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Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches


Midbrain dopamine (DA) neurons have diverse functions that can in part be explained by their heterogeneity. Although molecularly distinct subtypes of DA neurons have been identified by single-cell gene expression profiling, fundamental features such as their projection patterns have not been elucidated. Progress in this regard has been hindered by the lack of genetic tools for studying DA neuron subtypes. Here we develop intersectional genetic labeling strategies, based on combinatorial gene expression, to map the projections of molecularly defined DA neuron subtypes. We reveal distinct genetically defined dopaminergic pathways arising from the substantia nigra pars compacta and from the ventral tegmental area that innervate specific regions of the caudate putamen, nucleus accumbens and amygdala. Together, the genetic toolbox and DA neuron subtype projections presented here constitute a resource that will accelerate the investigation of this clinically significant neurotransmitter system.

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The authors wish to thank S. Ganguli, M. Jurado and I. Oksuz for technical assistance, S. Pieraut and A. Maximov for help with trimethropin injection protocol, and X. Zhuang (University of Chicago) and B. Lowell (Harvard) for sharing mouse strains. This work was supported by NIH grants R01NS06977 and R01NS047085 to C.S.C.; NIH grant R01MH110556-01A1 to D.A.D.; NIH grants R01MH110556-01A1, 1R21NS072703-01A1 and R01NS096240-01 and NARSAD and Paul Ruby Foundation grants to R.A.; and grants from MJFF and CIHR to J.-F.P.

Author information

J.-F.P. designed and performed most experiments, and wrote the manuscript; G.C. generated and validated the Sox6-FSF-Cre mouse; C.H., Q.C., and B.H. performed stereotaxic surgeries and some immunofluorescence staining; C.R. and K.D. provided the intersectional viral vectors; C.S.C. and D.A.D. provided intellectual and experimental guidance, and edited the manuscript; R.A. designed experiments, supervised the project and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Rajeshwar Awatramani.

Integrated supplementary information

  1. Supplementary Figure 1 Flp-dependent mCherry expression in the midbrain of a Th-2A-Flpo;RC::Frepe mouse.

    Scale bars = 100μm (apply to multiple panels).

  2. Supplementary Figure 2 Th mRNA is present in the posterior hypothalamus (PH) / rostral linear (RLi) region and the interpeduncular nucleus (IPN).

    At the level of the midbrain, we observed labeled neurons in the rostral linear (RLi) and posterior hypothalamic (PH) regions, as defined in the Allen Reference Atlas. Like all midbrain DA neurons, this population of neurons is derived from the midbrain floorplate and express the markers Dopa decarboxylase (Ddc mRNA), NURR1, FOXA2, and PITX3 (not shown). However, although Th mRNA is observed in this region in the adult, these neurons do not express detectable level of TH protein nor the dopamine transporter (Dat mRNA), and are not recombined with Dat-ires-Cre. Thus, labeled PH/RLi neurons share developmental origins with midbrain DA neurons, and harbor similar molecular profiles, apart from Dat expression. In addition to the PH/RLi, we also observed Flpo-induced recombination in the interpeduncular nucleus (IPN), another region in the vicinity of DA neurons, where Th mRNA is observed, and that is also recombined by Th-ires-Cre driver. However, IPN neurons have low/undetectable TH protein in adult brains. Further, these neurons are not related to midbrain DA neurons, since they do not express NURR1, FOXA2, PITX3 (not shown), Ddc mRNA, and are not derived from the midbrain floor plate.

  3. Supplementary Figure 3 Examples of brain regions with Th-2A-Flpo recombination.

    Comparison of Th mRNA expression (purple; Allen Brain Atlas), with TH protein and Th-2A-Flpo labeled cells (mCherry) distribution in a Th-2A-Flpo;RC::Frepe mouse brain. Some mCherry + neuron populations, such as cortical interneurons or medial forebrain neurons, displayed low or undetectable TH protein. However, since in Th-2A-Flpo the two coding sequences are separated by virtue of a ribosome skipping event that occurs at the glycyl-prolyl peptide bond at the C-terminus of the P2A peptide, effectively, an autocatalytic “cleavage”. By this design, TH proteins have to be translated for Flpo protein to be active. Scale bar: A-G = 100μm (applies to multiple panels).

  4. Supplementary Figure 4 Image processing pipeline for projection analysis.

    For this analysis, we first acquired images of four distinct rostrocaudal levels of the dorsal striatum based on Hintiryan et al.. These images were binarized, vectorized and superimposed onto reference sections of the Allen Reference Atlas. Depicted are projections of a Cck-Cre;Th-2A-Flpo mouse injected with AAV-CreON,FlpON-EYFP in the VTA.

  5. Supplementary Figure 5 Representative traces of DAergic projections of SNc subtypes.

    Ndnf and Sox6 projections densely cover most of the CPr, CPi, and CPc. This particular Sox6 experiment yielded less innervation of the dorsomedial striatum, but injections targeting the VTA in Sox6-FSF-Cre;Th-2A-Flpo, which also labeled the medial SNc (see experiment described in Fig. 4), resulted in labeling of the entire CPi (Table S1). The subtle differences between Sox6 and Ndnf projections might be explained by: 1) the limited diffusion of the virus did not permit the infection of all Sox6-expressing neurons, 2) the fact that while these genes are expressed principally in the same two subtypes, Ndnf expression is somewhat weaker in the Aldh1a1 + ventral tier neurons of the SNc compared to Sox6, resulting in a PBP/dorsal tier labeling bias. Aldh1a1 and Calb1 show somewhat complementary projection patterns in the CPr, CPi, and CPc. Vglut2 and Calb1 experiments show dense innervation of the CPt.

  6. Supplementary Figure 6 Characterization and validation of the Sox6-FSF-Cre driver.

    (A) Schematic of the Sox6-FSF-Cre allele. (B) We validated this strain by crossing it with a Cre reporter, after having removed the stop cassette with a Flp deleter mouse, which resulted in labeled βgal + DA and non-DA cells. Both TH + , and TH- (see arrowheads for example) neurons were observed in the midbrain, and the vast majority of recombined cells were SOX6 + . (C) SOX6 + DA neurons of the SNc were labeled by injection of AAV-CreON,FlpON-EYFP. EYFP + cells are TH + and SOX6 + . Scale bars: (B) low magnification = 100μm, high magnification = 50μm; (C) low magnification = 200μm, high magnification = 25μm.

  7. Supplementary Figure 7 Characterization and validation of the Aldh1a1-CreERT2 driver.

    (A) Schematic of the Aldh1a1-CreERT2 allele (black triangles represent FRT sites). (B) Recombination of the Cre reporter (tdTomato) by Aldh1a1-CreERT2 mouse injected with tamoxifen. (C) tdTomato + projections to the dorsolateral CP, ACB medial shell and lateral septum, but not the PFC, in the Aldh1a1-CreERT2;Th-2A-Flpo;Ai65 mouse. In the CP, tdTomato shows enrichment in several MOR + striosomes. (D) Colocalization of EYFP with ALDH1A1, OTX2, and SOX6 after viral injection of AAV-CreON-EYFP in the SNc or VTA of Aldh1a1-CreERT2 mice. Scale bars: B = 100 μm, C = 200 μm, D = 100 μm.

  8. Supplementary Figure 8 Calb1-Cre;Dat-tTA;Ai82 experiment.

    (A) Examples of EGFP + that are OTX2 + in the VTA (arrowheads). In the nigral region, most EGFP + cells in the medial SNc are SOX6 + (arrowheads), whereas most cells in the dorsolateral SNc are SOX6- (not shown). (B) Images of EGFP + fibers in the CPt and CPi. Since Calb1-Cre labels at least two populations (SOX6 + and SOX6-), our results can’t exclude that both these populations send projections to the medial CP. Scale bars: (A) low magnification = 200 μm, hign magnification = 50 μm; (B) low magnification = 200 μm.

  9. Supplementary Figure 9 A population of cells in the SNc coexpress Th mRNA and Vglut2 mRNA.

    Examples of DA neurons expressing Vglut2 mRNA are shown by arrowheads. The inset displayed cell in substantia nigra pars lateralis (SNpl) that are double positive. Scale bar: low magnification = 83 μm; high magnification = 40 μm.

  10. Supplementary Figure 10 Representative traces of DAergic projections to the nucleus accumbens (ACB).

    ACBr = rostral, ACBi = intermediate, ACBc = caudal.

  11. Supplementary Figure 11 Vip-Cre experiments using strategy I (A) and Strategy III (B).

    (A) tdTomato labeled cells in the PAG/DR region express TH, but not SOX6 and OTX2. (B) EGFP labeled neurons in the PAG/DR of a Vip-Cre;Dat-tTA;Ai82 mouse. EGFP + axons are observed in the lateral part of the central amygdala (CEAl) and oval nucleus of the bed nucleus of the stria terminalis (BSTov). Scale bars: (A) and (Β) low magnification = 100 μm; (A) high magnification = 40 μm.

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Fig. 1: Generation and validation of Th-2A-Flpo.
Fig. 2: Three intersectional strategies to genetically target dopamine neuron subtypes.
Fig. 3: Dopamine neuron subtype projections to the caudate putamen.
Fig. 4: Dopamine neuron subtype projections to the nucleus accumbens and olfactory tubercle.
Fig. 5: Dopamine neuron subtype projections to the amygdala.
Fig. 6: Dopamine neuron subtype projections to other brain regions.
Fig. 7: Remarkable specificity of genetically defined dopamine neuron subtype projections within target regions.
Supplementary Figure 1: Flp-dependent mCherry expression in the midbrain of a Th-2A-Flpo;RC::Frepe mouse.
Supplementary Figure 2: Th mRNA is present in the posterior hypothalamus (PH) / rostral linear (RLi) region and the interpeduncular nucleus (IPN).
Supplementary Figure 3: Examples of brain regions with Th-2A-Flpo recombination.
Supplementary Figure 4: Image processing pipeline for projection analysis.
Supplementary Figure 5: Representative traces of DAergic projections of SNc subtypes.
Supplementary Figure 6: Characterization and validation of the Sox6-FSF-Cre driver.
Supplementary Figure 7: Characterization and validation of the Aldh1a1-CreERT2 driver.
Supplementary Figure 8: Calb1-Cre;Dat-tTA;Ai82 experiment.
Supplementary Figure 9: A population of cells in the SNc coexpress Th mRNA and Vglut2 mRNA.
Supplementary Figure 10: Representative traces of DAergic projections to the nucleus accumbens (ACB).
Supplementary Figure 11: Vip-Cre experiments using strategy I (A) and Strategy III (B).