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.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Berke, J. D. What does dopamine mean? Nat. Neurosci. 21, 787–793 (2018).
Björklund, A. & Dunnett, S. B. Dopamine neuron systems in the brain: an update. Trends Neurosci. 30, 194–202 (2007).
Lammel, S., Lim, B. K. & Malenka, R. C. Reward and aversion in a heterogeneous midbrain dopamine system. Neuropharmacology 76 (Pt. B), 351–359 (2014).
Roeper, J. Dissecting the diversity of midbrain dopamine neurons. Trends Neurosci. 36, 336–342 (2013).
Morales, M. & Margolis, E. B. Ventral tegmental area: cellular heterogeneity, connectivity and behaviour. Nat. Rev. Neurosci. 18, 73–85 (2017).
Ikemoto, S. Dopamine reward circuitry: two projection systems from the ventral midbrain to the nucleus accumbens-olfactory tubercle complex. Brain Res. Rev. 56, 27–78 (2007).
Bromberg-Martin, E. S., Matsumoto, M. & Hikosaka, O. Dopamine in motivational control: rewarding, aversive, and alerting. Neuron 68, 815–834 (2010).
Poulin, J.-F. et al. Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep. 9, 930–943 (2014).
La Manno, G. et al. Molecular diversity of midbrain development in mouse, human, and stem cells. Cell 167, 566–580.e19 (2016).
Hook, P. W. et al. Single-cell RNA-seq of mouse dopaminergic neurons informs candidate gene selection for sporadic Parkinson disease. Am. J. Hum. Genet. 102, 427–446 (2018).
Poulin, J.-F., Tasic, B., Hjerling-Leffler, J., Trimarchi, J. M. & Awatramani, R. Disentangling neural cell diversity using single-cell transcriptomics. Nat. Neurosci. 19, 1131–1141 (2016).
Brignani, S. & Pasterkamp, R. J. Neuronal subset-specific migration and axonal wiring mechanisms in the developing midbrain dopamine system. Front. Neuroanat. 11, 55 (2017).
Dymecki, S. M., Ray, R. S. & Kim, J. C. Mapping cell fate and function using recombinase-based intersectional strategies. Methods Enzymol. 477, 183–213 (2010).
Awatramani, R., Soriano, P., Rodriguez, C., Mai, J. J. & Dymecki, S. M. Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation. Nat. Genet. 35, 70–75 (2003).
Cho, J. R. et al. Dorsal raphe dopamine neurons modulate arousal and promote wakefulness by salient stimuli. Neuron 94, 1205–1219.e8 (2017).
Lammel, S. et al. Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons. Neuron 85, 429–438 (2015).
Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).
Fenno, L. E. et al. Targeting cells with single vectors using multiple-feature Boolean logic. Nat. Methods 11, 763–772 (2014).
Chen, L., Xie, Z., Turkson, S. & Zhuang, X. A53T human α-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration. J. Neurosci. 35, 890–905 (2015).
Nouri, N. & Awatramani, R. A novel floor plate boundary defined by adjacent En1 and Dbx1 microdomains distinguishes midbrain dopamine and hypothalamic neurons. Development 144, 916–927 (2017).
Panman, L. et al. Sox6 and Otx2 control the specification of substantia nigra and ventral tegmental area dopamine neurons. Cell Rep. 8, 1018–1025 (2014).
Sgobio, C. et al. Aldehyde dehydrogenase 1-positive nigrostriatal dopaminergic fibers exhibit distinct projection pattern and dopamine release dynamics at mouse dorsal striatum. Sci. Rep. 7, 5283 (2017).
Trudeau, L.-E. et al. The multilingual nature of dopamine neurons. Prog. Brain Res. 211, 141–164 (2014).
Steinkellner, T. et al. Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons. J. Clin. Invest. 128, 774–788 (2018).
Menegas, W. et al. Dopamine neurons projecting to the posterior striatum form an anatomically distinct subclass. Elife 4, e10032 (2015).
Gerfen, C. R., Herkenham, M. & Thibault, J. The neostriatal mosaic: II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J. Neurosci. 7, 3915–3934 (1987).
Gangarossa, G. et al. Distribution and compartmental organization of GABAergic medium-sized spiny neurons in the mouse nucleus accumbens. Front. Neural Circuits 7, 22 (2013).
Mingote, S. et al. Functional connectome analysis of dopamine neuron glutamatergic connections in forebrain regions. J. Neurosci. 35, 16259–16271 (2015).
Hintiryan, H. et al. The mouse cortico-striatal projectome. Nat. Neurosci. 19, 1100–1114 (2016).
Oh, S. W. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014).
Yetnikoff, L., Lavezzi, H. N., Reichard, R. A. & Zahm, D. S. An update on the connections of the ventral mesencephalic dopaminergic complex. Neuroscience 282, 23–48 (2014).
Menegas, W., Babayan, B. M., Uchida, N. & Watabe-Uchida, M. Opposite initialization to novel cues in dopamine signaling in ventral and posterior striatum in mice. Elife 6, 988 (2017).
Lerner, T. N. et al. Intact-brain analyses reveal distinct information carried by SNc dopamine subcircuits. Cell 162, 635–647 (2015).
Howe, M. W. & Dombeck, D. A. Rapid signalling in distinct dopaminergic axons during locomotion and reward. Nature 535, 505–510 (2016).
Yin, H. H. et al. Dynamic reorganization of striatal circuits during the acquisition and consolidation of a skill. Nat. Neurosci. 12, 333–341 (2009).
Thorn, C. A. C., Atallah, H., Howe, M. & Graybiel, A. M. A. Differential dynamics of activity changes in dorsolateral and dorsomedial striatal loops during learning. Neuron 66, 781–795 (2010).
Gangarossa, G. et al. Spatial distribution of D1R- and D2R-expressing medium-sized spiny neurons differs along the rostro-caudal axis of the mouse dorsal striatum. Front. Neural Circuits 7, 124 (2013).
Di Salvio, M. et al. Otx2 controls neuron subtype identity in ventral tegmental area and antagonizes vulnerability to MPTP. Nat. Neurosci. 13, 1481–1488 (2010).
Khan, S. et al. Survival of a novel subset of midbrain dopaminergic neurons projecting to the lateral septum is dependent on NeuroD proteins. J. Neurosci. 37, 2305–2316 (2017).
Stuber, G. D., Hnasko, T. S., Britt, J. P., Edwards, R. H. & Bonci, A. Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum corelease glutamate. J. Neurosci. 30, 8229–8233 (2010).
Hnasko, T. S., Hjelmstad, G. O., Fields, H. L. & Edwards, R. H. Ventral tegmental area glutamate neurons: electrophysiological properties and projections. J. Neurosci. 32, 15076–15085 (2012).
Kabanova, A. et al. Function and developmental origin of a mesocortical inhibitory circuit. Nat. Neurosci. 18, 872–882 (2015).
Lammel, S. et al. Unique properties of mesoprefrontal neurons within a dual mesocorticolimbic dopamine system. Neuron 57, 760–773 (2008).
Matthews, G. A. et al. Dorsal raphe dopamine neurons represent the experience of social isolation. Cell 164, 617–631 (2016).
Beier, K. T. et al. Circuit architecture of VTA dopamine neurons revealed by systematic input-output mapping. Cell 162, 622–634 (2015).
Baimel, C., Lau, B. K., Qiao, M. & Borgland, S. L. Projection-target-defined effects of orexin and dynorphin on VTA dopamine neurons. Cell Rep. 18, 1346–1355 (2017).
Duan, B. et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell 159, 1417–1432 (2014).
Sciolino, N. R. et al. Recombinase-dependent mouse lines for chemogenetic activation of genetically defined cell types. Cell Rep. 15, 2563–2573 (2016).
Ray, R. S. et al. Impaired respiratory and body temperature control upon acute serotonergic neuron inhibition. Science 333, 637–642 (2011).
Bourane, S. et al. Gate control of mechanical itch by a subpopulation of spinal cord interneurons. Science 350, 550–554 (2015).
Skarnes, W. C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).
Osterwalder, M. et al. Dual RMCE for efficient re-engineering of mouse mutant alleles. Nat. Methods 7, 893–895 (2010).
Donnelly, M. L. et al. Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J. Gen. Virol. 82, 1013–1025 (2001).
Anastassiadis, K. et al. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis. Model. Mech. 2, 508–515 (2009).
Wallén, A. et al. Fate of mesencephalic AHD2-expressing dopamine progenitor cells in NURR1 mutant mice. Exp. Cell Res. 253, 737–746 (1999).
Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752–757 (1997).
Hsu, L. C., Chang, W. C., Hoffmann, I. & Duester, G. Molecular analysis of two closely related mouse aldehyde dehydrogenase genes: identification of a role for Aldh1, but not Aldh-pb, in the biosynthesis of retinoic acid. Biochem. J. 339, 387–395 (1999).
Meyers, E. N., Lewandoski, M. & Martin, G. R. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18, 136–141 (1998).
Tasic, B. et al. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016).
Jensen, P. et al. Redefining the serotonergic system by genetic lineage. Nat. Neurosci. 11, 417–419 (2008).
Chen, L. et al. Unregulated cytosolic dopamine causes neurodegeneration associated with oxidative stress in mice. J. Neurosci. 28, 425–433 (2008).
Sando, R. III et al. Inducible control of gene expression with destabilized Cre. Nat. Methods 10, 1085–1088 (2013).
Joksimovic, M. et al. Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools. Proc. Natl. Acad. Sci. USA 106, 19185–19190 (2009).
Anderegg, A. et al. An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool. PLoS Genet. 9, e1003973 (2013).
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.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
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).
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.
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).
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.
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.
(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.
(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.
(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.
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.
Supplementary Figure 10 Representative traces of DAergic projections to the nucleus accumbens (ACB).
ACBr = rostral, ACBi = intermediate, ACBc = caudal.
(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.
About this article
Cite this article
Poulin, J., Caronia, G., Hofer, C. et al. Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat Neurosci 21, 1260–1271 (2018). https://doi.org/10.1038/s41593-018-0203-4
Journal of Neuroscience Methods (2021)
Frontiers in Molecular Neuroscience (2020)
Nature Communications (2020)