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Cited by 238Single-cell and spatial transcriptomicsNeurotransmitter Receptor Influence on BehaviorNeuroscience and Neuropharmacology Research

Classification of midbrain dopamine neurons using single-cell gene expression profiling approaches

Open access
Poulin, Jean-Francois, Gaertner, Zachary, Moreno-Ramos, Oscar Andrés, Awatramani, Rajeshwar·2020·Trends in Neurosciences
DOI 10.1016/j.tins.2020.01.004PMC PMC7285906
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Abstract

Dysfunctional dopamine (DA) signaling has been associated with a broad spectrum of neuropsychiatric disorders, prompting investigations into how midbrain DA neuron heterogeneity may underpin this variety of behavioral symptoms. Emerging literature indeed points to functional heterogeneity even within anatomically defined DA clusters. Recognizing the need for a systematic classification scheme, several groups have used single-cell profiling to catalog DA neurons based on their gene expression profiles. Here we aim to synthesize points of congruence, but also highlight key differences, between the molecular classification schemes derived from these studies. In doing so, we hope to provide a common framework that will facilitate investigations into the functions of dopaminergic neuron subtypes in the healthy and diseased brain.

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Written from the paper’s full text.

TL;DR

This review synthesizes single-cell gene expression studies of mouse midbrain dopamine neurons and proposes seven putative molecular subtypes, four more robustly identified and three more ambiguous. It argues that molecular classification revises traditional anatomical schemes and provides a framework for linking dopamine neuron subtypes to function and disease.

Key findings

  • At least six independent groups profiled individual mouse midbrain dopamine neurons; the studies identified partially overlapping subtypes but differed in subtype number, defining markers, and naming conventions, partly due to differences in cell source, isolation, developmental stage, technical approach, and classification criteria.
  • The review proposes seven putative dopamine neuron subtypes defined by molecular signatures, including Aldh1a1+, Sox6+ ventral SNc neurons; Sox6+, Aldh1a1− SNc/PBP VTA/RR neurons; Vgat+ mostly VTA neurons; Otx2+, Aldh1a1+ ventromedial VTA neurons; Vip+ PAG/DR neurons; and an ambiguous Vglut2+, Calb1+ group likely encompassing two or more distinct populations.
  • Reported proportions include: Vgat+ dopamine neurons suggested to comprise less than 5% of all adult midbrain dopamine neurons in one study; Otx2+, Aldh1a1+ neurons roughly 12–36% of VTA dopamine neurons depending on quantification; Vip+ neurons about 49% of dopamine neurons in PAG/DR regions; and only 8% of Vglut2-Cre, Th-Flpo-labeled dorsal SNc/SNpl cells reported as Aldh1a1+.
  • The paper notes that human brain dopamine neurons number less than 500,000 cells and that dysfunctional dopamine signaling has been implicated in disorders including Parkinson’s disease, depression, chronic pain, drug addiction, schizophrenia, and ADHD.

Methods

  • This is a review and synthesis, not a primary experimental study. It compares single-cell profiling studies of mouse midbrain dopamine neurons, focusing almost exclusively on neurons expressing the dopamine transporter Dat/Slc6a3.
  • The authors synthesize points of congruence and difference across at least six independent single-cell studies, interpret classification schemes from studies that did not validate all clustering data, and propose a consensus classification correlated where possible with anatomical, projection, physiological, and developmental properties.

Limitations

  • Stated: single-cell transcriptomics has limitations (Box 1; reviewed in [10]); discrepancies across studies arise from different experimental parameters; several subtypes remain ambiguous or lack specific markers; some markers have not been validated by secondary methods; and functional/anatomical corroboration is still needed.
  • Stated: the Vglut2+, Calb1+ population may encompass multiple subtypes; Sox6+, Aldh1a1− neurons require additional markers and Cre drivers; Vip+ dopamine neuron function has never been examined in isolation; and NeuroD6-based drivers are expressed in non-dopamine VTA neurons, so experiments using them must be interpreted cautiously.
  • Evident: as a review, the proposed consensus taxonomy is interpretive and depends on reanalysis and best interpretation of prior studies rather than new primary data.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.tins.2020.01.004

Cites

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Statistics

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Claims the paper makes

0 of 121 cited sources checked · 0 supported · 0 flagged

  • Premisecitation

    The Aldh1a1+, Sox6+ dopamine neuron subtype also displays Aldh1a7 and Ndnf mRNA expression.

    “These cells also display Aldh1a7 [14,17] and Ndnf [13,14] mRNA expression.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [14] La Manno G (2016) Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566– 580.e19 27716510 PMC5055122 source

      Not checked in the free run.

    • cites [17] Kramer DJ (2018) Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability. eNeuro 5, ENEURO.0152–18.2018 PMC6104179 30135866 source

      Not checked in the free run.

    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

  • Premisecitation

    The majority of dopamine neurons are located in three anatomical clusters in the midbrain: the substantia nigra pars compacta, the ventral tegmental area, and the retrorubral area.

    “The majority of DA neurons are located in three anatomical clusters in the midbrain, namely the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA), and retrorubral area (RR) [2,3].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [2] Björklund A and Dunnett SB (2007) Dopamine neuron systems in the brain: an update. Trends Neurosci. 30, 194– 202 17408759 source

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    • cites [3] Dahlstroem A and Fuxe K (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. i. demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol Scand Suppl 14229500

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  • Premisecitation

    Molecular heterogeneity within the murine dopaminergic system has been investigated by at least six independent groups, all attempting to classify dopamine neurons based on gene expression.

    “Molecular heterogeneity within the murine DAergic system has been investigated by at least six independent groups, all attempting to classify DA neurons based on gene expression [13–18].”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

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    • cites [14] La Manno G (2016) Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566– 580.e19 27716510 PMC5055122 source

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    • cites [15] Tiklova K (2019) Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development. Nat Commun 10, 581– 12 30718509 PMC6362095 source

      Not checked in the free run.

    • cites [16] Hook PW (2018) Single-Cell RNA-Seq of Mouse Dopaminergic Neurons Informs Candidate Gene Selection for Sporadic Parkinson Disease. Am. J. Hum. Genet. 102, 427– 446 29499164 PMC5985341 source

      Not checked in the free run.

    • cites [17] Kramer DJ (2018) Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability. eNeuro 5, ENEURO.0152–18.2018 PMC6104179 30135866 source

      Not checked in the free run.

    • cites [18] Saunders AB (2018) Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell 174, 1015– 1030.e16 30096299 PMC6447408 source

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  • Interpretationcitation

    Aldh1a1 and Sox6 mRNAs are defining markers for a dopamine neuron subtype located in the ventral substantia nigra pars compacta.

    “Aldh1a1 and Sox6 mRNAs are the defining markers for this DA neuron subtype, a population overtly represented in two studies: DA1A in [13] and DA-SNC in [14].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

    • cites [14] La Manno G (2016) Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566– 580.e19 27716510 PMC5055122 source

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  • Premisecitation

    Identifying molecularly distinct neuronal populations requires measuring multiple markers in individual cells.

    “Indeed, the identification of molecularly distinct neuronal populations requires the measurement of multiple markers in individual cells (reviewed in [10–12]).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [10] Poulin J-F (2016) Disentangling neural cell diversity using single-cell transcriptomics. Nat. Neurosci. 19, 1131– 1141 27571192 source

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    • cites [11] Zeng H and Sanes JR (2017) Neuronal cell-type classification: challenges, opportunities and the path forward. Nat. Rev. Neurosci. 18, 530– 546 28775344 source

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    • cites [12] Tasic B (2018) Single cell transcriptomics in neuroscience: cell classification and beyond. Curr. Opin. Neurobiol. 50, 242– 249 29738987 source

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  • May combine two claims. 26
  • Classified as probably not a substantial claim. 5

  • May combine two claims.

    Premisecitation

    ALDH1A1+ dopamine neurons of the substantia nigra pars compacta project prominently to the dorsal caudate putamen, with fibers being denser in rostral sections and less dense caudally.

    “Prominent projections of ALDH1A1+ DA neurons of the SNc have been observed in the dorsal caudate putamen (CP; [20–22]), with fibers being more dense in rostral sections and less so in caudal regions.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [20] Poulin J-F (2018) Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat. Neurosci. 21, 1260– 1271 30104732 PMC6342021 source

      Not checked in the free run.

    • cites [21] Sgobio C (2017) Aldehyde dehydrogenase 1-positive nigrostriatal dopaminergic fibers exhibit distinct projection pattern and dopamine release dynamics at mouse dorsal striatum. Sci Rep 7, 5283– 13 28706191 PMC5509666 source

      Not checked in the free run.

    • cites [22] Wu J (2019) Distinct Connectivity and Functionality of Aldehyde Dehydrogenase 1a1-Positive Nigrostriatal Dopaminergic Neurons in Motor Learning. Cell Rep 28, 1167– 1181.e7 31365862 PMC6741442 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The single-cell classification studies identified partially overlapping dopamine neuron subtypes, some of which were located in a manner agnostic to traditional anatomical boundaries.

    “Although these studies differed significantly in their experimental parameters and analysis methods, they identified partially overlapping subtypes, some of which were located in a manner agnostic to traditional anatomical boundaries (SN, VTA, & RR).”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

    • cites [14] La Manno G (2016) Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566– 580.e19 27716510 PMC5055122 source

      Not checked in the free run.

    • cites [15] Tiklova K (2019) Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development. Nat Commun 10, 581– 12 30718509 PMC6362095 source

      Not checked in the free run.

    • cites [16] Hook PW (2018) Single-Cell RNA-Seq of Mouse Dopaminergic Neurons Informs Candidate Gene Selection for Sporadic Parkinson Disease. Am. J. Hum. Genet. 102, 427– 446 29499164 PMC5985341 source

      Not checked in the free run.

    • cites [17] Kramer DJ (2018) Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability. eNeuro 5, ENEURO.0152–18.2018 PMC6104179 30135866 source

      Not checked in the free run.

    • cites [18] Saunders AB (2018) Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell 174, 1015– 1030.e16 30096299 PMC6447408 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The review proposes seven putative dopamine neuron subtypes, each defined by a unique molecular signature and supported by multiple studies; four are more robustly identified by diagnostic markers, while three are more ambiguous.

    “After assessing the current literature, here we propose seven putative DA neuron subtypes, defined by a unique molecular signature, whose existence is supported by multiple studies. Four of these are more robustly identified by diagnostic markers, whereas the three others are more ambiguous.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The single-cell clustering data from different studies can be aligned and interpreted together to derive a common classification scheme, despite differences in experimental parameters, analysis methods, and naming schemes.

  • May combine two claims.

    Premisecitation

    Bulk profiling experiments comparing substantia nigra pars compacta and ventral tegmental area neurons yielded gene expression differences but could not distinguish heterogeneous populations within each anatomical cluster.

    “Bulk profiling experiments comparing SNc and VTA neurons did indeed yield some interesting gene expression differences [6–9], but suffered from the caveat that they could not distinguish heterogeneous populations within each anatomical cluster.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [6] Grimm J (2004) Molecular basis for catecholaminergic neuron diversity. Proc. Natl. Acad. Sci. U.S.A. 101, 13891– 13896 15353588 PMC518849 source

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    • cites [7] Chung CY (2005) Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum. Mol. Genet. 14, 1709– 1725 15888489 PMC2674782 source

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    • cites [8] Greene JG (2005) Gene expression profiling of rat midbrain dopamine neurons: implications for selective vulnerability in parkinsonism. 18, 19– 31 15649693

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    • cites [9] Brichta L (2015) Identification of neurodegenerative factors using translatome-regulatory network analysis. Nat. Neurosci. 18, 1325– 1333 26214373 PMC4763340 source

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

    Premisecitation

    Dysfunctional dopamine signaling has been implicated in numerous neuropsychiatric disorders, including depression, chronic pain, drug addiction, schizophrenia, and attention deficit and hyperactivity disorder.

    “Since then, dysfunctional DA signaling has been implicated in numerous neuropsychiatric disorders, including depression, chronic pain, drug addiction, schizophrenia, and attention deficit and hyperactivity disorder [2].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Björklund A and Dunnett SB (2007) Dopamine neuron systems in the brain: an update. Trends Neurosci. 30, 194– 202 17408759 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Vip+ dopamine neurons are Vglut2+ at neonatal timepoints, consistent with functional data showing glutamate co-transmission from periaqueductal gray/dorsal raphe dopamine neurons in the adult.

    “These neurons are also Vglut2+ at neonatal timepoints [13], consistent with functional data showing glutamate co-transmission from PAG/DR DA neurons in the adult [47,48].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

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    • cites [47] Matthews GA (2016) Dorsal Raphe Dopamine Neurons Represent the Experience of Social Isolation. Cell 164, 617– 631 26871628 PMC4752823 source

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    • cites [48] Cho JR (2017) Dorsal Raphe Dopamine Neurons Modulate Arousal and Promote Wakefulness by Salient Stimuli. Neuron 94, 1205– 1219.e8 28602690 source

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  • May combine two claims.

    Premisecitation

    Sox6+, Aldh1a1- dopamine neurons co-express high levels of Ndnf, Igf1, and Sncg, and some also express Calb1, Lypd1, Tacr3, and Cyp26b1 mRNAs.

    “These cells also co-express high levels of many of the mRNAs aforementioned in the previous population (Ndnf, Igf1 and Sncg), but in contrast, some of these neurons also express Calb1 [13,14], Lypd1 [14], Tacr3 [13,18], and Cyp26b1 [18] mRNAs.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

    • cites [14] La Manno G (2016) Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566– 580.e19 27716510 PMC5055122 source

      Not checked in the free run.

    • cites [18] Saunders AB (2018) Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell 174, 1015– 1030.e16 30096299 PMC6447408 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In the dorsal caudate putamen, ALDH1A1+ dopamine neurons preferentially innervate some mu-opioid receptor-positive patches, known as striosomes, although this preference is not exclusive.

    “In the dorsal CP, these neurons preferentially innervate some μ-opioid receptor+ (MOR) patches (commonly known as striosomes), although this is not exclusive [20–22].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [20] Poulin J-F (2018) Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat. Neurosci. 21, 1260– 1271 30104732 PMC6342021 source

      Not checked in the free run.

    • cites [21] Sgobio C (2017) Aldehyde dehydrogenase 1-positive nigrostriatal dopaminergic fibers exhibit distinct projection pattern and dopamine release dynamics at mouse dorsal striatum. Sci Rep 7, 5283– 13 28706191 PMC5509666 source

      Not checked in the free run.

    • cites [22] Wu J (2019) Distinct Connectivity and Functionality of Aldehyde Dehydrogenase 1a1-Positive Nigrostriatal Dopaminergic Neurons in Motor Learning. Cell Rep 28, 1167– 1181.e7 31365862 PMC6741442 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In Neurod6 knockout mice, dopamine projections to the lateral septum are reduced, suggesting a dependence on NEUROD6 function.

    “In Neurod6 KO mice, DA projections to the lateral septum are reduced, suggesting a dependence on NEUROD6 function [36].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Khan S (2017) Survival of a Novel Subset of Midbrain Dopaminergic Neurons Projecting to the Lateral Septum Is Dependent on NeuroD Proteins. J. Neurosci. 37, 2305– 2316 28130357 PMC5354344 source

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  • May combine two claims.

    Premisecitation

    Otx2+, Aldh1a1+ dopamine neurons direct major projections to the medial shell of the nucleus accumbens and minor projections to the lateral septum.

    “This population has been accessed using Aldh1a1 and NeuroD6 based drivers, and it appears to direct major projections to the medial shell of the ACB, and minor projections to the lateral septum [17,20,22,36].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [17] Kramer DJ (2018) Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability. eNeuro 5, ENEURO.0152–18.2018 PMC6104179 30135866 source

      Not checked in the free run.

    • cites [20] Poulin J-F (2018) Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat. Neurosci. 21, 1260– 1271 30104732 PMC6342021 source

      Not checked in the free run.

    • cites [22] Wu J (2019) Distinct Connectivity and Functionality of Aldehyde Dehydrogenase 1a1-Positive Nigrostriatal Dopaminergic Neurons in Motor Learning. Cell Rep 28, 1167– 1181.e7 31365862 PMC6741442 source

      Not checked in the free run.

    • cites [36] Khan S (2017) Survival of a Novel Subset of Midbrain Dopaminergic Neurons Projecting to the Lateral Septum Is Dependent on NeuroD Proteins. J. Neurosci. 37, 2305– 2316 28130357 PMC5354344 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Many Vgat+ dopamine neurons also express Vglut2 and have lower Dat and Th expression.

    “Many of these neurons also express Vglut2 (a.k.a. Slc17a6), a glutamate vesicular transporter [13,18], and also have lower Dat and Th expression [15].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

    • cites [18] Saunders AB (2018) Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell 174, 1015– 1030.e16 30096299 PMC6447408 source

      Not checked in the free run.

    • cites [15] Tiklova K (2019) Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development. Nat Commun 10, 581– 12 30718509 PMC6362095 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Dopamine neurons labeled with Neurod6-Cre have a smaller Ih, more depolarized membrane potential, higher membrane resistance, less pronounced afterhyperpolarization, and shorter action potential height compared to substantia nigra pars compacta neurons.

    “Physiologically, DA neurons labeled with Neurod6-Cre have a smaller Ih, more depolarized membrane potential, higher membrane resistance, less pronounced afterhyperpolarization, and shorter action potential height when compared to SNc neurons [17].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] Kramer DJ (2018) Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability. eNeuro 5, ENEURO.0152–18.2018 PMC6104179 30135866 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Aldh1a1+ dopamine neurons of the substantia nigra pars compacta are selectively decreased in a mouse model of Parkinson's disease and in human brains affected by the disease.

    “In addition, Aldh1a1+ DA neurons of the SNc are selectively decreased in a mouse model of PD [13], as well as in human brains affected by the disease [25].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

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    • cites [25] Liu G (2014) Aldehyde dehydrogenase 1 defines and protects a nigrostriatal dopaminergic neuron subpopulation. J. Clin. Invest. 124, 3032– 3046 24865427 PMC4071380 source

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  • May combine two claims.

    Interpretationcitation

    Vip+ dopamine neurons are the most caudally located population, found in the periaqueductal gray and dorsal raphe, where they account for about 49% of dopamine neurons, but are sparser in the ventral tegmental area.

    “Vip+ DA neurons are the most caudally located population”
    • Also stated in: § Vip+ DA neurons are the most caudally located population · plain
      “On a neuroanatomical basis, this subtype is located in the periaqueductal gray (PAG) and dorsal raphe (DR) regions, accounting for about 49% of DA neurons [13] in these regions, but more sparsely in the VTA [13–15,46]”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

    • cites [14] La Manno G (2016) Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566– 580.e19 27716510 PMC5055122 source

      Not checked in the free run.

    • cites [15] Tiklova K (2019) Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development. Nat Commun 10, 581– 12 30718509 PMC6362095 source

      Not checked in the free run.

    • cites [46] Dougalis AG (2012) Functional properties of dopamine neurons and co-expression of vasoactive intestinal polypeptide in the dorsal raphe nucleus and ventrolateral periaqueductal grey. Eur. J. Neurosci. 36, 3322– 3332 22925150 PMC4273537 source

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  • May combine two claims.

    Premisecitation

    Sox6+, Aldh1a1- dopamine neurons lack Aldh1a1, Anxa1, and Aldh1a7 expression.

    “In addition to lacking Aldh1a1 mRNA, they also do not express Anxa1 [14,17,18] and Aldh1a7 [14,17,18].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [14] La Manno G (2016) Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566– 580.e19 27716510 PMC5055122 source

      Not checked in the free run.

    • cites [17] Kramer DJ (2018) Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability. eNeuro 5, ENEURO.0152–18.2018 PMC6104179 30135866 source

      Not checked in the free run.

    • cites [18] Saunders AB (2018) Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell 174, 1015– 1030.e16 30096299 PMC6447408 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Vglut2+ dopamine neurons in the substantia nigra pars compacta are particularly resistant to degeneration in toxin models of Parkinson's disease, and VGLUT2 may play a neuroprotective role by inducing a BDNF/TRKB dependent signaling cascade.

    “In toxin models of PD, Vglut2+ DA neurons in the SNc are particularly resistant to degeneration [58,59], and VGLUT2 itself may play a neuroprotective role by inducing a BDNF/TRKB dependent signaling cascade [59].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [58] Steinkellner T (2018) Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons. J. Clin. Invest. 128, 774– 788 29337309 PMC5785252 source

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    • cites [59] Shen H (2018) Genetic deletion of vesicular glutamate transporter in dopamine neurons increases vulnerability to MPTP-induced neurotoxicity in mice. Proc. Natl. Acad. Sci. U.S.A. 115, E11532– E11541 30442663 PMC6298109 source

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  • May combine two claims.

    Interpretationcitation

    A Vglut2+, Calb1+ dopamine population is represented in at least three studies and is ambiguous, likely encompassing two or more distinct populations.

    “Vglut2+,Calb1+ DA neurons are ambiguous and likely encompass two or more distinct populations”
    • Also stated in: § Vglut2+,Calb1+ DA neurons are ambiguous and likely encompass two or more distinct populations · hedged
      “A Vglut2+, Calb1+ population is represented in at least three of the studies, although this remains somewhat ambiguous owing to the lack of more specific markers [13,16,18].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

    • cites [16] Hook PW (2018) Single-Cell RNA-Seq of Mouse Dopaminergic Neurons Informs Candidate Gene Selection for Sporadic Parkinson Disease. Am. J. Hum. Genet. 102, 427– 446 29499164 PMC5985341 source

      Not checked in the free run.

    • cites [18] Saunders AB (2018) Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell 174, 1015– 1030.e16 30096299 PMC6447408 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The boundaries of the dopamine primordium are defined by transcription factor expression: the caudal extent by the Otx2/Gbx2 boundary, the rostral extent by the En1/Dbx1 boundary, and the dorsal extent by the Lmx1a/Nkx6.1 boundary.

    “The caudal extent is defined by the Otx2/Gbx2 boundary coincident with the morphological mid-hindbrain boundary [74]. The rostral extent is defined by the En1/Dbx1 gene expression boundary which is located in the diencephalic region [75–78]. The dorsal extent is defined by the Lmx1a/Nkx6.1 gene expression boundary [71,79–81].”
    Quote in the paperNot a substantial claim0 of 9 sources checked
    • cites [74] Brodski C (2003) Location and size of dopaminergic and serotonergic cell populations are controlled by the position of the midbrain-hindbrain organizer. J. Neurosci. 23, 4199– 4207 12764108 PMC6741088 source

      Not checked in the free run.

    • cites [75] Nouri N and Awatramani RB (2017) A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons. Development 144, 916– 927 28174244 PMC5374351

      Not checked in the free run.

    • cites [76] Kee N (2017) Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages. Cell Stem Cell 20, 29– 40 28094018 source

      Not checked in the free run.

    • cites [77] Lahti L (2012) Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain. Development 139, 894– 905 22278924 source

      Not checked in the free run.

    • cites [78] Smits SM (2013) Molecular marker differences relate to developmental position and subsets of mesodiencephalic dopaminergic neurons. PLoS ONE 8, e76037 24116087 PMC3792114 source

      Not checked in the free run.

    • cites [71] Andersson E (2006) Identification of intrinsic determinants of midbrain dopamine neurons. Cell 124, 393– 405 16439212 source

      Not checked in the free run.

    • cites [79] Yan CH (2011) Lmx1a and lmx1b function cooperatively to regulate proliferation, specification, and differentiation of midbrain dopaminergic progenitors. J. Neurosci. 31, 12413– 12425 21880902 PMC6703256 source

      Not checked in the free run.

    • cites [80] Nakatani T (2010) Lmx1a and Lmx1b cooperate with Foxa2 to coordinate the specification of dopaminergic neurons and control of floor plate cell differentiation in the developing mesencephalon. Dev. Biol. 339, 101– 113 20035737 source

      Not checked in the free run.

    • cites [81] Anderegg AM (2013) An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool. PLoS Genet. 9, e1003973 24348261 PMC3861205 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Diversification of DA neurons starts early and likely proceeds in steps.

    “Taken together, it appears that DA neuron diversification begins to emerge early and probably occurs in a stepwise fashion.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Hook et al. identified two embryonic DA neuron types, E15.MB.1 and E15.MB.2; one corresponded to a neuroblast population and the other to a more mature DA neuron.

    “By contrast, [16] identified only two embryonic types (E15.MB.1 and E15.MB.2) representing a neuroblast population and a more mature DA neuron.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Hook PW (2018) Single-Cell RNA-Seq of Mouse Dopaminergic Neurons Informs Candidate Gene Selection for Sporadic Parkinson Disease. Am. J. Hum. Genet. 102, 427– 446 29499164 PMC5985341 source

      Not checked in the free run.

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

    Interpretationreasoning

    One possibility is that the Vglut2+, Calb1+ population encompasses at least two subtypes, one located in the substantia nigra pars compacta and the other in the ventral tegmental area.

    “One possibility is that the Vglut2+, Calb1+ population encompasses at least two subtypes, one located in the SNc and the other in the VTA.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A recent study suggested that striatal neuron contact may regulate Vglut2 expression in DA neurons.

    “Supporting the possibility of extrinsically driven refinement of gene expression, a recent study suggested that contact with striatal neurons could modulate the expression of Vglut2 in DA neurons [42].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Fortin GM (2019) Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells. FASEB J. 33, 400– 417 30011230 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Projections of Vip+ dopamine neurons are focal, mapped to the bed nucleus of the stria terminalis and central nucleus of the amygdala, and innervate a smaller target volume than other dopamine neurons.

    “The projections of these neurons are peculiar in that they are particularly focal – they have been mapped to the bed nucleus of the stria terminals (BST) and central nucleus of the amygdala [13,20], and appear to innervate a much smaller target volume than other DA neurons (e.g. [49] and [50]).”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [13] Poulin J-F (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9, 930– 943 25437550 PMC4251558 source

      Not checked in the free run.

    • cites [20] Poulin J-F (2018) Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat. Neurosci. 21, 1260– 1271 30104732 PMC6342021 source

      Not checked in the free run.

    • cites [49] Matsuda W (2009) Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum. J. Neurosci. 29, 444– 453 19144844 PMC6664950 source

      Not checked in the free run.

    • cites [50] Prensa L and Parent A (2001) The nigrostriatal pathway in the rat: A single-axon study of the relationship between dorsal and ventral tier nigral neurons and the striosome/matrix striatal compartments. J. Neurosci. 21, 7247– 7260 11549735 PMC6762986 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Although single-cell studies disagree on resulting subtypes, they have started to give a finer picture of molecular heterogeneity in the midbrain DAergic system, which is a major advance over anatomy-based definitions.

    “Nonetheless, and although single-cell studies have discrepancies in resulting subtypes, these studies have begun to provide a more granular picture of molecular heterogeneity in the midbrain DAergic system, a significant advance from anatomically based definitions.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Single-cell studies of DA neurons have discrepancies in the subtypes they identify.

  • May combine two claims.

    Interpretationreasoning

    Single-cell gene expression profiling has advanced the definition of DA neuron subtypes and revised traditional anatomy-based classification schemes.

    “Single-cell gene expression profiling has enabled progress in defining DA neuron subtypes, thus revising traditional anatomically based classification schemes.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Sox6 is expressed in the medial aspect of the dopamine progenitor pool, and this progenitor pool has been proposed to be the source of the majority of the substantia nigra pars compacta but not the ventral tegmental area.

    “More recently, Sox6 has been shown to be expressed in the medial aspect of the progenitor pool, and based on immunolabeling experiments, this progenitor pool has been proposed to be the source of the majority of the SNc but not the VTA [26].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Panman L (2014) Sox6 and Otx2 control the specification of substantia nigra and ventral tegmental area dopamine neurons. Cell Rep 8, 1018– 1025 25127144 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Comparing projections of Vglut2+, Aldh1a1- and Vglut2+, Aldh1a1+ ventral tegmental area cohorts shows significant overlap in the nucleus accumbens, olfactory tubercle, and lateral septum; projections in the basolateral amygdala, entorhinal cortex, and prefrontal cortex are exclusively from Aldh1a1-, Vglut2+ projections.

    “Comparing the projections of Vglut2+, Aldh1a1- and Vglut2+, Aldh1a1+ VTA cohorts, we observed significant overlap in the ACB, olfactory tubercle (OT), and lateral septum (LS). In comparison, we observed projections in the BLA, entorhinal and prefrontal cortex, exclusively from Aldh1a1-, Vglut2+ projections [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Poulin J-F (2018) Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat. Neurosci. 21, 1260– 1271 30104732 PMC6342021 source

      Not checked in the free run.

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

    Premisecitation

    Tiklova et al. suggested that some elements of DA neuron diversification may appear as early as E13, while further diversification continues through development.

    “Finally, [15] suggests that elements of DA neuron diversification might be present as early as E13, but further diversification continues throughout development.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Tiklova K (2019) Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development. Nat Commun 10, 581– 12 30718509 PMC6362095 source

      Not checked in the free run.

28 of 28

  • The paper relies on this without support.

    The single-cell clustering data from different studies can be aligned and interpreted together to derive a common classification scheme, despite differences in experimental parameters, analysis methods, and naming schemes.

    “In this review, we highlight congruencies and incongruencies in the putative DA neuron subtypes identified in these studies. In the two studies that did not validate all the single cell clustering data [17,18], we present our best interpretation of their classification scheme. We then undertake the challenging task of synthesizing a classification scheme for mouse midbrain DA neurons based on all”

  • The paper relies on this without support.

    Th mRNA+ neurons that do not express Dat do not produce functional amounts of dopamine, so they can be omitted from the classification.

    “While Th mRNA+ neurons that do not express Dat are also observed in the midbrain, it is not clear if these produce functional amounts of DA, and thus they have been omitted from this review [15,19,20]”

  • The paper relies on this without support.

    Midbrain DA neurons share a common developmental origin in the floor plate and express many terminal selector genes and their downstream cascades.

    “Parsing midbrain DA subtypes is a challenging endeavor since these neurons all share a common developmental origin in the floor plate and share the expression of many terminal selector genes and their downstream cascades.”

  • The paper relies on this without support.

    Single-cell studies of DA neurons have discrepancies in the subtypes they identify.

    “Nonetheless, and although single-cell studies have discrepancies in resulting subtypes, these studies have begun to provide a more granular picture of molecular heterogeneity in the midbrain DAergic system, a significant advance from anatomically based definitions.”
4 of 4

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?

  • Ventral midbrain dopamine neurons are diverse, project to different regions, and can release neurotransmitters besides dopamine.

    “Dopamine neurons of the ventral midbrain (DANs) comprise a diverse population of neurons targeting different regions and capable of releasing multiple neurotransmitters in addition to dopamine.31,103,104”

    From Synaptic and circuit functions of multitransmitter neurons in the mammalian brain (2023) · cites this paper as [104]

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Related

Authors
Poulin, J.-F., Gaertner, Z., Moreno-Ramos, O. A., & Awatramani, R.
Affiliations
  • Montreal Neurological Institute and Hospital (CA)
  • Northwestern University (US)
Corresponding author
Awatramani, Rajeshwar
ORCID
Venue
Trends in Neurosciences
Publisher
Elsevier BV
Published
2020-03
Volume
43
Issue
3
Pages
155-169
ISSN
0166-2236 (ISSN-L)
DOI
10.1016/j.tins.2020.01.004
PMC
PMC7285906
OpenAlex
W3006180028
Semantic Scholar
6ffbd478152a0fbd852f9f1312a2d8bc23e33290
Cited by
238
References
126
Citations by year
2026: 28 · 2025: 40 · 2024: 48 · 2023: 31 · 2022: 43 · 2021: 31 · 2020: 17
FWCI
2.92
Type
journal article
Language
en
Open access
Licence
Locations
  • Trends in Neurosciences (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
Foundation for the National Institutes of Health
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurotransmitter Receptor Influence on Behavior
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
midbrain dopaminergic neurons, single-cell gene expression profiling, molecular classification, neuropsychiatric disorders, dopamine signaling
MeSH terms
Brain, Dopamine, Mesencephalon, Gene Expression Profiling, 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 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

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