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Cited by 267Single-cell and spatial transcriptomicsNeuroscience and Neuropharmacology ResearchNeurotransmitter Receptor Influence on Behavior

Molecular and anatomical organization of the dorsal raphe nucleus

Open access
Huang, Kee Wui, Ochandarena, Nicole E, Philson, Adrienne C, Hyun, Minsuk, Birnbaum, Jaclyn E, Cicconet, Marcelo, Sabatini, Bernardo L·2019·eLife
DOI 10.7554/elife.46464PMC PMC6726424

Published version of a preprint

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Abstract

The dorsal raphe nucleus (DRN) is an important source of neuromodulators and has been implicated in a wide variety of behavioral and neurological disorders. The DRN is subdivided into distinct anatomical subregions comprised of multiple cell types, and its complex cellular organization has impeded efforts to investigate the distinct circuit and behavioral functions of its subdomains. Here we used single-cell RNA sequencing, in situ hybridization, anatomical tracing, and spatial correlation analysis to map the transcriptional and spatial profiles of cells from the mouse DRN. Our analysis of 39,411 single-cell transcriptomes revealed at least 18 distinct neuron subtypes and 5 serotonergic neuron subtypes with distinct molecular and anatomical properties, including a serotonergic neuron subtype that preferentially innervates the basal ganglia. Our study lays out the molecular organization of distinct serotonergic and non-serotonergic subsystems, and will facilitate the design of strategies for further dissection of the DRN and its diverse functions.

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Written from the paper’s full text.

TL;DR

This study maps the mouse dorsal raphe nucleus (DRN) using single-cell transcriptomics, in situ hybridization, and anatomical tracing, identifying at least 18 neuronal subtypes and 5 serotonergic subtypes. It finds that molecularly defined 5-HT subtypes occupy overlapping spatial domains and that a Pdyn-enriched subtype preferentially innervates basal ganglia circuits.

Key findings

  • scRNA-seq of 39,411 cells from 8 mice yielded 17 major clusters; 2,041 neurons resolved into 18 distinct neuronal clusters aggregated into 5 major classes, including 5 5-HT neuron subtypes, 3 DA subtypes, 5 glutamatergic subtypes, 3 GABAergic subtypes, and 1 GABA/Glu co-expressing cluster.
  • The 5 5-HT subtypes were 5-HT-I (n = 178), 5-HT-II (n = 186), 5-HT-III (n = 125), 5-HT-IV (n = 208), and 5-HT-V (n = 7); 333 differentially expressed genes separated them at a 5% false discovery rate, and their inferred distributions were distinct but overlapping, with 5-HT-V restricted to the posterior B6 tail and 5-HT-IV enriched ventrally.
  • Striatum-projecting 5-HT neurons were transcriptionally heterogeneous: 52% (39 of 75) expressed Pdyn, 46% (49 of 106) expressed Cbln2, 57% (70 of 123) expressed Slc17a8, and 4% (4 of 102) expressed Trh, supporting at least 2 molecular subtypes within this projection-defined population.
  • Pdyn+;Pet1+ (5-HT-III) axons densely innervated basal ganglia output nuclei including ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata, but not dLGN or hypothalamic nuclei, despite dense DRN Pet1+ input to those regions.

Methods

  • Droplet-based inDrop scRNA-seq was performed on microdissected DRN and surrounding tissue from acute coronal sections of 8 C57BL/6J mice (4 male, 4 female, age 8–10 weeks); after filtering, 39,411 cells were clustered using Seurat with canonical correlation analysis and SNN-based clustering, and differential expression was tested with MAST.
  • Spatial distributions were inferred by registering Allen Brain Atlas coronal in situ hybridization images to the Common Coordinate Framework, binning expression into 50 µm × 50 µm voxels, and calculating Pearson correlations between subtype gene-expression profiles and spatial bins; multiplexed RNAscope FISH validated subtype-enriched markers.
  • Anatomical tracing used retrograde rabies virus injections into basal ganglia, cortical, and sensory targets with manual registration of labeled cells, plus intersectional anterograde tracing in Pet1-Flpe;Pdyn-IRES-Cre;LSL-FSF-ReaChR:mCitrine mice.

Limitations

  • Stated: droplet-based scRNA-seq has low transcript capture efficiency and low reads per cell, with drop-out events making low-copy transcripts difficult to detect; the study therefore does not present a definitive catalogue of all DRN neuron subtypes and may not resolve finer heterogeneity such as 5-HT-I versus 5-HT-II.
  • Stated: missing data and tissue-processing artifacts in the Allen Brain Atlas dataset prevented analysis of the full 333 DE genes; spatial correlation analysis used 112 DE genes.
  • Evident: the 5-HT-V subtype is based on only 7 cells; spatial distributions are inferred by correlation with ISH rather than direct spatial transcriptomics; projection mapping relied on manual registration, and male and female data were pooled after no major transcriptional sex differences were observed.

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Registry

Crossref · 10.7554/elife.46464

Cites

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Claims the paper makes

0 of 52 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    DRN 5-HT neurons (n = 704 cells) separated into five distinct subtypes.

    “DRN 5-HT neurons (n = 704 cells) separated into five distinct subtypes.”
    Quote in the paperSubstantial claim
  • Findingown data

    The majority of cells in the dataset were non-neuronal cells that included astrocytes, oligodendrocyte precursor cells, differentiating and mature oligodendrocytes, ependymal cells, lymphocytes, microglia, perivascular macrophages, fibroblast-like or mesenchymal cells, endothelial cells, pericytes, and smooth muscle cells.

    “The majority of cells in the dataset were non-neuronal cells that included astrocytes, oligodendrocyte precursor cells (or polydendrocytes), differentiating and mature oligodendrocytes, ependymal cells of the cerebral aqueduct, lymphocytes, microglia, perivascular macrophages (pvMΦs), fibroblast-like or mesenchymal cells, endothelial cells, pericytes, and smooth muscle cells.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Molecularly defined subtypes of DRN 5-HT neurons are found in overlapping but different sets of anatomical subregions that do not match the spatial distribution of projection-defined subpopulations.

    “Molecularly defined subtypes of DRN 5-HT neurons are found in overlapping but different sets of anatomical subregions that do not match the spatial distribution of projection-defined subpopulations.”
    Quote in the paperSubstantial claim
  • Findingcitation

    Expression of Htr1b in perivascular macrophages is unique to the DRN and its close surroundings.

    “Examination of Htr1b expression in cortex, striatum, and ventral midbrain suggests that expression of this receptor in pvMΦs is unique to the DRN and its close surroundings (Hrvatin et al., 2018; Saunders et al., 2018; Zeisel et al., 2018).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Hrvatin et al., 2018 source

      Not checked in the free run.

    • cites Saunders et al., 2018 source

      Not checked in the free run.

    • cites Zeisel et al., 2018 source

      Not checked in the free run.

  • Findingown data

    The 5 major neuronal classes in decreasing order of abundance are 5-HT, dopaminergic, GABAergic, glutamatergic, and peptidergic neurons.

    “The 5 major neuronal classes (in decreasing order of abundance) are 5-HT, dopaminergic (DA), GABAergic, glutamatergic, and peptidergic neurons.”
    Quote in the paperSubstantial claim
  • May combine two claims. 55
  • Classified as probably not a substantial claim. 22

  • May combine two claims.

    Findingown data

    The authors used scRNA-seq to survey cell types in and around the DRN and identified at least 5 distinct subtypes of 5-HT neurons.

    “We used high-throughput single-cell RNA sequencing (scRNA-seq) to survey the cell types in and around the DRN and identified at least 5 distinct subtypes of 5-HT neurons, in addition to uncovering organizational principles for both neuronal and non-neuronal cell types based on gene expression profiles.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The processed dataset contained 39,411 cells from eight mice (4 male and 4 female, age range 8-10 weeks) with a median UMIFM count of 2,333 counts per cell and median gene detection rate of 1,034 genes per cell.

    “The processed dataset contained 39,411 cells from eight mice (4 male and 4 female, age range 8–10 weeks) with a median UMIFM count of 2,333 counts per cell (min. = 548; max. = 17,976; IQR = 1,903) and median gene detection rate of 1,034 genes per cell (min. = 216; max. = 5,765; IQR = 693).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The Viplow DA-I subtype showed more similarities in gene expression with the GABA/Glu neurons of CLi compared to the Viphigh DA-III subtype, indicating a corresponding topographical organization of these DA neuron subtypes along the anterior-posterior axis from the CLi to DRN.

    “The Viplow DA-I subtype showed more similarities in gene expression with the GABA/Glu neurons of CLi as compared to the Viphigh DA-III subtype, indicating a corresponding topographical organization of these DA neuron subtypes along the anterior-posterior axis from the CLi to DRN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DRN DA neurons lacking expression of nicotinic receptors separated into two subtypes, DA-II and DA-III, that differed in expression of genes related to neurotransmission including peptides such as Npw and the vesicular glutamate transporter Slc17a6, suggesting that DRN DA neurons are functionally heterogeneous.

    “The DRN DA neurons lacking expression of nicotinic receptors separated into two subtypes, DA-II and DA-III, that differed in expression of genes related to neurotransmission including peptides, such as Npw, and the vesicular glutamate transporter Slc17a6, suggesting that DRN DA neurons are functionally heterogeneous.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Subclustering conservatively separated the 2,041 neurons into 18 distinct clusters, which were aggregated into 5 major groups based on similarities in expression of genes, most notably those involved in synthesis and release of different neurotransmitters.

    “Subclustering conservatively separated the 2,041 neurons into 18 distinct clusters, which we aggregated into 5 major groups based on similarities in their expression of genes, most notably those involved in the synthesis and release of different neurotransmitters (Figure 3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Vip was found in all 3 DA neuron subtypes, as previously reported for DRN and CLi DA neurons, but Vip expression varied between the 3 subtypes.

    “Although Vip was found in all 3 DA neuron subtypes, as previously reported for DRN and CLi DA neurons (Dougalis et al., 2012; Poulin et al., 2018; Poulin et al., 2014), Vip expression varied between the 3 subtypes.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Dougalis et al., 2012 source

      Not checked in the free run.

    • cites Poulin et al., 2018 source

      Not checked in the free run.

    • cites Poulin et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Peptidergic neurons were depleted of genes for synthesis and release of small molecule neurotransmitters but highly enriched for genes encoding neuropeptides such as Cck, Cartpt, Ucn, and Postn.

    “Peptidergic neurons were depleted of genes for the synthesis and release of small molecule neurotransmitters (e.g. Slc6a4, Slc6a3, Slc32a1, Slc17a6, Slc17a8), but were highly enriched for the genes encoding neuropeptides such as Cck, Cartpt, Ucn, and Postn.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All major neuronal cell classes, with the exception of peptidergic neurons, were comprised of several subclusters that each represent a distinct neuron subtype; there were 5 5-HT neuron subtypes, 3 DA neuron subtypes, 5 glutamatergic neuron subtypes, 3 GABAergic neuron subtypes, and 1 cluster co-expressing markers for both GABAergic and glutamatergic neurotransmission.

    “All of the major neuronal cell classes, with the exception of the peptidergic neurons, were comprised of several subclusters that each represent a distinct neuron subtype (Figure 3B); we found 5 5-HT neuron subtypes, 3 DA neuron subtypes, 5 glutamatergic neuron subtypes, 3 GABAergic neuron subtypes, and 1 cluster co-expressing markers for both GABAergic and glutamatergic neurotransmission (GABA/G”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ependymal cells shared expression of the histamine synthesis gene Hdc, but were divided into distinct subtypes that differed in expression of cell adhesion and matrix-associated genes and signaling peptides such as Pdyn.

    “Ependymal cells shared expression of the histamine synthesis gene Hdc, but were further divided into distinct subtypes that differed in expression of cell adhesion and matrix-associated genes as well as signaling peptides such as Pdyn (Figure 1—figure supplement 2A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Molecularly distinct DRN 5-HT neuron subtypes are spatially segregated into distinct anatomical subdomains and innervate different but overlapping targets.

    “We present a physical map combining both transcriptional and anatomical features and show that molecularly distinct DRN 5-HT neuron subtypes are spatially segregated into distinct anatomical subdomains and innervate different but overlapping targets.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

  • May combine two claims.

    Findingown data

    5-HT subtypes have distinct spatial locations but also considerable overlaps in their distributions, particularly within the dorsomedial subregion in the anterior DRN that contains many basal ganglia-projecting 5-HT neurons.

    “Although we show that 5-HT subtypes have distinct spatial locations, we also found considerable overlaps in their distributions, particularly within the dorosmedial subregion in the anterior DRN that contains many basal ganglia-projecting 5-HT neurons.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

  • May combine two claims.

    Findingown data

    Although the 5-HT-III/IV group expressed genes for glutamatergic transmission, subtype 5-HT-IV expressed higher levels of the vesicular glutamate transporter Slc17a8 compared to 5-HT-III.

    “Although the 5-HT-III/IV group expressed genes for glutamatergic transmission, subtype 5-HT-IV expressed higher levels of the vesicular glutamate transporter Slc17a8 compared to 5-HT-III.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Most projection-defined subpopulations were located either ipsilateral to the injection site or along the midline, with the exception of habenula-projecting neurons, which were found in both ipsilateral and contralateral lateral wings of the DRN.

    “Most projection-defined subpopulations were located either ipsilateral to the injection site or along the midline, with the exception of the habenula-projecting neurons which were found in both ipsilateral and contralateral lateral wings of the DRN (Figure 6—figure supplement 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Subtypes 5-HT-I and 5-HT-II were intermingled in the dorsolateral DRN, which is enriched in Trh, a 5-HT-I enriched gene.

    “Subtypes 5-HT-I and 5-HT-II were intermingled in this region, which is enriched in Trh, a 5-HT-I enriched gene.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

  • May combine two claims.

    Findingown data

    Sensory structures such as dLGN and limbic structures such as hypothalamus were not innervated by Pdyn+;Pet1+ neurons, despite receiving dense input from DRN Pet1+ neurons.

    “In contrast, sensory structures, such as the dLGN (Figure 8E), and limbic structures, such as the hypothalamus (Figure 8F) were not innervated by Pdyn+;Pet1+ neurons, despite receiving dense input from DRN Pet1+ neurons.”
    • Also stated in: § Innervation of basal ganglia by a Pdyn-expressing 5-HT neuron subtype · plain
      “Pdyn+;Pet1+ axons were not found in structures that were functionally distant from basal ganglia circuits, such as dLGN (E2) and hypothalamic nuclei (F2), although these structures received dense inputs from DRN Pet1+ neurons (E1 and F1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was a crude separation of cortical- and subcortical-projecting subpopulations along the dorsal-ventral axis, but considerable overlaps between their distributions prevented the delineation of a clear boundary between cortical- and subcortical-projecting groups.

    “Although there was a crude separation of cortical- and subcortical-projecting subpopulations along the dorsal-ventral axis, there were considerable overlaps between their distributions, preventing the delineation of a clear boundary between cortical- and subcortical-projecting groups.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ISHs for genes enriched in the four subtypes revealed expression patterns consistent with the hypothesis that the two groups 5-HT-I/II and 5-HT-III/IV largely separated along the medial-lateral axis, with the added separation of 5-HT-IV from the other three subtypes along the dorsal-ventral axis.

    “ISHs for genes enriched in these four subtypes revealed expression patterns that were consistent with the hypothesis that the two groups 5-HT-I/II and 5-HT-III/IV largely separated along the medial-lateral axis, with the added separation of 5-HT-IV from the other three subtypes along the dorsal-ventral axis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    dLGN-projecting neurons were mostly located in dorsolateral subregions at more posterior levels, whereas Str-projecting neurons were mostly in the dorsomedial subregion at more anterior levels.

    “dLGN-projecting neurons were mostly located in dorsolateral subregions at more posterior levels, whereas Str-projecting neurons were mostly in the dorsomedial subregion at more anterior levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most 5-HT neuron subtypes are not confined to a specific DRN subregion, with the exception of subtype 5-HT-V.

    “Most subtypes were not confined to a specific DRN subregion, with the exception of subtype 5-HT-V (Figure 5, purple) that showed a tight distribution within the dorsomedial posterior B6 tail of the DRN.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Anatomical features are lost during dissociation of tissue for droplet-based scRNA-seq techniques.
    • Rests on an assumption: ISH against a small set of marker genes expressed exclusively in one cell type can be used to localize the putative location of cell types in complex tissue.

  • May combine two claims.

    Findingown data

    Within the cortical-projecting group, M1-projecting neurons were relatively confined to the anterior half of the DRN, whereas mPFC-projecting neurons were found in both anterior and posterior halves.

    “Within the cortical-projecting group, frontal- and prefrontal-projecting subpopulations were differentially distributed along the anterior-posterior axis of the DRN – M1-projecting neurons were relatively confined to the anterior half of the DRN, whereas mPFC-projecting neurons were found in both anterior and posterior halves.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither Str-projecting nor M1-projecting subpopulations were fully contained within the distribution of a single 5-HT neuron subtype.

    “However, neither Str-projecting nor M1-projecting subpopulations were fully contained within the distribution of a single 5-HT neuron subtype.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Subtypes 5-HT-III and 5-HT-IV showed higher correlations with voxels along the midline but were separated along the dorsal-ventral axis, with the Slc17a8-enriched 5-HT-IV subtype occupying most of the ventral DRN.

    “Both subtypes 5-HT-III and 5-HT-IV (Figure 5, blue) showed higher correlations with voxels along the midline, but were separated along the dorsal-ventral axis with the Slc17a8-enriched 5-HT-IV subtype occupying most of the ventral DRN.”
    • Also stated in: § Spatial distribution of 5-HT neuron subtypes in the DRN · plain
      “5-HT-IV was enriched in the ventral regions of the DRN”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Anatomical features are lost during dissociation of tissue for droplet-based scRNA-seq techniques.
    • Rests on an assumption: ISH against a small set of marker genes expressed exclusively in one cell type can be used to localize the putative location of cell types in complex tissue.

  • May combine two claims.

    Findingown data

    The difference between Str- and NAc-projecting distributions was largely driven by the presence of NAc-projecting neurons in posterior parts of the DRN where mPFC-projecting neurons were also located, whereas Str-projecting DRN neurons were relatively confined to the anterior half of the DRN.

    “This difference was largely driven by the presence of NAc-projecting neurons in posterior parts of the DRN where mPFC-projecting neurons were also located, whereas Str-projecting DRN neurons were relatively confined to the anterior half of the DRN (Figure 6—figure supplement 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Pdyn-expressing 5-HT-III subtype was localized to the dorsomedial subregion, while subtypes 5-HT-I and 5-HT-II were also found in this subregion in the anterior third of the DRN despite being distributed to dorsolateral subregions in more posterior sections.

    “While the Pdyn-expressing 5-HT-III subtype was localized to the dorsomedial subregion, subtypes 5-HT-I and 5-HT-II were also found in this subregion in the anterior third of the DRN despite being distributed to dorsolateral subregions in more posterior sections.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

  • May combine two claims.

    Findingown data

    The distribution of the Pdyn-enriched subtype 5-HT-III overlaps with the dorsomedial subregion and shows the highest correlations with voxels in the dorsomedial part of the DRN in its anterior half.

    “This overlaps with the distribution of the Pdyn-enriched subtype 5-HT-III (Figure 5, green), which showed the highest correlations with voxels in the dorsomedial part of the DRN in its anterior half.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Anatomical features are lost during dissociation of tissue for droplet-based scRNA-seq techniques.
    • Rests on an assumption: ISH against a small set of marker genes expressed exclusively in one cell type can be used to localize the putative location of cell types in complex tissue.

  • May combine two claims.

    Findingown data

    The smallest subtype cluster 5-HT-V (n = 7 cells) was the most transcriptionally distinct, was marked by expression of genes such as Met, P2ry1, and Trpc3, and lacked expression of genes for GABA co-transmission.

    “The cluster dendrogram for 5-HT neuron subtypes showed that the smallest subtype cluster 5-HT-V (n = 7 cells) was the most transcriptionally distinct, was marked by expression of genes such as Met, P2ry1, and Trpc3 (Figure 4—figure supplement 3C), and lacked expression of genes for GABA co-transmission.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Str-projecting neurons in the ventrolateral DRN fell within the distribution of subtype 5-HT-IV but not of subtype 5-HT-III.

    “Str-projecting neurons in the ventrolateral DRN (marked cyan in Figure 6D,F,G), where neurons innervating both Str and M1 are located, fell within the distribution of subtype 5-HT-IV but not of subtype 5-HT-III.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The remaining four 5-HT neuron subtypes are found within the larger anterior B7 part of the DRN and are segregated along both the medial-lateral axis and the dorsal-ventral axis.

    “The remaining four 5-HT neuron subtypes were found within the larger anterior B7 part of the DRN, and were segregated along both the medial-lateral axis and the dorsal-ventral axis.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Anatomical features are lost during dissociation of tissue for droplet-based scRNA-seq techniques.
    • Rests on an assumption: ISH against a small set of marker genes expressed exclusively in one cell type can be used to localize the putative location of cell types in complex tissue.

  • May combine two claims.

    Findingown data

    Of the five subtypes, 5-HT-V was the most transcriptionally distinct and spatially restricted, and was found in a tight dorsomedial cluster in the posterior B6 tail of the DRN.

    “Of the five subtypes, the 5-HT-V was the most transcriptionally distinct and spatially restricted, and was found in a tight dorsomedial cluster in the posterior B6 tail of the DRN.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

  • May combine two claims.

    Findingown data

    The distributions of subpopulations innervating related structures within the basal ganglia were more similar, with GPe- and Str-projecting subpopulations most similar, while Str- and NAc-projecting subpopulations showed a larger difference.

    “The distributions of subpopulations innervating related structures within the basal ganglia were more similar (Figure 6E) and, while the subpopulations innervating the external globus pallidus (GPe) and Str were most similar, the subpopulations innervating the Str and nucleus accumbens (NAc, also called the ventral striatum) showed a larger difference.”
    • Also stated in: § 5-HT neuron subtype distributions coincide with different projection-defined subpopulations · plain
      “Correlations between the Str-projecting subpopulation were higher with other subpopulations that also innervate nuclei of the basal ganglia, compared with subpopulations innervating functionally distinct targets such as M1 or dLGN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The expression of several genes in 5-HT-III appeared intermediate between 5-HT-I/II and 5-HT-IV; several genes enriched in 5-HT-III over 5-HT-IV were also expressed in subtypes 5-HT-I or 5-HT-II, including Pax5, Nxph4, Gad1, and Gad2.

    “However, the expression of several genes in 5-HT-III appeared to be intermediate between 5-HT-I/II and 5-HT-IV – while the 5-HT-III/IV group showed reduced expression of genes for GABAergic neurotransmission relative to subtypes 5-HT-I/II, several genes that were enriched in 5-HT-III over 5-HT-IV were also expressed in subtypes 5-HT-I or 5-HT-II, including Pax5, Nxph4, Gad1, and Gad2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Most subcortical-projecting neurons were located in the dorsal DRN, whereas most cortical-projecting neurons were located in the ventral DRN.

    “Consistent with previous reports (Ren et al., 2018; Waterhouse et al., 1986), we found that most subcortical-projecting neurons were located in the dorsal DRN, whereas most cortical-projecting neurons were located in the ventral DRN.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Ren et al., 2018 source

      Not checked in the free run.

    • cites Waterhouse et al., 1986 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    5-HT-IV subtype neurons, located in ventral subregions further from the aqueduct, do not express detectable levels of Hrh1 and express Hrh3 at very low levels.

    “In contrast, 5-HT-IV subtype neurons, which are in ventral subregions further from the aqueduct, do not express detectable levels of Hrh1 and express Hrh3 at very low levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Histamine receptors Hrh1 and Hrh3 are expressed in 5-HT subtypes 5-HT-I, 5-HT-II, and 5-HT-III, which are located in dorsal DRN subregions in close proximity to the cerebral aqueduct.

    “Notably, the histamine receptors Hrh1 and Hrh3 are expressed in 5-HT subtypes 5-HT-I, 5-HT-II, and 5-HT-III, which are all located in dorsal DRN subregions in close proximity to the cerebral aqueduct.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Changes in DRN 5-HT neuron function have been implicated in various neuropsychiatric diseases, including major depressive disorder, bipolar disorder, schizophrenia, obsessive-compulsive disorder, and movement disorders such as Parkinson's Disease.

    “Changes in DRN 5-HT neuron function have been implicated in various neuropsychiatric diseases, including major depressive disorder, bipolar disorder, schizophrenia, and obsessive-compulsive disorder, as well as movement disorders such as Parkinson’s Disease (Huot et al., 2011; Mahmood and Silverstone, 2001; Politis and Niccolini, 2015; Vaswani et al., 2003).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Huot et al., 2011 source

      Not checked in the free run.

    • cites Mahmood and Silverstone, 2001

      Not checked in the free run.

    • cites Politis and Niccolini, 2015

      Not checked in the free run.

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

    Premisecitation

    The dorsal raphe nucleus (DRN) is a major source of neuromodulators in the central nervous system and is the largest serotonergic nucleus, containing approximately one third of all serotonergic neurons in the brain.

    “The dorsal raphe nucleus (DRN) is a major source of neuromodulators in the central nervous system, and is the largest of the serotonergic nuclei, containing approximately a third of all serotonergic neurons (5-HT neurons) in the brain (Hornung, 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hornung, 2010 source

      Not checked in the free run.

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

    Premisecitation

    DRN 5-HT neurons share a common developmental lineage.

    “Although DRN 5-HT neurons share a common developmental lineage (Deneris and Gaspar, 2018; Jensen et al., 2008), DRN 5-HT neurons in different subregions express different G protein-coupled receptors and vesicular transporters, suggesting spatial clustering of functionally specialized neurons (Calizo et al., 2011; Fernandez et al., 2016; Lowry et al., 2000; Templin et al., 2012).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Deneris and Gaspar, 2018

      Not checked in the free run.

    • cites Jensen et al., 2008 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    These drugs affect multiple 5-HT pathways with distinct and contrasting effects on behavior.

    “This suggests these drugs affect multiple 5-HT pathways with distinct and contrasting effects on behavior and highlights the need for new approaches to target specific 5-HT neurons and their outputs (Marcinkiewcz et al., 2016; Urban et al., 2016).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Marcinkiewcz et al., 2016 source

      Not checked in the free run.

    • cites Urban et al., 2016 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The heterogeneity of 5-HT neurons suggests that they may be organized into distinct subsystems.

    “The heterogeneity of 5-HT neurons suggests that they may be organized into distinct subsystems.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Anatomical subregions fail to capture many features of 5-HT neurons, as most of the variation does not show subregional specificity.
    • Rests on an assumption: DRN 5-HT neurons vary in their axonal projection patterns, and the spatial distributions of projection-defined subpopulations are broad and do not map cleanly to DRN subregions.

  • May combine two claims.

    Study design and conductown data

    Cells from male and female mice were pooled for analysis as no major transcriptional sex differences were observed.

    “Cells from male and female mice were pooled for analysis as no major transcriptional sex differences were observed (Figure 1—figure supplement 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    5-HT can be released somatodendritically and from 5-HT axon terminals in the DRN.

    “As 5-HT can be released somatodendritically and from 5-HT axon terminals in the DRN (Colgan et al., 2012; de Kock et al., 2006; Kaushalya et al., 2008), we examined the expression patterns of 5-HT receptors in the dataset to infer local 5-HT signaling.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Colgan et al., 2012 source

      Not checked in the free run.

    • cites de Kock et al., 2006 source

      Not checked in the free run.

    • cites Kaushalya et al., 2008 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductcitation

    Inspection of rhombomere-specific marker gene expression in the 5-HT neuron cluster showed a lack of markers for R2 (Hoxa2) and R3/5 (Egr2) in contrast to expression of R1 markers (En1 and En2), indicating that the dissection excluded most of the median raphe nucleus.

    “Inspection of rhombomere-specific marker gene expression in the 5-HT neuron cluster showed a lack of markers for R2 (Hoxa2) and R3/5 (Egr2) derived neurons in contrast to the expression of R1 markers (En1 and En2), indicating that our dissection, as intended, excluded most of the median raphe nucleus (Okaty et al., 2015).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Okaty et al., 2015 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Functional differences exist between projection-defined subpopulations of DRN 5-HT neurons.

    “Functional differences exist between projection-defined subpopulations of DRN 5-HT neurons (Ren et al., 2018), but it is unknown if these subpopulations map onto molecularly defined subtypes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ren et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductcitation

    Inspection of ISHs from the Allen Brain Atlas indicated that peptidergic neurons were located in the Edinger-Westphal nucleus, adjacent to the DRN, confirming that the dissection region spanned most of the DRN along the anterior-posterior axis.

    “Inspection of ISHs from the Allen Brain Atlas (Lein et al., 2007) indicated that these neurons were located in the Edinger-Westphal nucleus, which is adjacent to the DRN, confirming that our dissection region spanned most of the DRN along the anterior-posterior axis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lein et al., 2007 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The localization of 5-HT-V to the dorsomedial posterior B6 tail of the DRN suggests that this subtype may innervate ventricular and subventricular regions and the choroid plexus and primarily target non-neuronal cells.

    “The localization of this subtype to the dorsomedial posterior B6 tail of the DRN also suggests that this subtype may innervate ventricular and subventriclar regions and the choroid plexus and primarily target non-neuronal cells (Muzerelle et al., 2016).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Muzerelle et al., 2016 source

      Not checked in the free run.

    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

  • May combine two claims.

    Interpretationown data

    Subtypes 5-HT-I and 5-HT-II occupied the dorsolateral subregions in the posterior half of the DRN and are likely spatially intermingled.

    “Subtypes 5-HT-I and 5-HT-II showed the greatest similarities in spatial distribution – both subtypes occupied the dorsolateral subregions in the posterior half of the DRN where they are likely spatially intermingled”
    • Also stated in: § Spatial distribution of 5-HT neuron subtypes in the DRN · hedged
      “Correlation maps for subtypes 5-HT-I and 5-HT-II suggest that they are largely intermingled in the dorsolateral DRN”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Anatomical features are lost during dissociation of tissue for droplet-based scRNA-seq techniques.
    • Rests on an assumption: ISH against a small set of marker genes expressed exclusively in one cell type can be used to localize the putative location of cell types in complex tissue.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Differential expression of many axon guidance and cell adhesion molecules between 5-HT neuron subtypes predicts that 5-HT subtypes innervate different brain regions.

    “Differential expression of many axon guidance and cell adhesion molecules between 5-HT neuron subtypes predicts that 5-HT subtypes innervate different brain regions.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

  • May combine two claims.

    Interpretationreasoning

    Str is a target of multiple molecular subtypes of 5-HT neurons, and some structures are innervated by multiple 5-HT neuron subtypes due to overlaps in the axonal projection fields of different subtypes.

    “This suggests that Str is a target of multiple molecular subtypes of 5-HT neurons, and supports the model that some structures are innervated by multiple 5-HT neuron subtypes due to overlaps in the axonal projection fields of different subtypes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Missing data in the Allen Brain Atlas dataset and artifacts introduced in tissue processing hampered alignment to the Common Coordinate Framework and prevented analysis of the full 333 DE genes.

    “Missing data in the Allen Brain Atlas dataset as well as artifacts apparently introduced in tissue processing that hampered alignment to the Common Coordinate Framework prevented us from analyzing the full 333 DE genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The Str-projecting DRN 5-HT neuron subpopulation is molecularly heterogeneous and is comprised of at least 2 transcriptionally distinct 5-HT neuron subtypes.

    “Therefore, our results support the hypothesis that the Str-projecting DRN 5-HT neuron subpopulation is molecularly heterogeneous, and is comprised of at least 2 transcriptionally distinct 5-HT neuron subtypes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The Pdyn+ subtype 5-HT-III neurons broadly but selectively innervate structures that are either a part of or functionally linked to the basal ganglia, and are unlikely to be involved in sensory modulation.

    “These results therefore demonstrate that the Pdyn+ subtype 5-HT-III neurons broadly but selectively innervate structures that are either a part of or functionally linked to the basal ganglia, and are unlikely to be involved in sensory modulation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Cortical-projecting subpopulations of DRN 5-HT neurons express Slc17a8.

    “It has been reported that cortical-projecting subpopulations of DRN 5-HT neurons express Slc17a8, in agreement with the extensive overlap between the spatial distribution of cortical-projecting neurons and Slc17a8 expression (Ren et al., 2018).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ren et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    A Pdyn-enriched 5-HT neuron subtype is well-positioned to broadly but specifically alter the input and output of basal ganglia circuits, potentially mediating the modulation of motor control, decision making, and reinforcement learning by internal state.

    “We also define a Pdyn-enriched 5-HT neuron subtype that is well-positioned to broadly but specifically alter the input and output of basal ganglia circuits, potentially mediating the modulation of motor control, decision making, and reinforcement learning by internal state.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: 5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

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

    Interpretationreasoning

    Ependymal cells may communicate via secretion of transmitters and peptides into the CSF.

    “Additionally, ependymal cells may communicate via secretion of these transmitters and peptides into the CSF.”
    Quote in the paperNot a substantial claim

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

    Interpretationown data

    Our results provide a resource for the design of intersectional strategies for the functional dissection of DRN cell types.

    “Our results provide a resource for the design of intersectional strategies for the functional dissection of DRN cell types.”
    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

    GO term enrichment scores were calculated using the Wallenius approximation method in goseq, and terms were filtered using Q values at a 5% false discovery rate with Benjamini-Hochberg correction.

    “Enrichment scores were calculated using the Wallenius approximation method in goseq, and terms were filtered using Q values at a 5% false discovery rate (Benjamini-Hochberg correction).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Fine alignment of each 2D histogram along the dorsal-ventral axis was adjusted manually using Tph2 ISH images as the reference, with anatomical landmarks visible in the Nissl or background stain used for the fine adjustment.

    “Fine alignment of each 2D histogram along the dorsal-ventral axis was adjusted manually using Tph2 ISH images as the reference. Anatomical landmarks visible in the Nissl or background stain, such as the cerebral aqueduct and fiber tracts, were used for the fine adjustment.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given

    Release of peptides such as Pdyn from a subset of ependymal cells can have differential effects dependent on the expression pattern of the κ-opioid receptor.

    “Release of peptides such as Pdyn from a subset of ependymal cells can also have differential effects dependent on the expression pattern of the κ-opioid receptor.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    Activation of Gi-coupled pathways on pvMΦs via 5-HT1B receptors is a potential mechanism for the regulation of macrophage polarization and inflammatory responses by tonic 5-HT release within the DRN.

    “Activation of Gi-coupled pathways on pvMΦs via 5-HT1B receptors is also a potential mechanism for the regulation of macrophage polarization and inflammatory responses by tonic 5-HT release within the DRN, which may be relevant to the increasing importance of neuroinflammatory pathways in the etiology of psychiatric disorders (Brisch et al., 2017; Howerton et al., 2014; Watkins et al., 2014).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Brisch et al., 2017 source

      Not checked in the free run.

    • cites Howerton et al., 2014 source

      Not checked in the free run.

    • cites Watkins et al., 2014 source

      Not checked in the free run.

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

    Study design and conductnone given

    P values for differential gene expression were corrected using the Benjamini-Hochberg method and filtered at a 5% false discovery rate (Q < 0.05).

    “P values were corrected using the Benjamini-Hochberg method and filtered a 5% false discovery rate (Q < 0.05).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Subtype-specific connections between 5-HT neurons and local GABAergic and glutamatergic neurons can mediate interactions between different subsystems and may contribute to the contrasting and opposing functions ascribed to the DRN as a whole.

    “Subtype-specific connections between 5-HT neurons and local GABAergic and glutamatergic neurons can mediate interactions between different subsystems and may contribute to the contrasting and opposing functions ascribed to the DRN as a whole.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Cell barcodes containing fewer than 250 total reads were discarded.

    “Cell barcodes containing fewer than 250 total reads were discarded”
    Quote in the paperNot a substantial claim

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

    Interpretationnone given

    The LSL-FSF-ReaChR:mCitrine intersectional reporter line used for conditional anterograde tracing will also be suitable for optogenetic perturbation experiments in vivo to investigate the circuit and behavioral functions of these distinct 5-HT subsystems.

    “The LSL-FSF-ReaChR:mCitrine intersectional reporter line that we used for conditional anterograde tracing will also be suitable for optogenetic perturbation experiments in vivo to investigate the circuit and behavioral functions of these distinct 5-HT subsystems.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Cells with fewer than 500 UMIFM counts were removed.

    “Cells with fewer than 500 UMIFM counts were removed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Enrichment of the Gi-coupled Hrh3 receptor in 5-HT-III suggests that histamine may inhibit this subtype while increasing the excitability of 5-HT-I and 5-HT-II neurons.

    “Enrichment of the Gi-coupled Hrh3 receptor in 5-HT-III further suggests that histamine may inhibit this subtype while increasing the excitability of 5-HT-I and 5-HT-II neurons.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All procedures were performed in accordance with protocols approved by the Harvard Standing Committee on Animal Care following guidelines described in the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.

    “All procedures were performed in accordance with protocols approved by the Harvard Standing Committee on Animal Care following guidelines described in the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Transmitters and peptides released into the ventricular system can have long range effects facilitated by the flow of CSF and signal to neuronal cells extending processes to the ventricular walls, including some 5-HT neurons, as well as non-neuronal cells including ventricular immune cells and choroid plexus cells.

    “Transmitters and peptides released into the ventricular system can have long range effects facilitated by the flow of CSF and signal to neuronal cells extending processes to the ventricular walls, including some 5-HT neurons, as well as non-neuronal cells including ventricular immune cells and choroid plexus cells.”
    Quote in the paperNot a substantial claim

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

    Interpretationnone given

    GABAergic and/or glutamatergic neurons may be excited by adjacent 5-HT neurons via activation of their 5-HT2C and 5-HT7 receptors to mediate indirect interactions between 5-HT neuron subtypes.

    “However, GABAergic and/or glutamatergic neurons may be excited by adjacent 5-HT neurons via activation of their 5-HT2C and 5-HT7 receptors to mediate indirect interactions between 5-HT neuron subtypes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationreasoning

    These interactions may be either synergistic if mediated by glutamatergic neurons innervating other 5-HT subtypes, or antagonistic in a form of lateral inhibition via GABAergic interneurons.

    “These interactions may be either synergistic if mediated by glutamatergic neurons innervating other 5-HT subtypes, or antagonistic in a form of lateral inhibition via GABAergic interneurons.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The spatial expression matrix was filtered in two stages: bins with no Tph2 expression were removed, and genes were filtered based on total expression and mean-normalized variance.

    “The spatial expression matrix (Genes x Bins) was further filtered in two stages: bins with no Tph2 expression were removed, and genes were filtered based on total expression and mean-normalized variance (Figure 5—figure supplement 1).”
    Quote in the paperSubstantial claim
70 of 70

  • The paper relies on this without support.

    Distinct neuronal cell types are spatially intermingled within the DRN and are difficult to separate anatomically.

    “Furthermore, these distinct neuronal cell types are spatially intermingled within the DRN and are difficult to separate anatomically.”

  • The paper relies on this without support.

    The density of 5-HT neurons varies gradually between DRN subregions, preventing the delineation of clear boundaries.

    “While these subregions can be roughly defined by the spatial clustering of 5-HT neurons, the density of 5-HT neurons varies gradually between the subregions preventing the delineation of clear boundaries.”

  • The paper relies on this without support.

    Anatomical subregions fail to capture many features of 5-HT neurons, as most of the variation does not show subregional specificity.

    “Nevertheless, these anatomical subregions fail to capture many features of 5-HT neurons, as most of the variation does not show subregional specificity.”

  • The paper relies on this without support.

    DRN 5-HT neurons vary in their axonal projection patterns, and the spatial distributions of projection-defined subpopulations are broad and do not map cleanly to DRN subregions.

    “Additionally, DRN 5-HT neurons vary in their axonal projection patterns, and the spatial distributions of these projection-defined subpopulations are broad and do not map cleanly to DRN subregions.”

  • The paper relies on this without support.

    There is no consensus on the definition of 5-HT neuron subsystems and only an incomplete understanding of correspondences between anatomically, molecularly, and functionally defined 5-HT neuron subtypes.

    “However, there is no consensus on the definition of these subsystems and only an incomplete understanding of correspondences between anatomically, molecularly, and functionally defined 5-HT neuron subtypes.”

  • The paper relies on this without support.

    It is unknown if projection-defined subpopulations of DRN 5-HT neurons map onto molecularly defined subtypes.

    “Functional differences exist between projection-defined subpopulations of DRN 5-HT neurons (Ren et al., 2018), but it is unknown if these subpopulations map onto molecularly defined subtypes.”

  • The paper relies on this without support.

    An unbiased census of DRN cell types and subtypes of 5-HT neurons is lacking.

    “Furthermore an unbiased census of DRN cell types and subtypes of 5-HT neurons is lacking, and presents a major obstacle in achieving a unified understanding of the diverse functions performed by these neurons.”

  • The paper relies on this without support.

    Anatomical features are lost during dissociation of tissue for droplet-based scRNA-seq techniques.

    “Anatomical features are lost during the dissociation of tissue for droplet-based scRNA-seq techniques.”

  • The paper relies on this without support.

    ISH against a small set of marker genes expressed exclusively in one cell type can be used to localize the putative location of cell types in complex tissue.

    “ISH against a small set of marker genes expressed exclusively in one cell type can be used to localize the putative location of cell types in complex tissue.”

  • The paper relies on this without support.

    5-HT neurons are organized into discrete subsystems that have distinct and dissociable behavioral functions and are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.

    “5-HT neurons are organized into discrete subsystems that (i) have distinct and dissociable behavioral functions, and (ii) are comprised of 5-HT neurons possessing unique molecular, physiological, and anatomical properties.”

  • The paper relies on this without support.

    The complex architecture of the DRN has impeded efforts to understand the interrelationship of molecular, physiological, and anatomical aspects of 5-HT diversity.

    “However, the complex architecture of the DRN has impeded efforts to understand the interrelationship of these three aspects of 5-HT diversity, as necessary to study the differential properties and functions of each subsystem and its distinct subtype of 5-HT neuron.”

  • The paper relies on this without support.

    Droplet-based scRNA-seq methods such as inDrop have relatively low transcript capture efficiency and low number of reads per cell.

    “Some key technical limitations of scRNA-seq, particularly for droplet-based methods such as the inDrop platform used in this study, are the relatively low transcript capture efficiency and low number of reads per cell.”

  • The paper relies on this without support.

    Expression patterns of known receptor-ligand pairs are informative about local interactions involving DRN cells.

    “Our scRNA-seq dataset yields predictions about local interactions involving DRN cells, including those with non-neuronal cells, based the expression patterns of known receptor-ligand pairs.”
13 of 13

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Authors
Huang, K. W., Ochandarena, N. E., Philson, A. C., Hyun, M., Birnbaum, J. E., Cicconet, M., & Sabatini, B. L.
Affiliations
  • Howard Hughes Medical Institute (US)
  • Harvard University (US)
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2019-08-14
Volume
8
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.46464
PMC
PMC6726424
OpenAlex
W2967535576
Semantic Scholar
8814ae691cf88141d9ce7886b00b93fc5b911095
Cited by
267
References
95
Citations by year
2026: 37 · 2025: 47 · 2024: 44 · 2023: 39 · 2022: 33 · 2021: 40 · 2020: 23 · 2019: 3
FWCI
8.19
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Harvard Dataverse
Funders
  • National Institute of Neurological Disorders and Stroke (NS103226)
  • Howard Hughes Medical Institute
  • National Institute of Mental Health (MH100568)
  • Harvard Brain Initiative (Bipolar Disorder Seed Grant)
  • Samsung (Samsung Scholarship)
  • Harvard Medical School (Lefler Center for the Study of Neurodegenerative Disorder)
  • Department of Neurobiology, Harvard Medical School (Graduate Fellowship, Stuart HQ and Victoria Quan Fellowship in Neurobiology)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurotransmitter Receptor Influence on Behavior
Keywords
dorsal raphe nucleus, serotonergic neurons, neuronal cell types, single-cell transcriptomics, spatial transcriptomics, neuromodulation, mouse brain
MeSH terms
Animals, Genotype, Neurons, Phenotype, In Situ Hybridization, Sequence Analysis, RNA, Gene Expression Profiling, Mice, Neuroanatomical Tract-Tracing Techniques, Spatial Analysis, Dorsal Raphe Nucleus

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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 2026-10-09.

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