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Cited by 508Single-cell and spatial transcriptomicsCircadian rhythm and melatoninHypothalamic control of reproductive hormones

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes

Open access
Romanov, Roman A, Zeisel, Amit, Bakker, Joanne, Girach, Fatima, Hellysaz, Arash, Tomer, Raju, Alpár, Alán, Mulder, Jan +23 more·2017·Nature Neuroscience
DOI 10.1038/nn.4462PMC PMC7615022
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Abstract

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

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Crossref · 10.1038/nn.4462

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Claims the paper makes

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  • Findingown data

    Differential mRNA expression subdivided Oxt and Avp neurosecretory cells into 3 and 4 subtypes, respectively.

    “Subsequent differential mRNA expression analysis subdivided Oxt and Avp neurosecretory cells, which project to the posterior lobe of the pituitary4, into 3 and 4 subtypes, respectively”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Swanson, L.W. & Kuypers, H.G. The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J. Comp. Neurol. 194, 555–570 (1980). source

      Not checked in the free run.

  • Findingown data

    The predicted 4th subtype of dopamine neurons (cluster #11; dopamine-4 cells) segregated from other dopamine clusters despite small sample size (6 neurons).

    “we set out to validate our data by choosing the predicted 4th subtype of dopamine neurons (cluster #11; “dopamine-4 cells”), which despite its small sample size (6 neurons) segregated from other dopamine clusters in our predictive model”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Small cluster size and lack of positional information can make single-cell RNA-seq data liable to false positives.
  • Findingown data

    Neuromedin S-containing neurons in the SCN co-express Vip (42%).

    “It is noteworthy that neuromedin S-containing neurons in the SCN co-express Vip (42%)”
    Quote in the paperNot a substantial claim
  • Findingown data

    Other Vip+ neurons that were Grp+/- and Nms- clustered separately.

    “Meanwhile, other Vip+ neurons that, however, were Grp+/- and Nms- clustered separately”
    Quote in the paperNot a substantial claim
  • Findingown data

    Multiple immunofluorescence labeling showed co-existence of VGLUT2 and GAD67 proteins in nerve endings at the median eminence.

    “we employed multiple immunofluorescence labeling to show the co-existence of VGLUT2 and GAD67 proteins in nerve endings at the median eminence (ME)”
    Quote in the paperSubstantial claim
  • May combine two claims. 46
  • Classified as probably not a substantial claim. 31
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Coincident detection of onecut-3 and phospho-Ser40-TH on a Th-GFP background revealed that these dopamine neurons populate the periventricular nucleus of the hypothalamus, with dendritic trees primarily oriented vertically.

    “Coincident detection of onecut-3 and enzymatically-active TH phosphorylated at residue Ser40 26 on a Th- GFP background revealed that these dopamine neurons populate the periventricular nucleus of the hypothalamus (Fig. 5d), with their dendritic trees being primarily oriented vertically”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Tekin, I., Roskoski, R. Jr., Carkaci-Salli, N. & Vrana, K.E. Complex molecular regulation of tyrosine hydroxylase. J. Neural Transm. 121, 1451–1481 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Vasopressin and VGLUT2, and vasopressin and GAD67, co-exist in nerve endings.

    “we revealed that even minute mRNA copy numbers can translate into meaningful biological modalities by showing the co-existence of vasopressin and VGLUT2 (Fig. 3e) and GAD67 (Fig. 3e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Glutamatergic neurons showed variability in neuropeptide gene expression; clusters #33-35 contained Trh, Bdnf and Adcyap1 mRNAs, while clusters #57-62 exhibited only marginal expression, particularly Cck.

    “glutamatergic neurons showed an apparent variability in their gene expression for neuropeptides since clusters #33-35 contained Trh, Bdnf and Adcyap1 mRNAs. In contrast, clusters #57-62 exhibited only marginal (if any) expression of genes for polypeptides (particularly Cck)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Across all 62 predicted neuronal clusters, Slc6a3 and Onecut3 were predominantly found in the dopamine-4 cell population, unlike redundant Th and Sst expression.

    “we broadened our specificity testing to all 62 predicted neuronal clusters and show that, as opposed to redundant Th and Sst expression, both Slc6a3 (DAT) and Onecut3 were predominantly found in this particular cell population”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Several neuronal clusters were dominated by neurons from male or female subjects, suggesting but not being causal to gender-specific coding.

    “Nevertheless, several neuronal clusters were dominated by neurons from male (e.g., #55) or female (#6) subjects, suggesting, but not being causal to, gender-specific coding”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Clusters #8-11 cells had low Gad1 and high Gad2, harbored Th, Slc18a2 and in some cases Slc6a3, qualifying them as dopamine neurons and suggesting dual dopamine/GABA neurotransmitter phenotypes.

    “This was particularly notable for clusters #8-11 cells (with low Gad1 and high Gad2) which also harbored Th, Slc18a2 (vesicular monoamine transporter 2, VMAT2) and in some cases, Slc6a3 (encoding dopamine transporter 1, DAT), qualifying them as dopamine neurons and suggesting the existence of dual dopamine/GABA neurotransmitter phenotypes”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Everitt, B.J., Hökfelt, T., Wu, J.Y. & Goldstein, M. Coexistence of tyrosine hydroxylase-like and gamma-aminobutyric acid-like immunoreactivities in neurons of the arcuate nucleus. Neuroendocrinology 39, 189–191 (1984). source

      Not checked in the free run.

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

    Findingown data

    Th and tyrosine hydroxylase protein in Oxt/Avp neurons appear to lack AADC, so their significance remains unclear.

    “The significance of Th and tyrosine hydroxylase protein in these neurons remains unclear since they appear to lack the gene for aromatic amino-acid decarboxylase (AADC”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Qrfp- and Npvf-expressing hypothalamic neurons are both glutamatergic and segregate into clusters #35 and #37, respectively.

    “Accordingly, Qrfp- and Npvf-expressing hypothalamic neurons are both glutamatergic and segregate into clusters #35 and #37, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cluster #62-enriched genes included Zfp458 and Ppp1r12b, whereas cluster #61 neurons were enriched in Pou2f2.

    “#62-enriched genes included Zfp458, Ppp1r12b, whereas neurons in #61 were enriched in Pou2f2”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Adcyap1r1 and Oprl1 are promiscuously expressed in most neuronal clusters, whereas Nmur2 and Nmbr are highly specific for dopamine-4 neurons.

    “Whereas Adcyap1r1 and Oprl1 are promiscuously expressed in most neuronal clusters (data not shown), Nmur2 and Nmbr are highly specific for dopamine-4 neurons”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Qrfp+ and Npvf hypothalamic neurons co-express Hcrt and Gal, respectively.

    “we resolved that Qrfp+ and Npvf hypothalamic neurons co-express the genes for orexin/hypocretin (Hcrt; Supplementary Fig. 6b) and galanin (Gal; Supplementary Fig. 6c), respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neurotransmitter identities included glutamatergic clusters #1-7 and #30-62, dopaminergic clusters #8-11, and GABAergic clusters #12-29.

    “Neurotransmitter identities included glutamatergic (clusters #1-7 and #30-62), dopaminergic (#8-11) and GABAergic (#12-29) cells.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Pomc gene expression depicted a clustering example.

    “As such, proopiomelanocortin (Pomc gene) depicted a clustering example”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Dopamine-4 neurons (A14, cluster #11) express DAT (Slc6a3), contrasting ventrolateral Arc/A12 neurons, but being similar to the dorsomedial A12 group.

    “We show that dopamine-4 neurons (A14, cluster #11) express the gene for DAT (Slc6a3; Fig. 6a), contrasting ventrolateral Arc/A12 neurons, but being in Slc6a3 expression similar to the dorsomedial A12 group 28.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Meister, B. & Elde, R. Dopamine transporter mRNA in neurons of the rat hypothalamus. Neuroendocrinology 58, 388–395 (1993). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Histochemical detection of TH using COLM demonstrated that most TH+ axons leaving the A14 area project laterally using the supraoptic decussation and upper lateral pathway and terminate outside the hypothalamus, likely in basolateral and central amygdaloid nuclei.

    “histochemical detection of TH using COLM technology demonstrated that most TH+ axons leaving the A14 area project laterally using the supraoptic decussation and upper lateral pathway and terminate outside the hypothalamus, likely in basolateral and central amygdaloid nuclei”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Lightsheet microscopy revealed 474 onecut-3+ cells in an adult mouse brain, while conventional neuroanatomy projected 500-1,000 cells/brain.

    “Lightsheet microscopy revealed 474 cells in an adult mouse brain (Fig. 5f, Supplementary Movies 1,2), while conventional neuroanatomy in serial sections projected 500-1,000 cells/brain.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Non-magnocellular cells are more heterogeneous and can be separated into GABAergic and glutamatergic subtypes.

    “In comparison, non-magnocellular cells present a more heterogeneous trait, and can be separated into “GABAergic” (Gad1/Gad2/Slc32a1) and “glutamatergic” (Slc17a6) subtypes20.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Krashes, M.J. et al. An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature 507, 238–242 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    TH+ onecut-3+ cells have significantly lower GFP signal than other TH+ cell groups, while their phospho-Ser40 levels are distinctly elevated.

    “When quantifying GFP intensity in these TH+ cells, we find that their GFP signal is significantly lower than that of other TH+ cell groups (Supplementary Fig. 7c), while their phospho-Ser40 levels are distinctly elevated”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dopamine-4 cells lacked neuropeptides abundant in other dopamine subclasses like Ghrh1 and Tac1, and partly Npy2r.

    “In turn, these cells lacked neuropeptides that were abundant in other dopamine subclasses like Ghrh1 and Tac1 (Fig. 5b), and partly Npy2r, the gene encoding neuropeptide Y receptor Y2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Npy6r mRNA is confined to Vip+/Nms+ circadian neurons (cluster #41) and is not detected in Vip-only cells in the adjacent circadian cluster (#40).

    “our data reveal that pancreatic polypeptide receptor (Npy6r) mRNA is confined to Vip+/Nms+ circadian neurons (cluster #41) leaving Vip-only neurons of the neighboring circadian cluster (#40) unchecked.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Lepr is a low-expressed gene (1 mRNA copy per cell) and is detected in only about 3% of hypothalamic neurons.

    “our analysis of leptin receptor (Lepr) expression whose activity upon binding white fat-derived leptin 41 is associated with satiety, shows that Lepr is a low-expressed gene (1 mRNA copy/per cell) and it is only detected in ~3% of hypothalamic neurons.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Friedman, J.M. & Halaas, J.L. Leptin and the regulation of body weight in mammals. Nature 395, 763–770 (1998). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Per2 was also expressed in cluster #42, where 23% of Per2+ neurons express Nms.

    “Significantly, Per2 was also expressed in cluster #42 (Fig. 8c, Supplementary Fig. 8f), where 23% of Per2+ neurons express Nms”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    These newly identified cells lie only in the medio-posterior periventricular area; they get Nms+ input from SCN clock neurons and clock genes are expressed in periventricular and Arc dopamine cells.

    “These newly distinguished neurons are exclusively situated at the medio-posterior subdivision of the periventricular area and receive Nms + synaptic afferents from SCN neurons regulating the circadian clock 32, 48, in addition to the expression of clock genes in the periventricular and Arc dopamine cells themselves 49.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [32] Mori, K. et al. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. EMBO J. 24, 325–335 (2005). source

      Not checked in the free run.

    • cites [48] DeMaria, J.E., Lerant, A.A. & Freeman, M.E. Prolactin activates all three populations of hypothalamic neuroendocrine dopaminergic neurons in ovariectomized rats. Brain Res. 837, 236–241 (1999). source

      Not checked in the free run.

    • cites [49] Lee, I.T. et al. Neuromedin s-producing neurons act as essential pacemakers in the suprachiasmatic nucleus to couple clock neurons and dictate circadian rhythms. Neuron 85, 1086–1102 (2015). source

      Not checked in the free run.

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

    Findingown data

    Some glutamatergic neurons were found to express Lepr mRNA, in agreement with earlier publications.

    “Moreover, we characterized Lepr mRNA expression in some glutamatergic neurons, corroborating published reports42.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Scott, M.M. et al. Leptin targets in the mouse brain. J. Comp. Neurol. 514, 518–532 (2009). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Neuromedin S co-localizes with VAMP2, marking presynapses, in close apposition to predominantly proximal dendrites of TH+/onecut-3+ periventricular neurons.

    “we find that neuromedin S co-localizes with vesicle-associated membrane protein 2 (VAMP2), marking presynapses, in close apposition to predominantly proximal dendrites of TH+/onecut-3+ periventricular neurons”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TH phosphorylation at Ser40, known to mark increased enzymatic activity and dopamine production, showed a distinct difference in the rising slope of cumulative distribution function (p = 0.0167), suggesting different levels of active TH enzyme at day and night periods.

    “This mark showed a distinct difference in the rising slope of cumulative distribution of function (CDF; p = 0.0167), suggesting different levels of active TH enzyme at day and night periods”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Tekin, I., Roskoski, R. Jr., Carkaci-Salli, N. & Vrana, K.E. Complex molecular regulation of tyrosine hydroxylase. J. Neural Transm. 121, 1451–1481 (2014). source

      Not checked in the free run.

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

    Findingown data

    A primary coarse filter identified 9,013 genes as neuronal by t-test enrichment with a 5% false discovery rate.

    “To remove noise prior to clustering, we first used a primary (coarse) filter and selected for genes, which were distinguished by t-test to be enriched in neurons using a false discovery rate (FDR) of 5% (results in 9,013 neuronal genes in total).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Level 2 analysis used 898 cells identified as neurons by level 1 clustering.

    “Data were from 898 cells, which qualified as neurons based on level 1 clustering.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Drop-seq data processing was performed on 220 neurons.

    “Drop-seq data processing of 220 neurons as output was performed using the Drop-seq Tools v1.12 software22.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Macosko, E.Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015). source

      Not checked in the free run.

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

    Findingown data

    The refinements resulted in 62 neuronal sub-clusters.

    “These refinements led to 62 neuronal sub-clusters being discerned.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Cells with more than 1,500 molecules per cell (excluding rRNA, mitochondrial RNA and repeats) were analyzed, resulting in a total of 3,131 cells.

    “Cells with more than 1,500 molecules/cell (excluding rRNA, mitochondrial RNA and repeats) were analyzed, and resulted in a total of 3,131 cells.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    BackSpinV2 distinguished 91 neuronal clusters.

    “This step distinguished 91 neuronal clusters (Fig. 1c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Neuromedin S is the endogenous agonist at neuromedin U receptor 2, with its production concentrated in the SCN.

    “Neuromedin S is the endogenous agonist at neuromedin U receptor 2, with its production being concentrated in the SCN32.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] Mori, K. et al. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. EMBO J. 24, 325–335 (2005). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Hypothalamic neurons might either co-release neurotransmitters, segregate them to subsets of synapses, or switch between modes of neurotransmission in response to specific inputs.

    “these data reinforce our conclusion that hypothalamic neurons might either co-release neurotransmitters, segregate them to subsets of synapses or switch between modes of neurotransmission in response to specific inputs”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [16] Pinto, S. et al. Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 304, 110–115 (2004). source

      Not checked in the free run.

    • cites [17] Cristino, L. et al. Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons. Proc. Natl. Acad. Sci. USA 110, E2229–E2238 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    A14 dopamine neurons are bona fide neurosecretory cells integrated into (extra-)hypothalamic neuronal networks.

    “Thus, we suggest that A14 dopamine neurons are bona fide neurosecretory cells (see also31), which are integrated into (extra-) hypothalamic neuronal networks.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Goudreau, J.L., Lindley, S.E., Lookingland, K.J. & Moore, K.E. Evidence that hypothalamic periventricular dopamine neurons innervate the intermediate lobe of the rat pituitary. Neuroendocrinology 56, 100–105 (1992). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Onecut-3+ dopamine neurons form a cell continuum spanning the SCN-Arc domain with an increasing cell gradient caudally, qualifying as A14 dopamine neurons.

    “we show that onecut-3+ dopamine neurons form a cell continuum spanning the SCN-Arc domain with an increasing cell gradient caudally, thus qualifying as “A14” dopamine neurons6, 27.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [6] Björklund, A., Moore, R.Y., Nobin, A. & Stenevi, U. The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain. Brain Res. 51, 171–191 (1973). source

      Not checked in the free run.

    • cites [27] Hökfelt, T., Martensson, R., Bjorklund, A., Kleinau, S. & Goldstein, M. Distribution maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. in Handbook of Chemical Neuroanatomy (eds. Bjorklund, A. & Hökfelt, T.) 277–379 (Elsevier, 1984).

      Not checked in the free run.

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

    Interpretationcitation

    Hypothalamic dopamine neurons release their contents either at the ME into the hypophyseal portal system or use DAT to remove superfluous dopamine at central synapses.

    “These data also recapitulate the known dichotomy of hypothalamic dopamine neurons releasing their contents either at the ME into the hypophyseal portal system 29 or using DAT to remove superfluous dopamine at central synapses.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Ben-Jonathan, N. & Hnasko, R. Dopamine as a prolactin (PRL) inhibitor. Endocr. Rev. 22, 724–763 (2001). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Qrfp and Npvf neurons are bona-fide excitatory neuropeptidergic subtypes.

    “we assign these neurons as bona-fide excitatory neuropeptidergic subtypes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The dorsomedial A12 neuronal pool contains GAD21 and is a source of dopamine inhibiting prolactin release.

    “The latter neuronal pool also contains GAD21 and is a source of dopamine inhibiting prolactin release 29.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Ben-Jonathan, N. & Hnasko, R. Dopamine as a prolactin (PRL) inhibitor. Endocr. Rev. 22, 724–763 (2001). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Neuromedin S release into the CSF would reconcile the difference between input concentration and Nmur2 expression and provide an alternative means of long-distance signaling.

    “neuromedin S release into the CSF would reconcile this difference and provide an alternative means of long-distance signaling.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The molecular profiling covered only the central column of the hypothalamus; lateral and caudal segments were not charted.

    “Our molecular profiling focused on the central column of the hypothalamus, leaving its most lateral and caudal segments uncharted.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    A gene may be expressed episodically at some point in a neuron's life, and this could be a usual phenomenon.

    “showing that episodic expression of any gene during the lifetime of a neuron might be considered a common feature.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The authors tried to reduce animal numbers and suffering in the experiments.

    “Particular effort was directed towards minimizing the number of animals used and their suffering during experiments.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A neuron is thought to have at least one fast neurotransmitter, possibly co-released with another, plus one or more peptide, gaseous or lipid neuromodulators.

    “By consensus definition, each neuron is expected to contain at least one fast neurotransmitter, which can be co-released with another neurotransmitter (e.g. dopamine), one or more peptide, gaseous or lipid neuromodulator20, 43–45.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [20] Krashes, M.J. et al. An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature 507, 238–242 (2014). source

      Not checked in the free run.

    • cites [43] Boulland, J.L. et al. Vesicular glutamate and GABA transporters sort to distinct sets of vesicles in a population of presynaptic terminals. Cereb. Cortex 19, 241–248 (2009). source

      Not checked in the free run.

    • cites [44] Dawson, T.M. & Snyder, S.H. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J. Neurosci. 14, 5147–5159 (1994). source

      Not checked in the free run.

    • cites [45] Jonas, P., Bischofberger, J. & Sandkühler, J. Corelease of two fast neurotransmitters at a central synapse. Science 281, 419–424 (1998). source

      Not checked in the free run.

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

    Premisecitation

    Cortical interneurons show higher mRNA transcript levels and express both Gad1 and Gad2.

    “When comparing single-cell RNA-seq data from cortical/hippocampal vs. hypothalamic neurons, the following differences apply: i) in the cerebral cortex, higher mRNA transcript levels are seen in interneurons, which also contain both Gad1 and Gad2 transcripts, and ii) cortical neurons rarely, if at all, produce mixed excitatory/inhibitory phenotypes 9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In the Arc (A12), Th+ parvocellular dorsomedial neurosecretory cells form large ME nerve endings, making up one-third of boutons in the lateral external layer, and release dopamine to tonically inhibit pituitary prolactin.

    “For dopamine, parvocellular dorsomedial neurosecretory cells (Th+) in the Arc (A12) give rise to large nerve endings in the ME, amassing 1/3 of all boutons in the lateral external layer 46, and release dopamine for the tonic inhibition of pituitary prolactin release29.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [46] Ajika, K. & Hökfelt, T. Ultrastructural identification of catecholamine neurones in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects. Brain Res. 57, 97–117 (1973). source

      Not checked in the free run.

    • cites [29] Ben-Jonathan, N. & Hnasko, R. Dopamine as a prolactin (PRL) inhibitor. Endocr. Rev. 22, 724–763 (2001). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    This circuit combines the circadian clock with hormonal output, including daily changes in circulating dopamine.

    “This synaptic circuit is significant, since it integrates the circadian pacemaker (characterized by Per genes) 33, 34and hormonal output, particularly diurnally fluctuating dopamine levels in the general circulation 7.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [33] Zhang, L. et al. A PERIOD3 variant causes a circadian phenotype and is associated with a seasonal mood trait. Proc. Natl. Acad. Sci. USA 113, E1536–E1544 (2016). source

      Not checked in the free run.

    • cites [34] Vitaterna, M.H. et al. Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc. Natl. Acad. Sci. USA 96, 12114–12119 (1999). source

      Not checked in the free run.

    • cites [7] Horvath, T.L. Suprachiasmatic efferents avoid phenestrated capillaries but innervate neuroendocrine cells, including those producing dopamine. Endocrinology 138, 1312–1320 (1997). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The transgenic reporter lines used for neuronal identity mapping were extensively validated.

    “The reporter lines GAD67+/gfp, GAD67+/gfp::CCKBAC/DsRedT3 52, 53, B6.B6D2-Tg(Th-EGFP)21-31Koba (developed by K. Kobayashi and available from Riken Bioresource Center), Crh-IRES-Cre::B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen 1)Hze/J (Ai6; both GFP reporters were from the Jackson laboratories, #012704 and #007906) and dopamine transporter (Dat1)-Cre::B6.Cg-Gt(ROSA)26Sortm14(CAG-dTomato)Hze/J mice were”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [52] Máté, Z. et al. Spatiotemporal expression pattern of DsRedT3/CCK gene construct during postnatal development of myenteric plexus in transgenic mice. Cell Tissue Res. 352, 199–206 (2013). source

      Not checked in the free run.

    • cites [53] Pierreux, C.E., Vanhorenbeeck, V., Jacquemin, P., Lemaigre, F.P. & Rousseau, G.G. The transcription factor hepatocyte nuclear factor-6/Onecut-1 controls the expression of its paralog Onecut-3 in developing mouse endoderm. J. Biol. Chem. 279, 51298–51304 (2004). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Cortical cells seldom show mixed excitatory and inhibitory phenotypes.

    “When comparing single-cell RNA-seq data from cortical/hippocampal vs. hypothalamic neurons, the following differences apply: i) in the cerebral cortex, higher mRNA transcript levels are seen in interneurons, which also contain both Gad1 and Gad2 transcripts, and ii) cortical neurons rarely, if at all, produce mixed excitatory/inhibitory phenotypes 9.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [9] Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015). source

      Not checked in the free run.

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

    Study design and conductown data

    Both sexes were used, and animals had free access to food.

    “An additional strength of our dataset is that it relies on both male and female animals kept under ad lib feeding conditions, suggesting that we resolved ground-state Lepr mRNA expression, relevant for behavioral output in both sexes (see also Supplementary Fig. 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    This Npy6r finding supports earlier work and improves understanding of receptor-dependent pancreatic polypeptide function.

    “This result lends significant support to previous data 40 and advances knowledge in receptor-dependent function determination for pancreatic polypeptide.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Yulyaningsih, E. et al. Pancreatic polypeptide controls energy homeostasis via Npy6r signaling in the suprachiasmatic nucleus in mice. Cell Metab. 19, 58–72 (2014). source

      Not checked in the free run.

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

    Study design and conductnone given

    Cells from males and females were processed in random order to prevent bias.

    “The processing of cells from male and female animals was random to avoid any methodological bias.”
    Quote in the paperNot a substantial claim

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

    Interpretationcitation

    Using alternative neurotransmitter pathways may cost less energy than rewiring.

    “These might be bioenergetically more favourable than rewiring strategies 16, 17.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [16] Pinto, S. et al. Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 304, 110–115 (2004). source

      Not checked in the free run.

    • cites [17] Cristino, L. et al. Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons. Proc. Natl. Acad. Sci. USA 110, E2229–E2238 (2013). source

      Not checked in the free run.

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

    Study design and conductcitation

    Sample sizes were not pre-determined statistically; they resemble those in cortex and hippocampus studies.

    “No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported for the cerebral cortex and hippocampus 9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015). source

      Not checked in the free run.

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

    Study design and conductown data

    Experimental protocols were approved by regional ethical committees and complied with applicable laws.

    “Experimental protocols were in accordance with the European Communities Council Directive (86/609/EEC), approved by regional ethical committees and regulated by applicable local laws (Stockholms Norra Djurförsöksetiska Nämnd; N512/12 (Sweden) and Tierversuchsgesetz 2012, BGBI, Nr. 114/2012 (Austria)).”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    Such an arrangement may help hypothalamic networks remain flexible under changing environmental conditions.

    “This arrangement could contribute to the flexibility of hypothalamic neuronal networks to adapt to ever-changing environmental stimuli.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The findings suggest a possible cell substrate for dopamine release into the hypophyseal portal system or intermediate lobe, which may inhibit pituitary prolactin at specific times.

    “Our data outline a potential cellular substrate that may participate in dopamine release into the hypophyseal portal system and/or the intermediate lobe for the chronospecific inhibition of pituitary prolactin secretion 29 (Fig. 6g,h).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Ben-Jonathan, N. & Hnasko, R. Dopamine as a prolactin (PRL) inhibitor. Endocr. Rev. 22, 724–763 (2001). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    After every capture, quality checks removed debris and doublets.

    “Capture quality for exclusion of debris or doublets was performed after each capture experiment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Combining single-cell RNA-seq with neuroanatomy can yield a molecular map of hypothalamic organization and help find functionally separate neuronal subclasses.

    “Cumulatively, we recognize onecut-3+ A14 dopamine neurons as distinctly different – at the level of molecular make-up and connectivity – from the dorsomedial A12 dopamine group; thus showcasing that single-cell RNA-seq in combination with neuroanatomy can produce a molecular protomap of hypothalamic neuronal organization and aid the future discovery of functionally-segregated neuronal subclasses.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Each tree junction was explained using two independent measures: the mean difference and the difference in the proportion of positive cells.

    “To this end, we used two independent criteria: the difference in the average and the difference in the fraction of positive (>0) cells (Table S3).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Clusters that showed a high percentage of similarity, with none of the key markers differing between the two cell populations, were merged.

    “and merged clusters that showed a high percentage of similarity, i.e. none of the key markers differed between the two cell populations.”
    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 conductown data

    Only cells passing visual QC were imported, with molecule counts.

    “Data from all cells that passed visual quality control were imported as molecule counts (including all metadata annotations).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Clusters identified as thalamus specific by known thalamic homeobox genes and transcription factors were excluded.

    “Moreover, we omitted clusters that appeared thalamus specific because of the presence of known thalamus-specific homeobox genes and transcription factors.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Counts included only neurons below the PVN and within 120 μm of the third ventricle's lateral wall, on both sides.

    “Only neurons situated ventral to the PVN and <120 μm from the lateral wall of the third ventricle were counted at both hemispheres (Fig. 5d, Supplementary Fig. 7d).”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Putative artifactual clusters that formed due to low or variable RNA quality were manually removed.

    “Next, we manually removed putative artifactual clusters that had formed due to low/variable quality of RNA”
    Quote in the paperNot a substantial claim

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

    Study design and conductcitation

    The BackSPIN algorithm has been extensively validated using computational and biological validation tools.

    “The algorithm has by now been extensively validated using both computational (including comparisons to PCA, and independent-component analysis, tSNE, and GP-LVM) biological validation tools 9, 63–66.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [9] Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015). source

      Not checked in the free run.

    • cites [63] Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011). source

      Not checked in the free run.

    • cites [64] Islam, S. et al. Quantitative single-cell RNA-seq with unique molecular identifiers. Nat. Methods 11, 163–166 (2014). source

      Not checked in the free run.

    • cites [65] Tsafrir, D. et al. Sorting points into neighborhoods (SPIN): data analysis and visualization by ordering distance matrices. Bioinformatics 21, 2301–2308 (2005). source

      Not checked in the free run.

    • cites [66] Marques, S. et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352, 1326–1329 (2016). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    The q value was the p value adjusted for multiple comparisons across the 62 clusters using the Benjamini-Hochberg method to control the false discovery rate.

    “The q value was calculated as the p value corrected for multiple testing using horizontal correction across the 62 clusters with the Benjamini-Hochberg procedure 75 to control the false discovery rate (FDR).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [75] Ose, A. et al. Functional characterization of mouse organic anion transporting peptide 1a4 in the uptake and efflux of drugs across the blood-brain barrier. Drug Metab. Dispos. 38, 168–176 (2010). source

      Not checked in the free run.

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

    Study design and conductnone given

    No experiment required blinded data collection.

    “None of the experiments required data collection in a blinded fashion.”
    Quote in the paperNot a substantial claim
68 of 68

  • The paper relies on this without support.

    The hypothalamus lacks distinct layering or stringent anatomical organizational principles.

    “In contrast to the cerebellum and cerebral cortex, the hypothalamus lacks distinct layering or other stringent anatomical, repetitive organizational principles.”

  • The paper relies on this without support.

    The degree of molecular diversity among hypothalamic dopamine neurons is unknown.

    “However, the degree of molecular diversity amongst dopamine neurons remains unknown.”

  • The paper relies on this without support.

    There is no consensus on or direct correlation between neurotransmitter and neuropeptide expression patterns.

    “However, there is no consensus on, or direct correlation between, neurotransmitter-neuropeptide expression patterns.”

  • The paper relies on this without support.

    Small cluster size and lack of positional information can make single-cell RNA-seq data liable to false positives.

    “However, the often small cluster size (< 10 cells) and the lack of positional information per se could render single-cell RNA-seq data liable to false positive outcomes.”

  • The paper relies on this without support.

    The hypothalamus has many neuronal subtypes that are functionally different yet intermingled in small volumes, which makes it hard to study.

    “In contrast, the study of brain regions that contain many functionally distinct, but positionally interspersed, neuronal subtypes in small volumes, such as the hypothalamus, remains a significant challenge.”

  • The paper relies on this without support.

    Any new method requires extensive validation.

    “Any new method requires extensive validation.”

  • The paper relies on this without support.

    Perfusion through the internal carotid and vertebral arteries preserves tissue integrity better than other fixation methods.

    “We have applied direct perfusion via the internal carotid and vertebral arteries, which facilitated the preservation of tissue integrity relative to alternative fixation methods.”
7 of 7

Claims other papers make about this one

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

  • Transcriptomically derived taxonomies in adult mammalian brain consistently show a hierarchical organization of cell types.

    “Transcriptomically derived cell type taxonomies in the adult mammalian brain, with majority of the studies conducted in mouse, have consistently revealed a hierarchical organization of cell types (Fig. 2) (Brain Initiative Cell Census Network, 2021; Macosko et al., 2015; Romanov et al., 2017; Russ et al., 2021; Saunders et al., 2018; Shekhar et al., 2016; Tasic et al., 2016; Tasic et al., 2018;”

    From What is a cell type and how to define it? (2022) · cites this paper as Romanov et al., 2017

  • Single-cell sequencing has the potential to identify cell-type-specific molecular targets for the development of new pharmaceuticals that would affect specific neural circuits rather than the entire brain.

    “Moreover, single-cell sequencing has the potential to identify cell-type-specific molecular targets for the development of new pharmaceuticals that would affect specific neural circuits rather than the entire brain [69].”

    From Translational opportunities for circuit-based social neuroscience: advancing 21st century psychiatry (2021) · cites this paper as [69]

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.

  • Glutamatergic cluster ranges disagree

    Minor

    One passage places clusters #28-29 among the glutamatergic cells, while the stated neurotransmitter classification assigns both to the GABAergic group. The claimed 33 glutamatergic clusters matches #30-62, not #28-62.

1 of 1

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Authors
Romanov, R. A., Zeisel, A., Bakker, J., Girach, F., Hellysaz, A., Tomer, R., Alpár, A., Mulder, J., Clotman, F., Keimpema, E., Hsueh, B., Crow, A. K., Martens, H., Schwindling, C., Calvigioni, D., Bains, J. S., Máté, Z., Szabó, G., Yanagawa, Y., Zhang, M.-D., Rendeiro, A., Farlik, M., Uhlén, M., Wulff, P., Bock, C., Broberger, C., Deisseroth, K., Hökfelt, T., Linnarsson, S., Horvath, T. L., & Harkany, T.
Affiliations
  • Medical University of Vienna (AT)
  • Karolinska Institutet (SE)
  • Stanford University (US)
  • Semmelweis University (HU)
  • Science for Life Laboratory (SE)
  • UCLouvain (BE)
  • University of Calgary (CA)
  • HUN-REN Institute of Experimental Medicine (HU)
  • Gunma University (JP)
  • Austrian Academy of Sciences (AT)
  • Christian-Albrechts-Universität zu Kiel (DE)
ORCID
Venue
Nature Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2016-12-19
Volume
20
Issue
2
Pages
176-188
ISSN
  • 1097-6256 (ISSN-L)
  • 1546-1726
DOI
10.1038/nn.4462
PMC
PMC7615022
OpenAlex
W2562003322
Cited by
502
References
80
Citations by year
2026: 25 · 2025: 54 · 2024: 42 · 2023: 75 · 2022: 58 · 2021: 58 · 2020: 56 · 2019: 37 · 2018: 66 · 2017: 29 · 2016: 1
FWCI
22.91
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Neuroscience (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Digital Access to Libraries (submittedVersion, other-oa)
Funders
  • Semmelweis Egyetem
  • Novo Nordisk Fonden (NNF17OC0027294, NNF15OC0015964, NNF14OC0011061, NNF16OC0021482, NNF14OC0010695, NNF15OC0016258)
  • National Institute on Aging (P01AG051459)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Neuroscience › Endocrine and Autonomic Systems › Circadian rhythm and melatonin
  • Health Sciences › Medicine › Reproductive Medicine › Hypothalamic control of reproductive hormones
Keywords
hypothalamus, dopaminergic neurons, neuronal cell types, single-cell RNA sequencing, neuropeptides, neurotransmitters, circadian clock, neuronal organization
MeSH terms
Animals, Dopamine, Hypothalamus, Immunohistochemistry, Mice, Inbred C57BL, Mice, Transgenic, Synaptic Transmission, Neuropeptides, Suprachiasmatic Nucleus, Tyrosine 3-Monooxygenase, Neurotransmitter Agents, Dopaminergic Neurons

About these checks

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

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

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

Statistics and arithmetic are recomputed, not judged.

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