sample.

“The axon of RCs usually emerged from the basal part of the soma and gave rise to very compact, dense axonal trees predominantly arborizing in layer 1 with tortuous collaterals having spindle-shaped boutons with diameters not seen in other types of human layer 1 interneurons in our sample.”

May combine two claims.

Cluster i2 is restricted to layer 2 because it expresses LHX6, which is not found in layer 1.

“cluster i2 represents a cell type restricted to layer 2 since it also expresses LHX6 which is not found in layer 1”

May combine two claims. The quote only partly states this claim.

The clusters corresponded to major neural cell classes expected to be captured, including microglia, astrocytes, OPCs, oligodendrocytes, one excitatory neuron type from upper layer 2, and eleven GABAergic neuron subtypes.

“clusters corresponded to all major classes of neural cell types that were expected to be captured”

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

Targeted recordings increased the number of rosehip cells in the database to n=120.

“Targeted recordings increased the number of RCs in our database (n=120)”

May combine two claims.

Rosehip cells have significantly smaller total axon length and maximal horizontal axon extent than neurogliaform and basket cells.

“Total length (11.13±1.99 mm) and maximal horizontal extent of axons (287.75±70.15 µm) of RCs were significantly smaller than those of NGFCs (24.74±8.90 mm, 648.68±202.60 µm, respectively; p<0.005 for both, MW U-test) and BCs (31.16±14.79 mm, p<0.009; 1102.76±296.99 µm, p<0.005, respectively, MW U-test).”

May combine two claims.

Rosehip cells received local excitatory inputs from layer 2-3 pyramidal cells sporadically (n=8, CR 5%) with monosynaptic EPSP amplitudes of 3.357±1.458 mV and paired pulse ratios of 0.68±0.12.

“RCs received local excitatory inputs from layer 2-3 pyramidal cells sporadically (n=8, CR 5%) with monosynaptic EPSP amplitudes of 3.357±1.458 mV and paired pulse ratios of 0.68±0.12.”

May combine two claims.

The rosehip cell (RC) has a highly distinctive transcriptomic signature, a highly distinctive morphological, physiological and connectional phenotype, and a strong correspondence between these properties.

“The RC represents a type with highly distinctive transcriptomic signature, a highly distinctive morphological, physiological and connectional phenotype, and a strong correspondence between these properties.”

May combine two claims.

Rosehip cells are immunopositive for CCK and negative for CNR1, SST, and CALB2.

“This revealed that RCs were immunopositive for CCK (n=10) but negative for CB1 cannabinoid receptor (CNR1, n=11), SST (n=9) and calretinin (CALB2; n=2; Fig.3A).”

May combine two claims.

Frequencies of maximal impedance in rosehip cells were significantly higher than in neurogliaform cells but not significantly different from unclassified interneurons.

“frequencies of maximal impedance (fmax) in RCs (4.17±1.1 Hz) were significantly higher than in NGFCs (1.98±1.04 Hz; p<0.045) but the difference was not significant compared to unclassified interneurons (2.47±1.47 Hz, p<0.142).”

May combine two claims.

Fast components of IPSPs arriving to rosehip cells from different presynaptic interneurons had similar amplitudes and showed paired pulse depression.

“Fast components of IPSPs arriving to RCs evoked by different presynaptic interneurons had similar amplitudes (0.982±0.705, 0.915±0.594 and 1.504±1.308 mV, respectively) and showed paired pulse depression with paired pulse ratios of (0.42±0.48, 0.27±0.04 and 0.71±0.26, respectively).”

May combine two claims.

Time constants of rosehip cells were similar to neurogliaform cells and faster than other cells.

“time constants of RCs (7.3±3.7 ms) were similar to neurogliaform (8.9±2.4 ms, p<0.001) and faster compared to other cells (11.1±12.5 ms, p<0.001).”

May combine two claims.

Application of the CB1 receptor antagonist AM251 was ineffective in modulating rosehip cell evoked IPSPs, supporting digital PCR, IHC, and ISH data.

“application of the CB1 receptor antagonist AM251 was ineffective in modulating RC evoked IPSPs (n=4, Fig.5H), supporting our results of single cell digital PCR, IHC and ISH data (Fig.3).”

May combine two claims.

Single-nucleus sequencing of human cortical layer 1 identified 10 GABAergic neuron types, which is more than the 8 types previously described for the entire human cortex.

“The targeted application of single nucleus sequencing reported here has demonstrated a significantly higher degree of GABAergic neuron complexity in just one layer of the human cerebral cortex (10 types) than what has previously been described in all of the cortex (8 types22).”

• cites [22] Lake, B. B. et al. Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain. Science 352, 1586–1590 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Rosehip cluster i5 had a weak reciprocal best match to mouse cluster Smad3 (r=0.34), while several other human clusters matched Smad3 almost as well (r>0.3).

“The rosehip cluster i5 had a weak (r=0.34) reciprocal best match to mouse cluster Smad3, although several other human clusters (i7, i1, i4, i3) matched Smad3 almost as well (r>0.3).”

May combine two claims.

Rosehip cells formed homologous electrical synapses between each other (n=5, CR 57%) and convergent heterologous electrical synapses with an unclassified layer 1 interneuron (n=2, CR 11%).

“RCs also formed homologous electrical synapses (n=5, CR 57%) between each other (Fig.5K) and established convergent heterologous electrical synapses (n=2, CR 11%) with an unclassified layer 1 interneuron.”

May combine two claims.

Input resistances of rosehip cells were similar to neurogliaform cells and lower than other interneurons.

“Input resistances of RCs (139.6±54.1 MΩ) were similar to those of NGFCs (160.1±55.9 MΩ) and lower compared to other interneurons (216.3±84.4 MΩ, p<0.001, Wilcoxon-test)”

May combine two claims.

Digital PCR on individual rosehip neurons showed positivity for CCK, CPLX3, SV2C, and TRPC3, and low CNR1 or absent NDNF.

“As predicted by the transcriptome data, RCs were positive for CCK, CPLX3, SV2C and TRPC3, and low (CNR1) or absent (NDNF) for genes not expressed by cells in that cluster”

May combine two claims.

ISH in human layer 1 shows expression of LAMP5, SV2C, EYA4, and CPLX3; in mouse layer 1, three of these four genes are also expressed, whereas Eya4-expressing cells are extremely rare.

“expression of LAMP5, SV2C, EYA4 and CPLX3 is seen by ISH in human layer 1 (Fig.3C); similarly, as predicted by transcriptomics three of these four genes are also expressed in mouse layer 1 whereas cells expressing Eya4 are extremely rare.”

May combine two claims.

Rosehip inputs simultaneous with backpropagating pyramidal action potentials suppressed Ca2+ signals only at sites neighboring (8±5 μm) the putative synapses between the two cells, with no effect at dendritic sites one step further (21±14 μm).

“Rosehip inputs simultaneous with backpropagating pyramidal action potentials were effective in suppressing Ca2+ signals only at sites that were neighboring (8±5 µm) to the putative synapses between the two cells. No effect of RCs was detected at dendritic sites one step further in distance (21±14 µm, Fig.6D-F).”

May combine two claims.

Triple fluorescent ISH on human MTG tissue found cells with predicted rosehip profiles, including CCK+/CNR1-/LAMP5+, CCK+/PDGFRA+/SOX13+, and CCK+/TRPC3+/CPLX3+ cells.

“we observed CCK+/CNR1-/LAMP5+, CCK+/PDGFRA+/SOX13+, and CCK+/TRPC3+/CPLX3+ cells, as well as cells where CCK was swapped with other positive rosehip markers”

May combine two claims.

Cross-species expression correlations between mouse and human clusters were low (r<0.5), and clusters could only be reliably grouped into broad cell classes.

“Expression correlations were quite low (r < 0.5), and clusters could only be reliably grouped into broad classes of cell types.”

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

Backpropagation of action potentials to dendrites of human neurons was confirmed by detecting dendritic Ca2+ responses following somatically elicited burst firing in layer 3 pyramidal cells.

“Backpropagation of action potentials to dendrites of human neurons has been shown in previous studies13,29 and we confirmed these results by detecting dendritic Ca2+ responses following somatically elicited burst firing (100 ms current injections, 4 spikes/burst) in layer 3 pyramidal cells.”

• cites [13] Verhoog, M. B. et al. Mechanisms underlying the rules for associative plasticity at adult human neocortical synapses. J. Neurosci. 33, 17197–17208 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [29] Kerekes, B. P. et al. Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro. Neurophotonics 1, 011013 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The remaining n=5 (11%) dendrites had no spines and received asymmetric synapses on the shaft, likely formed by interneurons.

“The remaining n=5 (11 %) dendrites had no spines and received asymmetric synapses on the shaft were likely to be formed by interneurons.”

May combine two claims.

GABAergic cells evoking IPSPs on rosehip cells included layer 1 NGFCs (n=10, CR 100%), rosehip cells (n=2, CR 17%), and unclassified interneurons (n=14, CR 40%); none of the tested layer 2 interneurons were connected to rosehip cells.

“GABAergic cells evoking IPSPs on RCs included layer 1 NGFCs (n=10, CR 100 %), RCs (n=2, CR 17%) and unclassified interneurons (n=14, CR 40%); however, none of the tested interneurons (n=9) having somata in layer 2 (defined as <70 µm below the layer 1/2 border) were connected to RCs.”

May combine two claims.

Ultrastructural analysis of postsynaptic dendrites (n=46) revealed dendritic spines and sparse innervation by symmetrical synapses on the shaft, suggesting that these dendrites belonged to pyramidal cells (n=41, 86%).

“further ultrastructural analysis of postsynaptic dendrites (n=46) revealed dendritic spines and sparse innervation by symmetrical synapses on the shaft, suggesting that these dendrites belonged to pyramidal cells (n=41, 86%, Fig.5J).”

May combine two claims.

Triple-positive cells for the GAD1/PDGFRA/TRPC3 combination were sparsely distributed across layer 1, though not restricted to this layer.

“The triple positive cells for this combination were sparsely distributed across layer 1, although not restricted to this layer (Suppl. Fig.5D).”

May combine two claims.

PDGFRA+ cells represent ~15% of GAD1+ cells, an upper bound for rosehip proportion.

“PDGFRA+ cells represent ~15% of GAD1+ cells, therefore an upper bound.”

May combine two claims.

Rosehip cells are immunopositive for GABA and NR2F2 and negative for parvalbumin, neuronal nitric oxide synthase, neuropeptide Y, calbindin, and choline acetyltransferase.

“Furthermore, RCs were immunopositive for gamma-aminobutyric acid (GABA; n=2), for chicken ovalbumin upstream promoter transcription factor II (NR2F2; n=2) and negative for parvalbumin (n=3), neuronal nitric oxide synthase (n=4), neuropeptide Y (n=2), calbindin (n=2), and choline acetyltransferase (n=3; Suppl. Fig.5A).”

May combine two claims.

Human clusters i5, i7, i1, i4, and i3 matched mouse Smad3+ and Ndnf+ clusters; i6, i8, i9, i11, and i10 matched mouse Vip+ clusters; and i2 matched mouse Sst+ clusters.

“Human clusters i5, i7, i1, i4, and i3 matched mouse Smad3+ and Ndnf+ clusters. Clusters i6, i8, i9, i11, and i10 matched mouse Vip+ clusters, and cluster i2 matched mouse Sst+ clusters.”

Classified as probably not a substantial claim.

Human neuron membrane properties remained stable without significant changes for as long as 20 hours after slicing.

“Membrane properties of human neurons did not show significant changes for up to 20 h after slicing”

May combine two claims.

RCs are CCK-positive but cannabinoid receptor-negative, and they appear to selectively target distal dendrites of pyramidal neurons.

“In contrast, RCs are CCK-positive but cannabinoid receptor-negative, and appear to selectively target distal dendrites of pyramidal neurons.”

May combine two claims.

The transcriptomic clusters were consistent across the two donors and were not attributable to technical artifacts.

“confirming that transcriptomic clusters were consistent between donors and not driven by technical artifacts.”

Classified as probably not a substantial claim.

In the multiplex FISH quantification, 408 GAD1-positive cells from two individuals were used to calculate the proportion of GAD1-positive cells that expressed PDGFRA and TRPC3.

“A total of 408 GAD1+ cells were identified from two individuals for this quantification.”

May combine two claims.

There is evidence for significant neuronal differences between rodents and humans, including distinct membrane and synaptic properties and dendritic complexity of human neurons that might contribute to human-specific signal processing.

“Indeed there is evidence for significant neuronal differences between rodents and human; for example, distinct membrane8,9 and synaptic10–14 properties and dendritic complexity15–17 of human neurons might contribute to human specific signal processing.”

• cites [8] Eyal, G. et al. Unique membrane properties and enhanced signal processing in human neocortical neurons. eLife 5, e16553 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [9] Wang, B. et al. A subtype of inhibitory interneuron with intrinsic persistent activity in human and monkey neocortex. Cell Rep. 10, 1450–1458 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [10] Molnár, G. et al. Complex events initiated by individual spikes in the human cerebral cortex. PLoS Biol. 6, e222 (2008). sourceNot checked yet

  Not checked in the free run.

• cites [11] Molnár, G. et al. Human pyramidal to interneuron synapses are mediated by multi-vesicular release and multiple docked vesicles. eLife 5, e18167 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [12] Testa-Silva, G. et al. Human synapses show a wide temporal window for spike-timing-dependent plasticity. Front. Synaptic Neurosci. 2, 12 (2010).Not checked yet

  Not checked in the free run.

• cites [13] Verhoog, M. B. et al. Mechanisms underlying the rules for associative plasticity at adult human neocortical synapses. J. Neurosci. 33, 17197–17208 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [14] Szegedi, V. et al. Plasticity in single axon glutamatergic connection interneurons regulates complex events in the human neocortex to GABAergic. PLoS Biol. 14, e2000237 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [15] Elston, G. N. Pyramidal cells of the frontal lobe: all the more spinous to think with. J. Neurosci. 20, RC95 (2000). sourceNot checked yet

  Not checked in the free run.

• cites [16] Morales, J. et al. Random positions of dendritic spines in human cerebral cortex. J. Neurosci. 34, 10078–10084 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [17] Mohan, H. et al. Dendritic and axonal architecture of individual pyramidal neurons across layers of adult human neocortex. Cereb. Cortex 25, 4839–4853 (2015). sourceNot checked yet

  Not checked in the free run.

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

Recent systematic efforts suggest several dozen inhibitory and excitatory cell types in rodent neocortical circuits.

“Recent systematic efforts have provided insight into the cellular composition and organization of rodent neocortical circuits, suggesting the presence of several dozen inhibitory and excitatory cell types3–5,7.”

• cites [3] 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 [4] 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 [5] Zeisel, A. et al. Brain structure. 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 [7] 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.

May combine two claims.

The data strongly link the anatomically defined rosehip phenotype with a distinctive transcriptomic signature found in human but apparently not in mouse layer 1.

“Together, these data strongly link the anatomically-defined rosehip phenotype with a highly distinctive transcriptomic cell type signature that is found in human but apparently not in mouse layer 1.”

May combine two claims.

Regional differences between human temporal cortex and mouse visual cortex are unlikely to account for the transcriptomic difference, because the anatomically defined rosehip type was found in multiple human regions.

“Although the transcriptomic comparison is between human temporal cortex and mouse visual cortex, regional differences seem unlikely to account for this difference as we found the anatomically defined rosehip type in multiple human regions.”

May combine two claims.

Quantification of cell proportions using marker expression is complicated because markers for one cell type are often expressed in others and individual markers are often not expressed in every cell in a cluster.

“Quantification of cell proportions using marker expression is complicated by two factors; first, markers for one cell type are often expressed in others, and second, individual markers are often not expressed in every cell in a cluster.”

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

In rodents, CCK-positive cells selectively express high levels of cannabinoid receptors and are involved in perisomatic inhibition.

“It is widely accepted28 that CCK-positive cells in the rodent show selectively high expression of cannabinoid receptors and are involved in perisomatic inhibition.”

• cites [28] Katona, I. & Freund, T. F. Multiple functions of endocannabinoid signaling in the brain. Annu. Rev. Neurosci. 35, 529–558 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Local inputs to rosehip cells appear predominantly GABAergic, with potential underrepresentation of pyramidal cell triggered EPSPs because some axon collaterals were cut during slicing.

“Thus, local inputs to RCs appear to be predominantly GABAergic, acknowledging that some axon collaterals of pyramidal cells were cut during the slicing procedure (Fig.5C) leading to a potential underrepresentation of pyramidal cell triggered EPSPs.”

May combine two claims.

The combined single-nucleus transcriptomics and human slice physiology approaches provide convergent evidence for robust cell type identities and for species conservation or specialization.

“Together these approaches provide convergent evidence for robust cell type identities and concomitant evidence for species conservation or specialization.”

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

Specimens used for RNA-sequencing were screened for RNA quality and had an RNA integrity number (RIN) of at least 7.

“Specimens were further screened for RNA quality and had an RNA integrity number (RIN) ≥7.”

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

Every human specimen was a nonpathological control and was assigned to the same experimental group.

“All human specimens were controls (nonpathological) and were therefore allocated into the same experimental group.”

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

Positive controls (10 nuclei pools and/or 10 pg and 1 pg total RNA) were included on every 96-well plate of sorted nuclei.

“Positive controls (10 nuclei pools and/or 10 pg and 1 pg total RNA) were included on every 96-well plate of sorted nuclei.”

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

The Random Forest classification model correctly classified all 196 samples in the training set with high confidence scores.

“Using this Random Forest classification model, all 196 samples in the training set were classified correctly with high confidence scores.”

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

For the interspike-interval standard deviation calculation, sweeps containing fewer than five spikes were excluded.

“Sweeps were discarded with less than 5 spikes for the calculation of ISI SD.”

May combine two claims.

Action potentials backpropagate to distal dendrites of human pyramidal cells and can be attenuated by RC activation.

“Action potentials backpropagate to distal dendrites of human pyramidal cells and can be attenuated by RC activation.”

May combine two claims.

The Random Forest model achieved a classification accuracy of 95% on an independent test set, with only 8 misclassifications (4.9%).

“Application of the random forest model to these test Pass and Fail samples resulted in only 8 misclassifications (4.9%), for a classification accuracy of 95%.”

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

The sample sizes used in this study are comparable to those in earlier publications.

“our sample sizes are similar to those reported in previous publications10,11,14.”

• cites [10] Molnár, G. et al. Complex events initiated by individual spikes in the human cerebral cortex. PLoS Biol. 6, e222 (2008). sourceNot checked yet

  Not checked in the free run.

• cites [11] Molnár, G. et al. Human pyramidal to interneuron synapses are mediated by multi-vesicular release and multiple docked vesicles. eLife 5, e18167 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [14] Szegedi, V. et al. Plasticity in single axon glutamatergic connection interneurons regulates complex events in the human neocortex to GABAergic. PLoS Biol. 14, e2000237 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The bouton morphology and/or compact axonal field of RCs resembles that of cat cortex cell types (dendrite targeting and clutch cells) that innervate relatively proximal dendrites.

“The bouton morphology and/or the compact axonal field of RCs resembles that of cell types described in deeper layers of the cat cortex that innervate relatively proximal dendrites (dendrite targeting and clutch cells)40,41.”

• cites [40] Tamás, G., Buhl, E. H. & Somogyi, P. Fast IPSPs elicited via multiple synaptic release sites by different types of GABAergic neuron in the cat visual cortex. J. Physiol. (Lond.) 500, 715–738 (1997). sourceNot checked yet

  Not checked in the free run.

• cites [41] Kisvárday, Z. F., Martin, K. A. C., Whitteridge, D. & Somogyi, P. Synaptic connections of intracellularly filled clutch cells: a type of small basket cell in the visual cortex of the cat. J. Comp. Neurol. 241, 111–137 (1985). sourceNot checked yet

  Not checked in the free run.

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

The study did not blind data collection or analysis to experimental conditions.

“Data collection and analysis were not performed blind to the conditions of the experiments.”

• Also stated in: § Statistics · plain

  “data collection and analysis were not performed blind to the conditions of the experiments.”

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

In total, 443 out of 470 nuclei (94.3%) were defined as core.

“In total, 443/470 (94.3%) of nuclei were defined as core.”

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

Positive control probes (POLR2A, UBC, PPIB) were applied to every tissue sample in the multiplex FISH experiments to ensure RNA quality.

“Positive controls (POLR2A, UBC and PPIB) were used on each tissue sample to ensure RNA quality (ACD Bio, 320861).”

• cites ACD Bio, 320861Not checked yet

  Not checked in the free run.

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

The tissue specimens were pre-screened for known neuropsychiatric and neuropathological history and underwent routine serological testing and toxicology screening.

“The tissue specimens used in this study were pre-screened for known neuropsychiatric and neuropathological history, and underwent routine serological testing and toxicology screening.”

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

No data that passed quality control were excluded from the analyses.

“No data with successful quality control were excluded from the analyses.”

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

The sharp theta-range resonance detected in individual RCs and its potential spread through gap junctions to a rosehip network could phase-selectively interact with long-range inputs, similar to mechanisms suggested for oscillation-dependent memory consolidation.

“The sharp resonance in the theta-range detected in individual RCs and its potential spread through gap junctions to a rosehip network could phase-selectively interact with long range inputs similarly to mechanisms suggested for example in oscillation dependent memory consolidation30,46.”

• cites [30] Freund, T. F. & Buzsáki, G. Interneurons of the hippocampus. Hippocampus 6, 347–470 (1996). sourceNot checked yet

  Not checked in the free run.

• cites [46] Klausberger, T. & Somogyi, P. Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321, 53–57 (2008). sourceNot checked yet

  Not checked in the free run.

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

The recordings that entered the analysis were stopped at 15 hours after slice preparation.

“recordings included in the analysis were arbitrarily terminated 15 h after slice preparation.”

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

Tissue collection complied with the Uniform Anatomical Gift Act and other applicable laws and regulations.

“All tissue collection was performed in accordance with the provisions of the Uniform Anatomical Gift Act described in Health and Safety Code §§ 7150, et seq., and other applicable state and federal laws and regulations.”

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

A better understanding of human cellular and circuit organization may help counteract the current lack of success in translating promising rodent results to effective treatment against human neuropsychiatric disorders.

“A better understanding of human cellular and circuit organization may help counteract the current lack of success in translating promising rodent results to effective treatment against human neuropsychiatric disorders49,50.”

• cites [49] Mak, I. W., Evaniew, N. & Ghert, M. Lost in translation: animal models and clinical trials in cancer treatment. Am. J. Transl. Res. 6, 114–118 (2014).Not checked yet

  Not checked in the free run.

• cites [50] Cavanaugh, S. E., Pippin, J. J. & Barnard, N. D. Animal models of Alzheimer disease: historical pitfalls and a path forward. ALTEX 31, 279–302 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Human pyramidal cells have uniquely small membrane capacitance, which promotes backpropagation of action potentials and increases excitability in human dendrites relative to rodent dendrites with larger capacitance.

“The uniquely small membrane capacitance (Cm) found in human pyramidal cells8 promotes backpropagation of action potentials and increases excitability in human dendrites13,29 relative to rodent dendrites having larger Cm.”

• cites [8] Eyal, G. et al. Unique membrane properties and enhanced signal processing in human neocortical neurons. eLife 5, e16553 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [13] Verhoog, M. B. et al. Mechanisms underlying the rules for associative plasticity at adult human neocortical synapses. J. Neurosci. 33, 17197–17208 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [29] Kerekes, B. P. et al. Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro. Neurophotonics 1, 011013 (2014). sourceNot checked yet

  Not checked in the free run.

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

The function of neuron types specific to the human circuit could be important for understanding pathological alterations of network functions.

“The function of neuron types specific to the human circuit could be important in understanding pathological alterations of network functions.”

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

The study did not perform a sample-size calculation.

“No sample-size calculation was performed”

• Also stated in: § Statistics · plain

  “No statistical methods were used to pre-determine sample sizes”

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

A Random Forest machine learning classification approach was implemented to remove low-quality nuclei samples prior to downstream analysis.

“To remove data from low quality nuclei samples prior to downstream analysis, we implemented a Random Forest machine learning classification approach as described in detail in Aevermann et al. 58.”

• cites [58] Aevermann, B. et al. Production of a preliminary quality control pipeline for single nuclei RNA-seq and its application in the analysis of cell type diversity of post-mortem human brain neocortex. Pac. Symp. Biocomput. 22, 564–575 (2017).Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

To assess the robustness of clusters, the iterative clustering procedure was repeated 100 times for random subsamples of 80% of nuclei.

“To assess the robustness of clusters, the iterative clustering procedure described above was repeated 100 times for random subsamples of 80% of nuclei.”

Classified as probably not a substantial claim.

Low-quality nuclei samples were removed before downstream analysis by a Random Forest machine-learning classification method.

“Data from low quality nuclei samples was removed prior to downstream analysis, using a Random Forest machine learning classification approach as described58.”

• cites [58] Aevermann, B. et al. Production of a preliminary quality control pipeline for single nuclei RNA-seq and its application in the analysis of cell type diversity of post-mortem human brain neocortex. Pac. Symp. Biocomput. 22, 564–575 (2017).Not checked yet

  Not checked in the free run.

May combine two claims.

The addition of new human cell types, or specialization of existing types through major modification of cellular features, would be expected to alter circuit function and therefore cannot be studied in rodents.

“Addition of new human cell types, or specialization of existing types through major modification of cellular features, would be expected to alter circuit function3,43,44, and therefore cannot be studied in rodents.”

• cites [3] 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 [43] 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 [44] Gjorgjieva, J., Drion, G. & Marder, E. Computational implications of biophysical diversity and multiple timescales in neurons and synapses for circuit performance. Curr. Opin. Neurobiol. 37, 44–52 (2016). sourceNot checked yet

  Not checked in the free run.

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

In the digital PCR analysis, reactions with CT values below 23 or above 33 were treated as primer dimers or background and removed from the dataset.

“reactions having CT values less than 23 or greater than 33 were considered primer dimers or background signals, respectively, and excluded from the data set.”

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

The Western Institutional Review Board determined that tissue collection processes did not constitute human subjects research requiring IRB review.

“The Western Institutional Review Board reviewed tissue collection processes and determined that they did not constitute human subjects research requiring IRB review.”

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

The study did not use randomization.

“Randomization was not used.”

May combine two claims.

RCs may provide supplementary inhibitory control required to balance the potentially higher excitability in human dendrites and modulate interactions between long-range excitatory connections arriving to layer 1 and backpropagating action potentials.

“Thus, RCs may provide supplementary inhibitory control required to balance the potentially higher excitability in human dendrites8 and modulate interactions between long range excitatory connections arriving to layer 1 and backpropagating action potentials suggested to participate in interhemispheric modulation45.”

• cites [8] Eyal, G. et al. Unique membrane properties and enhanced signal processing in human neocortical neurons. eLife 5, e16553 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [45] Palmer, L. M. et al. The cellular basis of GABA(B)-mediated interhemispheric inhibition. Science 335, 989–993 (2012). sourceNot checked yet

  Not checked in the free run.

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

Several highly selective markers for RCs have been implicated as risk factors for neuropsychiatric disease, including netrin G1 (NTNG1) for Rett syndrome and neurotrypsin (PRSS12) for mental retardation.

“For example, several highly selective markers for RCs have been implicated as risk factors for neuropsychiatric disease, including netrin G1 (NTNG1) for Rett syndrome47 and neurotrypsin (PRSS12) for mental retardation48.”

• cites [47] Borg, I. et al. Disruption of Netrin G1 by a balanced chromosome translocation in a girl with Rett syndrome. Eur. J. Hum. Genet. 13, 921–927 (2005). sourceNot checked yet

  Not checked in the free run.

• cites [48] Molinari, F. et al. Truncating neurotrypsin mutation in autosomal recessive nonsyndromic mental retardation. Science 298, 1779–1781 (2002). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In rodent layer 1, high-throughput electrophysiology found two interneuron types and two canonical pathways involving feedforward interneuron-to-interneuron connections.

“Moreover, when assessing layer 1 canonical inhibitory pathways in rodent with high throughput electrophysiology capable of sampling all cell types in layer 1, Lee at al.42 found two interneuron types and two canonical pathways involving feed forward interneuron-to-interneuron connections.”

• cites [42] Lee, A. J. et al. Canonical organization of layer 1 neuron-led cortical inhibitory and disinhibitory interneuronal circuits. Cereb. Cortex 25, 2114–2126 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Focal intralayer inhibition restricted by the compact axonal arbor of RCs to distal dendrites of a column of pyramidal cells is missing from the rodent; instead, mouse feedforward inhibitory connections are vertically spread to all somatodendritic domains.

“Furthermore, focal intralayer inhibition restricted by the compact axonal arbor of RCs to distal dendrites of a column of pyramidal cells is also missing from the rodent; rather, mouse feedforward inhibitory connections are vertically spread to all somatodendritic domains42.”

• cites [42] Lee, A. J. et al. Canonical organization of layer 1 neuron-led cortical inhibitory and disinhibitory interneuronal circuits. Cereb. Cortex 25, 2114–2126 (2015). sourceNot checked yet

  Not checked in the free run.

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

Individuals with a history of epilepsy were excluded from the electrophysiological experiments.

“Patients with a history of epilepsy were excluded from this study.”