CoChat
Sign Up
Cited by 235Virus-based gene therapy researchNeuroscience and Neuropharmacology ResearchCRISPR and Genetic Engineering

Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans

Open access
Vormstein-Schneider, Douglas, Lin, Jessica D., Pelkey, Kenneth A., Chittajallu, Ramesh, Guo, Baolin, Arias-Garcia, Mario A., Allaway, Kathryn, Sakopoulos, Sofia +30 more·2020·Nature Neuroscience
DOI 10.1038/s41593-020-0692-9PMC PMC8015416

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.

Integrity signals

Registry

Crossref · 10.1038/s41593-020-0692-9

Cites

60 of 60 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 51 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Additional PV-specific enhancers with high specificity in distinct brain regions were characterized.

    “characterize additional PV-specific enhancers with exquisite specificity within distinct brain regions.”
    Quote in the paperSubstantial claim
  • Findingown data

    The element allows selective targeting of PV cINs during development and across rodents, non-human primates, and humans.

    “this element allows for the selective targeting of PV cINs both during development and across species including rodents, non-human primates and humans.”
    Quote in the paperSubstantial claim
  • Findingown data

    Three enhancers collectively target the breadth of neuronal populations expressing Scn1a.

    “we identified three enhancers that collectively target the breadth of neuronal populations expressing Scn1a.”
    Quote in the paperSubstantial claim
  • Findingown data

    The PV-specific enhancer enabled selective targeting and manipulation of PV neurons across vertebrate species, including humans.

    “the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans.”
    Quote in the paperSubstantial claim
  • Findingown data

    The authors developed a strategy that identified multiple new enhancers for targeting functionally distinct neuronal subtypes.

    “we developed a strategy that led to the identification of multiple novel enhancers to target functionally distinct neuronal subtypes.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 20
  • May combine two claims. 32
  • The quote only partly states this claim. 1

  • Classified as probably not a substantial claim.

    Findingreasoning

    Twenty-five regulatory elements near seven additional genes enriched in PV INs were selected.

    “we further selected twenty-five regulatory elements in the vicinity of seven additional genes whose expression is enriched in PV INs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The element was validated in synaptic tagging, calcium imaging, and opto- and chemogenetic approaches, both ex vivo and in vivo.

    “we validated it in a variety of contexts, including synaptic tagging, calcium imaging, as well as opto- and chemo-genic approaches, both ex vivo and in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Quantification using coronal sections with highest reporter density showed sensitivity for PV was 87% for E2, 85% for E29, and 40% for E22.

    “we could show that the sensitivity for PV (i.e. the percentage of PV cINs that express the viral reporter) appears highest for both E2 and E29 but lower for E22 (capturing 87%, 85 % and 40% of PV cINs, respectively - extended data figure 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    E5 sparsely labeled interneurons across all layers and had notable enrichment for layer 5 pyramidal neurons.

    “the E5 regulatory element, while sparsely labeling interneurons across all layers, had a notable enrichment for pyramidal neurons in layer 5”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    E2 reporter selectivity for PV cINs was greater than 50% with P1 injection, 67% with P7 injection, and over 80% with P10 injection.

    “This revealed that we could obtain greater than 50% selectivity for PV cINs upon injection at P1, increasing to 67% by P7 injection and over 80% after P10 injection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Local injection of rAAV-E2-dTomato at various titers showed higher titers increased reporter expression but did not significantly alter specificity.

    “The results show that while higher titers had increased levels of reporter expression, no significant alteration of specificity was observed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On average 75% of PV cINs expressed the viral reporter, with a maximum of 93%.

    “Conversely, on average 75% of PV cINs expressed the viral reporter, reaching a maximum of 93%”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pyramidal neuron activity near virally labeled PV cINs was interrupted by laser stimulation, and this effect was abolished by picrotoxin.

    “pyramidal neuron activity in the vicinity of virally labeled PV cINs was consistently interrupted by laser stimulation. Notably, this effect was abolished by treatment with picrotoxin”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    E2 driving synaptophysin-tdTomato restricted reporter expression presynaptically to PV cINs, with terminals peri-somatically located onto pyramidal neurons.

    “When E2 was used to drive a synaptophysin-tdTomato fusion gene36, reporter expression was restricted pre-synaptically to PV cINs, with terminals peri-somatically located onto pyramidal neurons”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] Daigle, T. L. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174, 465–480 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The E2 enhancer was useful for investigating PV cIN connectivity, excitability, and for manipulating their activity with chemogenetic and optogenetic effectors.

    “This enhancer also proved useful for investigating various aspects of PV cIN function including connectivity, excitability, and for manipulating their activity with chemogenetic and optogenetic effectors.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using Lhx6-Cre/Intact mice, specificity for PV cINs was 72% with P4-P7 and 78% with P7-P10 time courses.

    “We obtained 72% and 78% specificity for PV cINs with a P4-P7 or a P7-P10 time course, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Adult mice systemically injected with rAAV-E2-dTomato showed detectable reporter expression after one week and high stable expression after three weeks.

    “Adult mice systemically-injected with rAAV-E2-dTomato showed detectable expression of the viral reporter after one week and reached a high and stable level of expression after 3 weeks.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In vivo optogenetic stimulation of PV cINs increased inhibitory interneuron firing rates and silenced excitatory neuronal firing.

    “Reliably, inhibitory interneuron firing rates were increased by laser stimulation, while excitatory neuronal firing was silenced”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A total of 92 genes showed significant enrichment in PV cINs in both Tasic et al. 2016 and Saunders and Macosko et al. 2018 scRNAseq datasets, with average expression at least 2x higher in PV cINs than other interneurons and 5x higher than pyramidal or non-neuronal cells, adjusted p<0.01.

    “A total of 92 genes showed significant enrichment in PV cINs in both datasets (i.e. the average expression of the genes was at least 2 times higher in PV cINs compared to other interneurons meta-clusters and 5 times higher compared to pyramidal or non-neuronal cell types - with an adjusted p-value below 0.01 based on pairwise comparison of each meta-clusters using t-test”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Tasic et al., 2016 source

      Not checked in the free run.

    • cites Saunders and Macosko et al, 2018

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Adult mice injected systemically with rAAV-E2-dTomato had virally labeled neurons in S1 cortex and PFC, some of which showed stereotypical fast-spiking properties.

    “Adult mice were injected systemically with rAAV-E2-dTomato. (a,b) Slice recording of the intrinsic properties of virally labeled neurons in S1 cortex and PFC. The left panels show plots of recorded cells with the indicated intrinsic properties. The blue dots represent cells with stereotypical fast-spiking properties.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Virally labeled chandelier cells were observed in adult mice injected systemically with rAAV-E2-dTomato.

    “Representative image of virally labeled chandelier cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The approach led to identification of 10 enhancers with various degrees of specificity, including three that collectively recapitulate Scn1a expression in cortex.

    “This approach led to the identification of 10 enhancers with various degrees of specificity, including three that collectively recapitulate the expression of Scn1a in the cortex.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Single-cell transcriptomic and epigenetic technologies have accelerated systematic enhancer discovery.

    “systematic enhancer discovery has been accelerated by the recent development of technologies allowing for transcriptomic and epigenetic studies at single-cell resolution6–12.”
    Quote in the paperNot a substantial claim0 of 7 sources checked
    • cites [6] Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 12, 1743–1749 (2016). source

      Not checked in the free run.

    • cites [7] Hrvatin, S. et al. A scalable platform for the development of cell-type-specific viral drivers. eLife 8, e48089 (2019). source

      Not checked in the free run.

    • cites [8] Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M. & Sah, D. W. Y. Gene therapy for neurological disorders: progress and prospects. Nat. Rev. Drug Discov. 9, 641–659 (2018). source

      Not checked in the free run.

    • cites [9] de Leeuw, C. N. et al. rAAV-compatible mini-romoters for restricted expression in the brain and eye. Mol. Brain 9, 52 (2016). source

      Not checked in the free run.

    • cites [10] Jüttner, J. et al. Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans. Nat. Neurosci. 22, 1345–1356 (2019). source

      Not checked in the free run.

    • cites [11] Blankvoort, S., Witter, M. P., Noonan, J., Cotney, J. & Kentros, C. Marked diversity of unique cortical enhancers enables neuron-specific tools by enhancer-driven gene expression. Curr. Biol. 13, 2103–2114 (2018). source

      Not checked in the free run.

    • cites [12] Mehta, P. et al. Functional access to neuron subclasses in rodent and primate forebrain. Cell Rep. 26, 2818–2832 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Short regulatory elements can restrict viral expression to broad neuronal classes in the cerebral cortex or to a subpopulation of a subtype.

    “We and others have identified short regulatory elements capable of restricting viral expression to broad neuronal classes within the cerebral cortex (i.e. pan-pyramidal5 and pan-interneuron6) or a select subpopulation of a particular subtype (i.e subset of Sst within layer five7).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] Bedbrook, C. N., Deverman, B. E. & Gradinaru, V. Viral strategies for targeting the central and peripheral nervous systems. Annu. Rev. Neurosci. 41, 323–348 (2018). source

      Not checked in the free run.

    • cites [6] Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 12, 1743–1749 (2016). source

      Not checked in the free run.

    • cites [7] Hrvatin, S. et al. A scalable platform for the development of cell-type-specific viral drivers. eLife 8, e48089 (2019). source

      Not checked in the free run.

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

    Premisecitation

    Haploinsufficiency or pathogenic variants of Scn1a cause Dravet Syndrome, a common intractable epileptic encephalopathy with early onset seizures.

    “Haploinsufficiency or pathogenic variants of Scn1a cause Dravet Syndrome, a common and intractable form of epileptic encephalopathy characterized by the early onset of seizures17–19.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [17] Cheah, C. S. et al. Specific deletion of Nav1.1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome. Proc. Natl Acad. Sci. USA 109, 14646–14651 (2012). source

      Not checked in the free run.

    • cites [18] Dutton, S. B. et al. Preferential inactivation of Scn1a in parvalbumin interneurons increases seizure susceptibility. Neurobiol. Dis. 49, 211–220 (2013). source

      Not checked in the free run.

    • cites [19] Yu, F. H. et al. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat. Neurosci. 9, 1142–1149 (2006). source

      Not checked in the free run.

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

    Premisecitation

    Enhancer proximity to the transcriptional start site scales directly with expression level.

    “it has been posited that the proximity of the enhancer to the transcriptional start site (TSS) of a gene scales directly to the level of expression20.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Fulco, C. P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    rAAVs are the preferred gene delivery method in the nervous system but have limited genomic payload and are not intrinsically selective for particular neuronal populations.

    “Recombinant adeno-associated viruses (rAAVs) are the method of choice for gene delivery in the nervous system but have a limited genomic payload and are not intrinsically selective for particular neuronal populations5.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] Bedbrook, C. N., Deverman, B. E. & Gradinaru, V. Viral strategies for targeting the central and peripheral nervous systems. Annu. Rev. Neurosci. 41, 323–348 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The findings demonstrate generalizability of the enhancer selection method and utility of functionally tested tools across animal models.

    “Together our findings demonstrate the generalizability of our enhancer selection method, as well as the utility of a variety of functionally-tested tools that can be immediately utilized across animal models.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Scn1a is expressed in distinct neuronal populations and its disruption is associated with severe epilepsy.

    “Scn1a, a gene expressed in distinct neuronal populations and whose disruption is associated with severe epilepsy13.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Griffin, A. et al. Preclinical animal models for dravet syndrome: seizure phenotypes, comorbidities and drug screening. Front. Pharmacol. 9, 573 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    E2 and E29 are capable of targeting the majority of PV cINs, while E22 might be restricted to a subset in non-human primates.

    “These numbers indicate E2 and E29 are capable of targeting the majority PV cINs, while E22 might be restricted to a subset of the population in non-human primates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Despite systemic delivery, the E2 vector can selectively target PV-expressing neurons in various brain regions with insignificant off-target expression outside the CNS.

    “This shows that despite systemic delivery, the E2 vector can be used to selectively target PV-expressing neurons in various brain regions, with insignificant off-target expression outside of the central nervous system.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    scATAC-seq of GFP+ nuclei from Dlx6a-cre::Sun1-eGFP mouse V1 interneurons recovered 3,374 nuclei with average 107,000 reads and 74,100 fragments per cell, 62% of loaded nuclei.

    “we recovered 3,374 nuclei with on average 107,000 reads and 74,100 fragments detected per cell (62% of the total number of nuclei loaded on the 10X chip).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The location of active enhancers within a cell type correlates with chromatin accessibility.

    “the location of active enhancers within a given cell type correlates with chromatin accessibility21,22.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [21] Mo, A. et al. Epigenomic signatures of neuronal diversity in the mammalian brain. Neuron 86, 1369–1384 (2015). source

      Not checked in the free run.

    • cites [22] Luo, C. et al. Robust single-cell DNA methylome profiling with snmC-seq2. Nat. Commun. 9, 3824 (2018). source

      Not checked in the free run.

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

    Premisecitation

    Regulatory elements are subject to positive selection pressure, so sequences with highest conservation across mammals were identified.

    “as regulatory elements are subject to positive selection pressure, we identified sequences showing the highest conservation across mammalian species, including humans25–29.”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [25] Bejerano, G. et al. Ultraconserved elements in the human genome. Science 304, 1321–1325 (2004). source

      Not checked in the free run.

    • cites [26] Dimitrieva, S. & Bucher, P. UCNEbase—a database of ultraconserved non-coding elements and genomic regulatory blocks. Nucleic Acids Res. 41(Database issue), D101–D109 (2013). source

      Not checked in the free run.

    • cites [27] Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014). source

      Not checked in the free run.

    • cites [28] Dousse, A., Junier, T. & Zdobnov, E. M. CEGA—a catalog of conserved elements from genomic alignments. Nucleic Acids Res. 44, 96–100 (2016). source

      Not checked in the free run.

    • cites [29] Dickel, D. E. et al. Ultraconserved enhancers are required for normal development. Cell 172, 491–499 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductreasoning

    Estimating the percentage of target population infected is non-trivial; the only practical solution is saturation injections, which is currently impossible in primates.

    “Estimating this is non-trivial. In fact, the only practical solution to achieving this is by performing viral injections that reach saturation (i.e. all cells are infected by at least one viral particle). This is currently impossible in primates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    PV cINs exert strong inhibitory control over local networks and their dysfunction is implicated in Dravet syndrome, focal epilepsy, ASD, and schizophrenia.

    “These neurons exert a strong level of inhibitory control over local networks and their disfunction has been directly implicated in neurological and neuropsychiatric disorders, including Dravet syndrome, focal epilepsy, ASD and schizophrenia 31–34.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [31] Batista-Brito, R. et al. The cell-intrinsic requirement of Sox6 for cortical interneuron development. Neuron 63, 466–481 (2009). source

      Not checked in the free run.

    • cites [32] Rossignol, E., Kruglikov, I., van den Maagdenberg, A. M., Rudy, B. & Fishell, G. CaV2.1 ablation in cortical interneurons selectively impairs fast-spiking basket cells and causes generalized seizures. Ann. Neurol. 74, 209–222 (2013). source

      Not checked in the free run.

    • cites [33] Gandal, M. J., Nesbitt, A. M., McCurdy, R. M. & Alter, M. D. Measuring the maturity of the fast-spiking interneuron transcriptional program in autism, schizophrenia, and bipolar disorder. PLoS ONE 7, e41215 (2012). source

      Not checked in the free run.

    • cites [34] Barnes, S. A. et al. Disruption of mGluR5 in parvalbumin-positive interneurons induces core features of neurodevelopmental disorders. Mol. Psychiatry 20, 1161–1172 (2015). source

      Not checked in the free run.

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

    Study design and conductreasoning

    The data for systemic viral injection in a marmoset and local injection in macaques came from a single animal and were not repeated, for ethical and economic reasons.

    “For ethical and economic reasons, the data generated for systemic viral injection in a marmoset and for local injection in macaques come from a unique animal and was not repeated.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Estimates of viral specificity in human brain tissues were made within regions of cortex and subiculum where staining density reflected the known distribution and density of PV cells.

    “As such, estimates of viral specificity were made within regions of cortex and subiculum where staining density was reflective of the known distribution and density of these cells.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The more distal an enhancer, the harder it is to predict which gene it regulates.

    “As a consequence, the more distal the enhancer the harder it is to predict what gene it regulates.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Enhancers can regulate gene expression up to 1 Mb away from the transcriptional start site.

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

    Study design and conductcitation

    No statistical methods were used to predetermine sample sizes, but the sample sizes were similar to those in previous publications (6).

    “No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications (6).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 12, 1743–1749 (2016). source

      Not checked in the free run.

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

    Study design and conductnone given

    Only stable electrophysiological recordings with less than 20% change in series resistance were included.

    “Upon break-in, series resistance (typically 15–25 MΩ) was compensated and only stable recordings (<20% change) were included.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The images in figures 2a, 3a, 4a, 4b and extended data figures 1b, 2d, 2f, 3a show representative data obtained from at least three independent injections of the indicated virus in the indicated species.

    “The images presented in the figures 2a, 3a, 4a, 4b and the extended data figures 1b, 2d, 2f, 3a show representative data that were obtained from at least 3 independent injections from the indicated virus in the indicated species.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The specificity of E2 at developmental stages and simplicity of viral injection over transgenic animals provides means to study normal development and role in disease.

    “The specificity of the E2 enhancer at these developmental stages and the simplicity of the viral injection over the use of transgenic animals provides the means to study both their normal development and role in disease.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Equal variance between the two populations was not tested.

    “Equal variance between the two populations was not tested.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The image for the marmoset injection in figure 4a is representative of the specificity and cellular density observed across 12 sections from the same animal.

    “The image for the marmoset injection on figure 4a is representative of the specificity and cellular density observed across 12 sections from the same animal.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Data collection and analysis were not blinded to experimental conditions, but experimenters from different research groups performed the quantification.

    “Data collection and analysis were not performed blind to the conditions of the experiments, but experimenters from different research groups performed the quantification.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    In all cases where the number of biological replicates was not above two, the replicates were highly consistent.

    “In all cases where the number of biological replicates was not above 2, the replicates were highly consistent.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All antibodies used in the study were commercially available and had been validated by their manufacturer.

    “All antibodies used in this study are commercially available and have been validated by the manufacturer.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The three characterized enhancers are located within the first intron, perhaps indicating these SNPs represent mutations affecting Scn1a expression.

    “The three enhancers we characterized were located within this region, perhaps indicating that these SNPs represent mutations affecting the expression of Scn1a.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    All rAAV batches produced had titers between 10^11 and 10^13 viral genomes per ml.

    “All batches produced were in the range of 10E+11 to 10E+13 viral genomes per ml.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Quantifications in this study used at least two independent biological replicates per data point.

    “Quantifications were performed using a minimum of two independent biological replicates.”
    • Also stated in: § Quantifications, statistics and reproducibility. · plain
      “At least two biological replicates per data point were included for all quantification presented in this study.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Sections where PV-IHC was not reflective of the known distribution of PV cells were excluded from the study.

    “Sections where the PV-IHC was not reflective of the know distribution of these cells were excluded from the study.”
    Quote in the paperNot a substantial claim
47 of 47

  • The paper relies on this without support.

    The search space for enhancer selection is very large and prior success has been limited.

    “Despite these advances, the search space for enhancer selection remains enormous and to date success has been limited.”

  • The paper relies on this without support.

    PV cINs constitute 40% of all cortical interneurons.

    “fast-spiking neurons that constitute 40% of all cortical interneurons.”

  • The paper relies on this without support.

    The proportion of a given cellular population infected by a given rAAV depends on the mode of administration and the absolute number of viral particles injected.

    “the proportion of a given cellular population infected by a given rAAV depends on the mode of administration and the absolute number of viral particles injected.”

  • The paper relies on this without support.

    Diffusion of AAVs in parenchymal tissue upon local injection is limited.

    “As the diffusion of AAVs in parenchymal tissue upon local injection is limited”

  • The paper relies on this without support.

    Enhancers can regulate gene expression up to 1 Mb away from the transcriptional start site.

    “it has been shown that enhancers can regulate gene expression up to 1Mb away from the transcriptional start site.”

  • The authors state this as an assumption.

    The functional elements responsible for the specificity of the selected enhancers are nested within highly conserved sub-regions of the enhancer.

    “These results corroborate the initial assumption that the functional elements responsible for the specificity of the selected enhancers are nested within highly conserved sub-regions of the enhancer.”

  • The paper relies on this without support.

    None of the macaque local injections achieved 100% infectivity.

    “we strongly suspect that in none of these cases 100% infectivity was achieved.”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Molecular classification of cell types enables genetic access to specific cell types using transgenic mice and enhancer-based viral vectors.

    “Finally, molecular classification of cell types enables genetic access to specific cell types using transgenic mice25–27 and, more recently, enhancer-based viral vectors28–32.”

    From A multimodal cell census and atlas of the mammalian primary motor cortex (2021) · cites this paper as [29]

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Vormstein-Schneider, D., Lin, J. D., Pelkey, K. A., Chittajallu, R., Guo, B., Arias-Garcia, M. A., Allaway, K., Sakopoulos, S., Schneider, G., Stevenson, O., Vergara, J., Sharma, J., Zhang, Q., Franken, T. P., Smith, J., Ibrahim, L. A., Mastro, K. J., Sabri, E., Huang, S., Favuzzi, E., Burbridge, T., Xu, Q., Guo, L., Vogel, I., Sanchez, V., Saldi, G. A., Gorissen, B. L., Yuan, X., Zaghloul, K. A., Devinsky, O., Sabatini, B. L., Batista-Brito, R., Reynolds, J., Feng, G., Fu, Z., McBain, C. J., Fishell, G., & Dimidschstein, J.
Affiliations
  • Broad Institute (US)
  • National Institutes of Health (US)
  • McGovern Institute for Brain Research (US)
  • Salk Institute for Biological Studies (US)
  • Albert Einstein College of Medicine (US)
  • New York University Abu Dhabi (AE)
  • New York University (US)
Corresponding author
Dimidschstein, Jordane
ORCID
Venue
Nature Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2020-08-17
Volume
23
Issue
12
Pages
1629-1636
ISSN
  • 1097-6256 (ISSN-L)
  • 1546-1726
DOI
10.1038/s41593-020-0692-9
PMC
PMC8015416
OpenAlex
W3048977247
Cited by
235
References
60
Citations by year
2026: 28 · 2025: 46 · 2024: 49 · 2023: 40 · 2022: 32 · 2021: 32 · 2020: 6 · 2019: 1
FWCI
16.10
Type
journal article
Language
en
Update notices
correction (2022-08-09)
Open access
Licence
Locations
  • Nature Neuroscience (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
Funders
  • U.S. Department of Health & Human Services | NIH | Center for Information Technology (K99NS106528)
  • Simons Foundation (566615)
  • U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (R01-MH111529, UG3MH120096, MH071679)
  • U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NS081297, NS074972)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
cell type-specific gene expression, adeno-associated virus vectors, enhancer identification, parvalbumin interneurons, vasoactive intestinal peptide interneurons, SCN1A, cerebral cortex, neuronal cell types, gene regulatory elements
MeSH terms
Dependovirus, Animals, Callithrix, Cerebral Cortex, Female, Genetic Vectors, Humans, Interneurons, Macaca mulatta, Mice, Inbred C57BL, Neurons, Parvalbumins, Species Specificity, Vasoactive Intestinal Peptide, Rats, Sprague-Dawley, Mice, Rats, NAV1.1 Voltage-Gated Sodium Channel

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review