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Cited by 304Single-cell and spatial transcriptomicsNeuroscience and Neuropharmacology ResearchNeurotransmitter Receptor Influence on Behavior

Single-cell transcriptomes and whole-brain projections of serotonin neurons in the mouse dorsal and median raphe nuclei

Open access
Ren, Jing, Isakova, Alina, Friedmann, Drew, Zeng, Jiawei, Grutzner, Sophie M, Pun, Albert, Zhao, Grace Q, Kolluru, Sai Saroja +9 more·2019·eLife
DOI 10.7554/elife.49424PMC PMC6812963

Published version of a preprint

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Abstract

Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed diverse and segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

AI snapshot

Written from the paper’s full text.

TL;DR

Plate-based single-cell RNA-sequencing of 999 mouse dorsal and median raphe serotonin neurons identified 11 transcriptomic clusters with distinct spatial, molecular, and projection features. Intersectional viral tracing and single-neuron reconstruction showed that Vglut3+ and Trh+ DR serotonin neurons preferentially innervate cortical versus subcortical targets, while individual DR serotonin neurons have highly diverse whole-brain arborization patterns.

Key findings

  • scRNA-seq of 999 Tph2+ serotonin neurons from 14 adult mouse brains resolved 11 transcriptomic clusters: six principal DR clusters (DR-1–6), one caudal DR cluster (cDR), and four median raphe clusters (MR-1–4); HCR-smFISH with 16 markers mapped these clusters to pDR, cDR, or MR locations.
  • Co-transmitter and neuropeptide expression was heterogeneous: 65% of serotonin neurons expressed Vglut3, 35% expressed Gad1, Gad2, or both, 13% expressed Vglut3 plus Gad1/Gad2, and 14% expressed none of these glutamate/GABA markers; Trh was expressed in 14% and Crh in 7% of serotonin neurons.
  • Intersectional Sert-Flp with Vglut3-Cre or Trh-Cre showed that Vglut3+ pDR serotonin neurons preferentially innervate cortical regions, whereas Trh+ pDR serotonin neurons innervate subcortical regions including anterior/medial hypothalamus and thalamic nuclei; whole-brain mapping used n = 3 mice per genotype.
  • Reconstruction of 50 individual DR serotonin neurons revealed total axon lengths from 1.9 cm to 26.3 cm, with cortex-projecting neurons having the longest axons; 37/50 projections were unilateral, and 34/46 forebrain-projecting neurons had >80% of forebrain axon length in either cortical or subcortical regions.

Methods

  • Serotonin neurons were labeled using Sert-Cre;Ai14 mice, acutely dissociated from brain slices of postnatal day 40–45 mice (six females, eight males), FACS-sorted, and processed with Smart-seq2; 709 cells came from eight MR/pDR/cDR samples and 290 cells from six pDR-only samples, yielding 999 cells after QC.
  • Clustering used Seurat v3.0 with PCA and Louvain clustering; HCR-based smFISH against 16 cluster markers plus Tph2 was used to map transcriptomic clusters anatomically.
  • For projection mapping, Sert-Flp mice were crossed with Vglut3-Cre or Trh-Cre and injected with AAV-CreON/FlpON-mGFP; brains were cleared with AdipoClear, imaged by light-sheet microscopy, segmented with a 3D U-Net, and aligned to the Allen Common Coordinate Framework. Single-neuron reconstructions used dual-AAV sparse labeling and fMOST in 14 Sert-Cre mice.

Limitations

  • Stated: single-neuron reconstruction was limited in scope to 50 reconstructed cells out of approximately 9000 DR serotonin neurons, and larger-scale reconstruction integrated with molecular identity and function is needed.
  • Stated: whole-brain tracing showed inter-individual variability in total labeled axons and detailed projection patterns, and heterogeneity within Vglut3+ or Trh+ populations may contribute; larger experiments or single-cluster Cre lines are required.
  • Stated/evident: the Sert-Flp line can drive reporter expression outside raphe serotonin neurons due to transient developmental Sert expression; the paper also notes that whether Gad1/Gad2+ serotonin neurons actually release GABA remains to be determined, and MR cluster anatomical organization was less obvious with clusters more intermingled.

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Integrity signals

Registry

Crossref · 10.7554/elife.49424

Cites

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Statistics

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Claims the paper makes

0 of 66 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    HCR-smFISH showed that markers for Clusters 1-6, Ret, Trh, Gad1, Npas1, Syt2, and C1ql2 were mostly restricted to the pDR.

    “Specifically, we found that the distribution of markers for Clusters 1–6, Ret, Trh, Gad1, Npas1, Syt2, and C1ql2 (Figure 1D), were mostly restricted to the pDR.”
    Quote in the paperSubstantial claim
  • Findingown data

    The 999 MR and DR serotonin neurons comprised 11 transcriptomic clusters.

    “These 999 MR and DR serotonin neurons comprised 11 clusters (Figure 1B; Figure 1—figure supplement 2).”
    • Also stated in: § Abstract · plain
      “we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters.”
    Quote in the paperSubstantial claim
  • Findingown data

    Each of the 11 clusters contained cells from both sexes, indicating few sex-specific differences at the level of transcriptomic clusters.

    “Each cluster contained cells from both sexes after removing genes located on the Y chromosome, indicating that there were few sex-specific differences at the level of transcriptomic clusters (Figure 1—figure supplement 1D).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Six of the 11 transcriptomic clusters (Cluster 1-6) consisted only of serotonin neurons dissected from the pDR.

    “Of the 11 transcriptomic clusters, six (Cluster 1–6) consisted of only serotonin neurons dissected from the pDR.”
    Quote in the paperSubstantial claim
  • Findingown data

    Each of the 11 clusters expressed a set of cluster-discriminatory genes, including markers for neurotransmitter systems such as Vglut3, Gad1, and Gad2.

    “Each of the 11 clusters expressed a set of cluster-discriminatory genes, including markers for specific neurotransmitter systems, such as Vglut3, Gad1, and Gad2 (Figure 1C,D; Figure 1—figure supplement 1–6).”
    Quote in the paperSubstantial claim
  • May combine two claims. 66
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 16

  • May combine two claims.

    Findingown data

    Npas1 and Npas3 are preferentially expressed in DR-6, and Aff2 is enriched within DR-3.

    “TFs associated with neurodevelopmental disorders, such as Npas1 and Npas3 (Erbel-Sieler et al., 2004), are preferentially expressed in DR-6, and Aff2 (Mondal et al., 2012) is enriched within DR-3.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Crh is highly expressed in the DR-3 cluster but much less elsewhere (7% of serotonin neurons).

    “Corticotropin-releasing hormone (Crh) is highly expressed in the DR-3 cluster but much less everywhere else (7% serotonin neurons).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cxcl12 and Cdh6 are preferentially expressed in MR-1 and DR-4 clusters, respectively.

    “The chemokine ligand-12 (Cxcl12) and cadherin-6 (Cdh6) are preferentially expressed in MR-1 and DR-4 clusters, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Npas3 is specifically expressed in DR-6 and highly correlated with Htr1d.

    “Npas3 is specifically expressed in the DR-6 cluster and is highly correlated with Htr1d, suggesting a critical role of maintaining the characteristics of DR-6 serotonin neurons.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The majority of clusters express Slc17a8 (Vglut3), including almost all cDR serotonin neurons, the majority of MR clusters, and DR-3-6 clusters.

    “The majority of the clusters express Slc17a8, which encodes vesicular glutamate transporter-3 (Vglut3). These include almost all serotonin neurons from the cDR, the majority from MR clusters (Domonkos et al., 2016), and DR-3–6 clusters (Figure 1D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In contrast to DR, the anatomical organization of molecular features defining MR clusters is less obvious and different clusters appear more intermingled.

    “In contrast to DR, the anatomical organization of the molecular features that define MR clusters is less obvious and different clusters appear more intermingled.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cluster 6 marker Npas1+ was largely excluded from the densest portion of Tph2 expression at the midline and was found scattered in the more rostral and ventral portion of the lateral wings.

    “Cluster 6 marker Npas1+ was largely excluded from the densest portion of Tph2 expression at the midline and instead was found scattered in the more rostral and ventral portion of the lateral wings.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All serotonin neurons express at least one type of serotonin receptor, with Htr1a being the most widely expressed.

    “Finally, all serotonin neurons express at least one type of serotonin receptors, with the Gi-coupled Htr1 subfamily in particular Htr1a being the most widely expressed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Two MR clusters express Gad2 but not Gad1, and cDR expresses neither.

    “Two MR clusters express Gad2 but not Gad1, and cDR expresses neither.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Htr1a is highly expressed in the MR-4 cluster, which does not express other serotonin receptors.

    “Htr1a is highly expressed in the MR-4 cluster, which does not express other serotonin receptors.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Within the DR, Trh+, Gad1+, and Gad2+ serotonin neurons were mainly located in the dorsal DR, whereas Vglut3+ and Syt2+ serotonin neurons were mainly located in the ventral DR and cDR.

    “Within the DR, Trh+, Gad1+, and Gad2+ serotonin neurons were mainly located in the dorsal DR, whereas Slc17a8+ (also known as Vglut3, used hereafter) and Syt2+ serotonin neurons were mainly located in the ventral DR and cDR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Bcl11a and Bcl11b are highly enriched in DR-3 and MR-4, respectively.

    “For example, Bcl11a and Bcl11b (also named Ctip1 and Ctip2) (Chen et al., 2008; Wiegreffe et al., 2015) are highly enriched in DR-3 and MR-4, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Crhr2 was localized preferentially near the midline and was absent from the lateral wing.

    “On the other hand, Crhr2, which should be expressed in all DR serotonin neuron clusters except Cluster 1 and 6 (Figure 1D), was localized preferentially near the midline and was absent from the lateral wing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MR-3 expresses Tacr3, Cnr1, Sstr1, Prlhr, and Nmbr.

    “Another neuropeptide receptor enriched cluster is MR-3, expressing Tacr3, cannabinoid receptor 1(Cnr1), somatostatin receptor-1 (Sstr1), prolactin-releasing hormone receptor (Prlhr), and neuromedin B receptor (Nmbr).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the pDR, Vglut3+ serotonin neurons were mainly located ventrally, whereas Gad2+ serotonin neurons were mainly located dorsally.

    “In the pDR, Vglut3+ serotonin neurons were mainly located ventrally, whereas Gad2+ serotonin neurons were mainly located dorsally, consistent with our previous study (Ren et al., 2018) and the HCR-smFISH results (Figure 2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Trh+ serotonin axons project to the anterior and medial hypothalamus, posterior amygdala, and lateral geniculate nucleus in the thalamus, none of which were targeted by Vglut3+ axons.

    “Instead, Trh+ serotonin axons project to the anterior and medial hypothalamus, posterior amygdala, and the lateral geniculate nucleus in the thalamus, none of which were targeted by Vglut3+ axons (Figure 6D).”
    • Also stated in: § Abstract · plain
      “Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Two hypothalamus-projecting cells had substantial branches to the striatum, and one to the pons (>10% axon length).

    “Two cells had substantial branches to the striatum, and one to the pons (>10% axon length).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Complete morphological reconstruction revealed that the projection pattern of individual DR serotonin neurons was highly diverse yet follow some general patterns.

    “Complete morphological reconstruction revealed that the projection pattern of individual DR serotonin neurons was highly diverse yet follow some general patterns”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Two cortex-projecting serotonin neurons sent substantial branches to the pons (>10% of axon length) and also innervated the cerebellum.

    “two cortex-projecting serotonin neurons also sent substantial branches to the pons (>10% of axon length), and also innervated the cerebellum.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Subcortical regions primarily in thalamus (zona incerta and medial geniculate) and hypothalamus (anterior and dorsomedial nuclei) were preferentially targeted by Trh+ axons and largely avoided by the Vglut3+ population.

    “subcortical regions primarily in thalamus (zona incerta and medial geniculate) and hypothalamus (anterior and dorsomedial nuclei) were preferentially targeted by Trh+ axons and largely avoided by the Vglut3+ population”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    None of the amygdala-projecting cells had collateralization in the cortical regions.

    “None of them had collateralization in the cortical regions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Trh+ axons originated preferentially from the lateral wing of pDR and were largely absent from Vglut3+ target structures.

    “By contrast, Trh+ axons, which originated preferentially from the lateral wing of pDR, were largely absent from these structures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Vglut3+Sert+ GFP cells were mostly located in the ventral pDR, whereas Trh+Sert+ cells were located in the dorsal pDR.

    “As predicted, Vglut3+Sert+ GFP cells were mostly located in the ventral pDR (Figure 6B), whereas Trh+Sert+ cells were located in the dorsal pDR (Figure 6C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cortex-projecting group had the most complex branching pattern, with various combinations of target regions.

    “this group had the most complex branching pattern, with various combinations of target regions”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Regions targeted densely but exclusively in one or two individual brains include anterior bed nucleus of stria terminalis (BNST), posterior amygdala, and globus pallidus external segment (GPe) in Trh+ projections, as well as the lateral central amygdala (CeA) and dentate nucleus of the cerebellum for Vglut3+ projections.

    “Notable examples include anterior bed nucleus of stria terminalis (BNST), posterior amygdala, and globus pallidus external segment (GPe) in Trh+ projections, as well as the lateral central amygdala (CeA) and dentate nucleus of the cerebellum for Vglut3+ projections.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Axons from both populations followed similar initial trajectories and were observed in shared structures along the median forebrain bundle.

    “Axons from both populations followed similar initial trajectories and were observed in shared structures along the length of the median forebrain bundle (Figure 7C,D; Figure 7—figure supplements 1–3; Figure 7—Video 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    One striatum-projecting cell had substantial branches to the cortical regions and two to the hypothalamus.

    “One cell had substantial branches to the cortical regions and two to the hypothalamus.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Individual brains from the same genotype exhibit considerable variation in total axons labeled and in detailed projection patterns.

    “individual brains from the same genotype exhibit considerable variation in total axons labeled (Figure 7E top) and in detailed projection patterns”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Serotonin neurons from DR-1-3 clusters are preferentially localized in dorsal pDR, whereas those from DR-4-6 in ventral pDR, with DR-6 neurons preferentially localized to the ventral lateral wings.

    “serotonin neurons from DR-1–3 clusters are preferentially localized in dorsal pDR, whereas those from DR-4–6 in ventral pDR, with DR-6 neurons preferentially localized to the ventral lateral wings.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Comparing marker genes enriched in R1-R3-derived serotonin neurons from Okaty et al. and MR-1-MR-4 transcriptomic clusters from current study suggests an approximate correspondence: R1-derived serotonin neurons correspond to MR-1 cluster, R2 to MR-4 cluster, and R3 to both MR-2 and MR-3 clusters.

    “Comparing marker genes enriched in R1–R3-derived serotonin neurons from Okaty et al. and MR-1–MR-4 transcriptomic clusters from our current study (Figure 3—figure supplement 4) indeed suggests an approximate correspondence: R1-derived serotonin neurons correspond to our MR-1 cluster, R2 to MR-4 cluster, and R3 to both MR-2 and MR-3 clusters.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Okaty et al., 2015 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Cortical-projecting serotonin neurons can have elaborate arborization patterns across multiple cortical areas and the longest total axon lengths per cell.

    “The cortical-projecting serotonin neurons can have elaborate arborization patterns across multiple cortical areas (e.g., Figure 8—figure supplement 1C) and the longest total axon lengths per cell (Figure 8H).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Using viral-genetic intersectional approaches, they found that Vglut3+ pDR serotonin neurons project profusely to cover much of the neocortex, as well as the olfactory bulb, cortical amygdala, and lateral hypothalamus.

    “Using viral-genetic intersectional approaches to access specifically Vglut3+ pDR serotonin neurons in combination with staining in histological sections (Figure 6) and iDISCO-based whole brain imaging (Figure 7), we found that these neurons project profusely to cover much of the neocortex, as well as the olfactory bulb, cortical amygdala, and lateral hypothalamus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All cDR serotonin neurons co-express Vglut3, and express several unique markers.

    “All cDR serotonin neurons co-express Vglut3, and express several unique markers (Figure 3—figure supplement 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transcriptome data suggest that each DR/MR serotonin neuron type can be distinguished from others by the expression of two marker genes, supporting the view that neuronal subtypes are generally specified by unique combination of genes rather than single genes (Li et al., 2017).

    “Our transcriptome data suggest that each DR/MR serotonin neuron type can be distinguished from others by the expression of two marker genes (Figure 1D; Figure 3—figure supplement 1–3), supporting the view that neuronal subtypes are generally specified by unique combination of genes rather than single genes (Li et al., 2017).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Li et al., 2017

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Single-cell transcriptomic analysis indicated that serotonin neurons in the cDR are strikingly homogenous and profoundly different from both the pDR and MR at the molecular level.

    “Our single-cell transcriptomic analysis indicated that serotonin neurons in the cDR are strikingly homogenous and profoundly different from both the pDR and MR at the molecular level (Figure 1, Figure 4, Figure 4—figure supplement 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Based on tissue source and fluorescent in situ hybridization using transcriptomic cluster markers, six types were assigned to principal DR (pDR), one type to caudal DR (cDR), and four types to MR.

    “Based on tissue source from which scRNA-seq data was collected and fluorescent in situ hybridization using transcriptomic cluster markers, we were able to assign six types to principal DR (pDR), one type to caudal DR (cDR), and four types to MR (Figure 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Trh+ serotonin neurons predominantly project to subcortical regions, most notably anterior and medial nuclei of the hypothalamus and several thalamic nuclei, a pattern mostly complementary to the Vglut3+ population.

    “We found that Trh+ serotonin neurons predominantly project to subcortical regions, most notably anterior and medial nuclei of the hypothalamus and several thalamic nuclei, a pattern mostly complementary to the Vglut3+ population (Figures 6–7, Figure 7—Video 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    They could not discern obvious difference in spatial distributions among the four MR types, despite the fact that MR serotonin neurons have heterogenous developmental origin (Jensen et al., 2008).

    “By contrast, we could not discern obvious difference in spatial distributions among the four MR types, despite the fact that MR serotonin neurons have heterogenous developmental origin (Jensen et al., 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Jensen et al., 2008 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The rest of the DR serotonin neurons were divided into thalamus-projecting and brainstem/cerebellum-projecting groups based on the highest density of axons, one of which likely projecting to the spinal cord (brain 36) as previously reported.

    “The rest of the DR serotonin neurons were divided into thalamus-projecting and brainstem/cerebellum-projecting groups based on the highest density of axons (Figure 8—figure supplement 3B,C), one of which likely projecting to the spinal cord (brain 36) as previously reported (Bowker et al., 1981; Bowker et al., 1982).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Bowker et al., 1981 source

      Not checked in the free run.

    • cites Bowker et al., 1982 source

      Not checked in the free run.

  • May combine two claims.

    Findingreasoning

    Most forebrain-projecting DR serotonin neurons pass through the lateral hypothalamus to reach their targets; it is difficult to determine whether axons in the lateral hypothalamus play a local function.

    “As a specific example, most forebrain-projecting DR serotonin neurons pass through the lateral hypothalamus to reach their targets; it is thus difficult to determine whether axons in the lateral hypothalamus play a local function.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    One amygdala-projecting cell had axons almost equally distributed in the bed nucleus of stria terminalis (BNST), basal lateral amygdala, basal medial amygdala, and medial amygdala (MeA), and had substantial branches to the hypothalamus (>10% axon length).

    “One cell had axons that were almost equally distributed in the bed nucleus of stria terminalis (BNST), basal lateral amygdala, basal medial amygdala, and medial amygdala (MeA), and had substantial branches to the hypothalamus (>10% axon length).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Gene expression patterns of specific serotonin neuron clusters can allow genetic access to these subpopulations for anatomical tracing, physiological recording, and functional perturbation.

    “Gene expression patterns of specific serotonin neuron clusters can in principle allow genetic access to these specific subpopulations for anatomical tracing, physiological recording, and functional perturbation (Luo et al., 2018).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Luo et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    HCR-smFISH experiments support that Clusters 1-6 correspond to pDR serotonin neurons, Cluster 7 to cDR serotonin neurons, and Clusters 8-11 to MR serotonin neurons.

    “In summary, our HCR-smFISH experiments support the notion that Clusters 1–6 correspond to pDR serotonin neurons, Cluster 7 corresponds to cDR serotonin neurons, and Clusters 8–11 correspond to MR serotonin neurons.”
    • Also stated in: § Abstract · plain
      “Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    DR and MR serotonin neurons have diverse expression patterns of neuropeptide receptors, indicating they are subject to complex modulation by neuropeptides.

    “In summary, the DR and MR serotonin neurons have diverse expression pattern of neuropeptide receptors, indicating that they are subject to complex modulation by a host of neuropeptides.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Vesicular monoamine transporters can transport GABA into synaptic vesicles.

    “It has been reported that vesicular monoamine transporters (Slc8a1 for Vmat1; Slc8a2 for Vmat2) can transport GABA into synaptic vesicles (Stensrud et al., 2014), and virtually all serotonin neurons expressed Vmat2 (Figure 1—figure supplement 1D; Figure 4A left).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Stensrud et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Clusters 1-3 correspond to the dorsal DR and Clusters 4-6 to the ventral DR.

    “These data suggest that Clusters 1–3 correspond to the dorsal DR and Cluster 4–6 to the ventral DR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Vglut3+ and Trh+ pDR serotonin neurons consist of largely complementary cell types at the transcriptomic level and have a distinct distribution along the dorsal-ventral axis in the pDR.

    “Based on our scRNA-seq and HCR-smFISH results, the Vglut3+ and Trh+ pDR serotonin neurons consist of largely complementary cell types at the transcriptomic level and have a distinct distribution along the dorsal–ventral axis in the pDR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    In Sert-Flp;H11-CAG-FRT-stop-FRT-EGFP mice, 98.5% of GFP+ neurons are Tph2+ and 100% of Tph2+ neurons are GFP+.

    “Anti-Tph2 staining on the brain slices containing pDR showed that 98.5% GFP+ neurons are Tph2+ and 100% Tph2+ neurons are GFP+ (Figure 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    DR and MR contain GABAergic and glutamatergic neurons that do not express Tph2, some of which may project to the same target regions.

    “However, DR and MR contain not only serotonin neurons but also GABAergic and glutamatergic neurons that do not express Tph2 (and hence do not release serotonin), some of which may project to the same target regions (McDevitt et al., 2014).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites McDevitt et al., 2014 source

      Not checked in the free run.

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

    Premisecitation

    Transcription factors are the best molecular feature for cell type definition.

    “Transcription factors (TFs) have been shown to be the best molecular feature for cell type definition (Schaum et al., 2018).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Schaum et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    HCR-smFISH observations support the anatomical breakdown suggested by dissection of primary tissue and provide finer boundaries.

    “Thus, these observations support the anatomical breakdown suggested by the dissection of primary tissue, and additionally provide a more granular and detailed description about finer boundaries.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    DR serotonin neurons derive entirely from rhombomere 1, whereas MR serotonin neurons derive predominantly from rhombomeres 1, 2, and 3.

    “DR serotonin neurons derive entirely from rhombomere 1 of the developing mouse brain, whereas MR serotonin neurons derive predominantly from rhombomeres 1, 2, and 3 (Alonso et al., 2013; Bang et al., 2012; Jensen et al., 2008).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Alonso et al., 2013 source

      Not checked in the free run.

    • cites Bang et al., 2012

      Not checked in the free run.

    • cites Jensen et al., 2008 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The DR and MR serotonin systems differ in developmental origin, connectivity, physiology, and behavioral function.

    “Evidence has suggested that the DR and MR serotonin systems differ in developmental origin, connectivity, physiology, and behavioral function (Alonso et al., 2013; Calizo et al., 2011; Okaty et al., 2019).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Alonso et al., 2013 source

      Not checked in the free run.

    • cites Calizo et al., 2011 source

      Not checked in the free run.

    • cites Okaty et al., 2019 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The MR serotonin neurons project to structures near the midline, whereas the DR serotonin neurons target more lateral regions.

    “The MR serotonin neurons project to structures near the midline, whereas the DR serotonin neurons target more lateral regions (Jacobs and Azmitia, 1992; Muzerelle et al., 2016; Vertes, 1991; Vertes et al., 1999).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Jacobs and Azmitia, 1992

      Not checked in the free run.

    • cites Muzerelle et al., 2016 source

      Not checked in the free run.

    • cites Vertes, 1991 source

      Not checked in the free run.

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

    Premisecitation

    Groups B1-B3 are located in the medulla and provide descending serotonergic innervation to the spinal cord and other parts of the medulla; Groups B4-B9 are located in the pons and midbrain, including the DR and MR, and provide ascending innervation to the forebrain and midbrain.

    “Groups B1–B3 are located in the medulla and provide descending serotonergic innervation to the spinal cord and other parts of the medulla. Groups B4–B9 are located in the pons and midbrain, including the dorsal raphe (DR; groups B6 and B7) and median raphe (MR; groups B5 and B8) nucleus, and provide ascending innervation to the forebrain and midbrain.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Dahlström and Fuxe, 1964

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    DR serotonin neurons that project to frontal cortex and amygdala constitute two sub-systems with distinct cell body locations, axonal collateralization patterns, biased inputs, physiological response properties, and behavioral functions.

    “As a specific example, we recently found that DR serotonin neurons that project to frontal cortex and amygdala constitute two sub-systems with distinct cell body locations, axonal collateralization patterns, biased inputs, physiological response properties, and behavioral functions (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.

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

    Premisecitation

    In neural circuits where cell types have been well studied by anatomical and physiological methods, there is an excellent correspondence between cell types defined by transcriptomes and the classical methods.

    “In neural circuits where cell types have been well studied by anatomical and physiological methods, there is an excellent correspondence between cell types defined by transcriptomes and the classical methods (Li et al., 2017; Shekhar et al., 2016).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Li et al., 2017

      Not checked in the free run.

    • cites Shekhar et al., 2016 source

      Not checked in the free run.

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

    Premisecitation

    Serotonin neurons in the central nervous system are spatially clustered in the brainstem, originally designated as groups B1-B9.

    “Serotonin neurons in the central nervous system are spatially clustered in the brainstem, originally designated as groups B1–B9 (Dahlström and Fuxe, 1964).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Dahlström and Fuxe, 1964

      Not checked in the free run.

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

    Premisecitation

    Collectively, ~12,000 serotonin neurons from the DR and MR in mice (~0.015% of all CNS neurons) innervate the entire forebrain (Ishimura et al., 1988) and modulate diverse physiological and behavioral functions.

    “Collectively,~12,000 serotonin neurons from the DR and MR in mice (~0.015% of all CNS neurons) innervate the entire forebrain (Ishimura et al., 1988) and modulate diverse physiological and behavioral functions.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ishimura et al., 1988 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MR serotonin neurons derive from three separate rhombomeres (R1-R3), and Okaty et al. sampled these three populations separately, allowing the question of whether developmental origins correspond to transcriptomic clusters in adult.

    “The MR serotonin neurons derive from three separate rhombomeres (R1–R3), and Okaty et al. sampled these three populations separately, allowing us to ask the question of whether developmental origins correspond to transcriptomic clusters in adult.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Okaty et al., 2015 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Kast et al. (2017) found that the cDR expressed distinct gene markers compared to pDR, and tracing from these neurons showed cDR was different from the MR and DR in efferent innervation.

    “By searching Allen Brain Institute development brain database and using multiplex fluorescent in situ hybridization (ML-FISH), Kast et al. (2017) found that the cDR expressed distinct gene markers comparing to pDR, and tracing from these neurons showed cDR was different from the MR and DR in efferent innervation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kast et al., 2017 source

      Not checked in the free run.

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

    Premisecitation

    The cDR has been suggested to be more similar to the MR than to the pDR in afferent innervation (Commons, 2015).

    “The cDR has been suggested to be more similar to the MR than to the pDR in afferent innervation (Commons, 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Commons, 2015 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    In previous study, they characterized two projection-defined parallel DR serotonin sub-systems.

    “In our previous study, we characterized two projection-defined parallel DR serotonin sub-systems.”
    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.

    Premisecitation

    Serotonin neurons that project to the orbitofrontal cortex (OFC) and central amygdala (CeA) differ in input and output patterns, physiological response properties, and behavioral functions (Ren et al., 2018).

    “We found that serotonin neurons that project to the orbitofrontal cortex (OFC) and central amygdala (CeA) differ in input and output patterns, physiological response properties, and behavioral functions (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.

    Interpretationown data

    Compared to droplet-based platform of the two studies, our FACS-based scRNA-seq platform allowed more sequencing depth per cell and sequencing larger number of cells, accounting for our finer classification of transcriptomic types of serotonin neurons.

    “Compared to droplet-based platform of the two studies, our FACS-based scRNA-seq platform allowed more sequencing depth per cell (Figure 1—figure supplement 1B) and sequencing larger number of cells, accounting for our finer classification of transcriptomic types of serotonin neurons.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Single-cell reconstruction of DR serotonin neurons revealed many more than six branching patterns, indicating that there must be diverse collateralization patterns even within the same transcriptomic cluster.

    “However, our single-cell reconstruction of DR serotonin neurons revealed many more than six branching patterns (e.g., Figure 8G), indicating that there must be diverse collateralization patterns even within the same transcriptomic cluster, and highlighting the complexity of individual serotonin neuron projections.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    These data support and extend previous finding (Ren et al., 2018) for the preferential ventral pDR location of Vglut3+ serotonin neurons, which is highly expressed in DR-4 and DR-5 clusters.

    “These data support and extend our previous finding (Ren et al., 2018) for the preferential ventral pDR location of Vglut3+ serotonin neurons, which is highly expressed in DR-4 and DR-5 clusters.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ren et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Because DR serotonin neurons derive exclusively from rhombomere 1 (R1), and the single-cell RNA-seq data was obtained from just 8 cells, the Okaty et al. dataset is limited in providing information about heterogeneity within DR serotonin neurons.

    “Because DR serotonin neurons derive exclusively from rhombomere 1 (R1), and the single-cell RNA-seq data was obtained from just 8 cells, the Okaty et al. dataset is limited in providing information about heterogeneity within DR serotonin neurons.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Okaty et al., 2015 source

      Not checked in the free run.

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

    Premisecitation

    Forebrain-projecting serotonin neurons have been estimated to have very long axons based on the total serotonin fiber density and number of serotonin neurons (Wu et al., 2014).

    “Forebrain-projecting serotonin neurons have been estimated to have very long axons based on the total serotonin fiber density and number of serotonin neurons (Wu et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wu et al., 2014

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Given that CeA-projecting DR serotonin neurons do not innervate most of the hypothalamus (Ren et al., 2018), and Trh+ serotonin neurons only partially innervate CeA, these two populations are at most partially overlapping.

    “Given that CeA-projecting DR serotonin neurons do not innervate most of the hypothalamus (Ren et al., 2018), and Trh+ serotonin neurons only partially innervate CeA, these two populations are at most partially overlapping.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ren et al., 2018 source

      Not checked in the free run.

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

    Premisecitation

    Several studies have reported that subgroups of serotonin neurons in the MR and DR express Vglut3, and subsequent slice recording confirmed that serotonin terminals can co-release glutamate and serotonin.

    “For example, several studies have reported that subgroups of serotonin neurons in the MR and DR express Vglut3 and indeed, subsequent slice recording confirmed that serotonin terminals can co-release glutamate and serotonin (Domonkos et al., 2016; Liu et al., 2014; Ren et al., 2018; Sengupta et al., 2017; Varga et al., 2009; Wang et al., 2019).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Domonkos et al., 2016 source

      Not checked in the free run.

    • cites Liu et al., 2014 source

      Not checked in the free run.

    • cites Ren et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Molecular features of these distinct serotonin groups reflect their anatomical organization and provide tools for future exploration of the full projection map of molecularly defined serotonin groups.

    “We have shown that the molecular features of these distinct serotonin groups reflect their anatomical organization and provide tools for future exploration of the full projection map of molecularly defined serotonin groups.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The reported projection and arborization complexity of individual DR neurons in Gagnon and Parent appear qualitatively similar to our findings, but these studies cannot be directly compared because Gagnon and Parent did not validate the serotonin identity of individually traced DR neurons, which could include Tph2-negative long-distance projecting GABAergic and glutamatergic neurons (Bang and Com

    “While the reported projection and arborization complexity of individual DR neurons appear qualitatively similar to our findings, these studies cannot be directly compared because the Gagnon and Parent study did not validate the serotonin identity of individually traced DR neurons, which could include Tph2-negative long-distance projecting GABAergic and glutamatergic neurons (Bang and Commons, 2012”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Gagnon and Parent, 2014

      Not checked in the free run.

    • cites Bang and Commons, 2012

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    These serotonin neurons use glutamate as co-transmitter in their cortical targets, and are in turn modulated by corticotropin-releasing hormone and neuropeptide Y.

    “which suggests that these serotonin neurons use glutamate as co-transmitter in their cortical targets, and are in turn modulated by corticotropin-releasing hormone and neuropeptide Y.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Previous study suggests that DR serotonin sub-systems have biased input but segregated output (Ren et al., 2018).

    “Our previous study suggests that DR serotonin sub-systems have biased input but segregated output (Ren et al., 2018).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ren et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Comprehensive transcriptomic dataset and identification of 11 distinct groups of the DR and MR serotonin neurons have revealed the molecular heterogeneity of the forebrain-projecting serotonin system.

    “In conclusion, our comprehensive transcriptomic dataset and identification of 11 distinct groups of the DR and MR serotonin neurons have revealed the molecular heterogeneity of the forebrain-projecting serotonin system.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All procedures followed animal care and biosafety guidelines approved by Stanford University's Administrative Panel on Laboratory Animal Care and Administrative Panel of Biosafety in accordance with NIH guidelines.

    “All procedures followed animal care and biosafety guidelines approved by Stanford University’s Administrative Panel on Laboratory Animal Care and Administrative Panel of Biosafety in accordance with NIH guidelines.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The reconstructed neurons were checked by another person.

    “The reconstructed neurons were checked by one another person.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    The molecular architecture of serotonin system lays the foundation for integrating anatomical, neurochemical, physiological, and behavioral functions.

    “The molecular architecture of serotonin system lays the foundation for integrating anatomical, neurochemical, physiological, and behavioral functions.”
    Quote in the paperNot a substantial claim
79 of 79

  • The paper relies on this without support.

    The heterogeneity of DR and MR serotonin neurons must be reflected at the molecular level.

    “Ultimately, the heterogeneity of DR and MR serotonin neurons must be reflected at the molecular level.”

  • The paper relies on this without support.

    The DR contains approximately 9000 serotonin neurons.

    “our study is still limited by the scope (50 reconstructed cells out of 9000 DR serotonin neurons).”
2 of 2

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?

  • Dorsal raphe 5-HT/vGluT3+ neurons project widely throughout the cortex and increase or decrease their activity to reward and punish, respectively.

    “Those in the dorsal raphe project widely throughout the cortex and increase or decrease their activity to reward and punish, respectively.76,78”

    From Synaptic and circuit functions of multitransmitter neurons in the mammalian brain (2023) · cites this paper as [76]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Vglut3 is assigned two different gene symbols

    Minor

    The results identify Vglut3 as Slc17a8, but the Animals section calls the Vglut3-Cre line Slc18a8-Cre.

1 of 1

Cites

Flagged first, then by citations

2 of the 97 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Çiçek, 2016, Medical Image Computing and Computer-Assisted Intervention – MICCAI 2016. MICCAI 2016. Lecture Notes in Computer Science, 9901

Cited by

Sorted by citations

Related

Authors
Ren, J., Isakova, A., Friedmann, D., Zeng, J., Grutzner, S. M., Pun, A., Zhao, G. Q., Kolluru, S. S., Wang, R., Lin, R., Li, P., Li, A., Raymond, J. L., Luo, Q., Luo, M., Quake, S. R., & Luo, L.
Affiliations
  • Howard Hughes Medical Institute (US)
  • Stanford University (US)
  • National Institute of Biological Sciences, Beijing (CN)
  • Stanford Medicine (US)
  • Wuhan National Laboratory for Optoelectronics (CN)
  • Chan Zuckerberg Biohub San Francisco (US)
Corresponding author
  • Ren, Jing
  • Quake, Stephen R
  • Luo, Liqun
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2019-10-24
Volume
8
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.49424
PMC
PMC6812963
OpenAlex
W2982239238
Semantic Scholar
73c6ef27934225685f1623abe2f5530230da5ad5
Cited by
304
References
97
Citations by year
2026: 32 · 2025: 52 · 2024: 50 · 2023: 59 · 2022: 42 · 2021: 41 · 2020: 25 · 2019: 2 · 2018: 1
FWCI
9.10
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health (R01 NS104698, NS057488)
  • National Science Foundation (NeuroNex)
  • Ministry of Science and Technology of the People's Republic of China (2015BAI08B02)
  • National Natural Science Foundation of China (91432114, 91632302)
  • Beijing Municipal Government
  • NSFC (61721092, 81827901)
  • Swiss National Science Foundation (PostDoc Mobility Fellowship)
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
serotonergic neurons, dorsal raphe nucleus, median raphe nucleus, single-cell transcriptomics, neuronal heterogeneity, VGLUT3, thyrotropin-releasing hormone, hypothalamus, neuropeptides, single-neuron reconstruction, mice
MeSH terms
Animals, Brain Mapping, Neural Pathways, Raphe Nuclei, Sequence Analysis, RNA, Mice, Single-Cell Analysis, Serotonergic Neurons, Transcriptome

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Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

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