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Function and developmental origin of a mesocortical inhibitory circuit

Nature Neuroscience volume 18, pages 872–882 (2015)Cite this article

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Abstract

Midbrain ventral tegmental neurons project to the prefrontal cortex and modulate cognitive functions. Using viral tracing, optogenetics and electrophysiology, we found that mesocortical neurons in the mouse ventrotegmental area provide fast glutamatergic excitation of GABAergic interneurons in the prefrontal cortex and inhibit prefrontal cortical pyramidal neurons in a robust and reliable manner. These mesocortical neurons were derived from a subset of dopaminergic progenitors, which were dependent on prolonged Sonic Hedgehog signaling for their induction. Loss of these progenitors resulted in the loss of the mesocortical inhibitory circuit and an increase in perseverative behavior, whereas mesolimbic and mesostriatal dopaminergic projections, as well as impulsivity and attentional function, were largely spared. Thus, we identified a previously uncharacterized mesocortical circuit contributing to perseverative behaviors and found that the diversity of dopaminergic neurons begins to be established during their progenitor phase.

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Figure 1: Shh is required for the induction of the lateral MbDN progenitor domain after E9.0.
Figure 2: Severe loss of Calbindin-positive MbDNs in Gli2ΔMb>E9.0 mice.
Figure 3: MbDN mesocortical projections are severely reduced in Gli2ΔMb>E9.0 mice.
Figure 4: Retrograde tracing of mesocortical and mesolimbic projections.
Figure 5: Expression of ChR2-eYFP in VTA neurons.
Figure 6: Optogenetic stimulation of mPFC-projections from the VTA activated a subset of mPFC neurons.
Figure 7: Mesocortical MbDNs in control mice inhibited mPFC pyramidal cells via interneuron activation.
Figure 8: Loss of an inhibitory mesocortical motif and increased perseverative behavior in Gli2ΔMb>E9.0 mice.

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References

  1. Iversen, L., Iversen, S., Dunnett, S. & Bjorklund, A. Dopamine Handbook (Oxford University Press, 2009).

  2. Björklund, A. & Dunnett, S.B. Dopamine neuron systems in the brain: an update. Trends Neurosci. 30, 194–202 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Roeper, J. Dissecting the diversity of midbrain dopamine neurons. Trends Neurosci. 36, 336–342 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Lammel, S. et al. Input-specific control of reward and aversion in the ventral tegmental area. Nature 491, 212–217 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Niwa, M. et al. Adolescent stress-induced epigenetic control of dopaminergic neurons via glucocorticoids. Science 339, 335–339 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chaudhury, D. et al. Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons. Nature 493, 532–536 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Stamatakis, A.M. et al. A unique population of ventral tegmental area neurons inhibits the lateral habenula to promote reward. Neuron 80, 1039–1053 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Goldman-Rakic, P.S. Cellular basis of working memory. Neuron 14, 477 (1995).

    Article  CAS  PubMed  Google Scholar 

  9. Sandson, J. & Albert, M.L. Varieties of perseveration. Neuropsychologia 22, 715–732 (1984).

    Article  CAS  PubMed  Google Scholar 

  10. Kawano, M. et al. Particular subpopulations of midbrain and hypothalamic dopamine neurons express vesicular glutamate transporter 2 in the rat brain. J. Comp. Neurol. 498, 581–592 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Yamaguchi, T., Wang, H.-L., Li, X., Ng, T.H. & Morales, M. Mesocorticolimbic glutamatergic pathway. J. Neurosci. 31, 8476–8490 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gorelova, N., Mulholland, P.J., Chandler, L.J. & Seamans, J.K. The glutamatergic component of the mesocortical pathway emanating from different subregions of the ventral midbrain. Cereb. Cortex 22, 327–336 (2012).

    Article  PubMed  Google Scholar 

  13. Stuber, G.D., Hnasko, T.S. & Bonci, A. Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum corelease glutamate. J. Neurosci. 30, 8229–8233 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tecuapetla, F. et al. Glutamatergic signaling by mesolimbic dopamine neurons in the nucleus accumbens. J. Neurosci. 30, 7105–7110 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Blaess, S. & Ang, S.-L. Genetic control of midbrain dopaminergic neuron development. Wiley Interdiscip. Rev. Dev. Biol. 4, 113–134 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Andersson, E. et al. Identification of intrinsic determinants of midbrain dopamine neurons. Cell 124, 393–405 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Ono, Y. et al. Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells. Development 134, 3213–3225 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Blaess, S. et al. Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei. Neural Dev. 6, 29 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Hayes, L., Zhang, Z., Albert, P., Zervas, M. & Ahn, S. Timing of Sonic hedgehog and Gli1 expression segregates midbrain dopamine neurons. J. Comp. Neurol. 519, 3001–3018 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Panman, L. et al. Sox6 and Otx2 control the specification of substantia nigra and ventral tegmental area dopamine neurons. Cell Reports 8, 1018–1025 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Lammel, S. et al. Unique properties of mesoprefrontal neurons within a dual mesocorticolimbic dopamine system. Neuron 57, 760–773 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Blaess, S., Corrales, J.D. & Joyner, A.L. Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region. Development 133, 1799–1809 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Matise, M.P., Epstein, D.J., Park, H.L., Platt, K.A. & Joyner, A.L. Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development 125, 2759–2770 (1998).

    CAS  PubMed  Google Scholar 

  24. Kele, J. et al. Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons. Development 133, 495–505 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Pickel, V.M., Joh, T.H., Field, P.M., Becker, C.G. & Reis, D.J. Cellular localization of tyrosine hydroxylase by immunohistochemistry. J. Histochem. Cytochem. 23, 1–12 (1975).

    Article  CAS  PubMed  Google Scholar 

  26. Kittappa, R., Chang, W.W., Awatramani, R.B. & McKay, R.D.G. The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age. PLoS Biol. 5, e325 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zetterström, R.H. et al. Dopamine neuron agenesis in Nurr1-deficient mice. Science 276, 248–250 (1997).

    Article  PubMed  Google Scholar 

  28. Afonso-Oramas, D. et al. Dopamine transporter glycosylation correlates with the vulnerability of midbrain dopaminergic cells in Parkinson's disease. Neurobiol. Dis. 36, 494–508 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Cardin, J.A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663–667 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sohal, V.S., Zhang, F., Yizhar, O. & Deisseroth, K. Parvalbumin neurons and gamma rhythms enhance cortical circuit performance. Nature 459, 698–702 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Simon, H.H., Saueressig, H., Wurst, W., Goulding, M.D. & O'Leary, D.D. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J. Neurosci. 21, 3126–3134 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Brown, M.T.C. et al. Ventral tegmental area GABA projections pause accumbal cholinergic interneurons to enhance associative learning. Nature 492, 452–456 (2012).

    Article  CAS  PubMed  Google Scholar 

  33. Tritsch, N.X., Ding, J.B. & Sabatini, B.L. Dopaminergic neurons inhibit striatal output through non-canonical release of GABA. Nature 490, 262–266 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Robbins, T.W. From arousal to cognition: the integrative position of the prefrontal cortex. Prog. Brain Res. 126, 469–483 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Bari, A., Dalley, J.W. & Robbins, T.W. The application of the 5-choice serial reaction time task for the assessment of visual attentional processes and impulse control in rats. Nat. Protoc. 3, 759–767 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Swanson, L.W. The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res. Bull. 9, 321–353 (1982).

    Article  CAS  PubMed  Google Scholar 

  38. Dalle Torre di Sanguinetto, S.A., Dasen, J.S. & Arber, S. Transcriptional mechanisms controlling motor neuron diversity and connectivity. Curr. Opin. Neurobiol. 18, 36–43 (2008).

    Article  CAS  Google Scholar 

  39. Greig, L.C., Woodworth, M.B., Galazo, M.J., Padmanabhan, H. & Macklis, J.D. Molecular logic of neocortical projection neuron specification, development and diversity. Nat. Rev. Neurosci. 14, 755–769 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Zhang, S. et al. Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons. Nat. Neurosci. 18, 386–392 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lavin, A. et al. Mesocortical dopamine neurons operate in distinct temporal domains using multimodal signaling. J. Neurosci. 25, 5013–5023 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Taylor, S.R. et al. GABAergic and glutamatergic efferents of the mouse ventral tegmental area. J. Comp. Neurol. 522, 3308–3334 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gorelova, N., Seamans, J.K. & Yang, C.R. Mechanisms of dopamine activation of fast-spiking interneurons that exert inhibition in rat prefrontal cortex. J. Neurophysiol. 88, 3150–3166 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Matsuda, Y., Marzo, A. & Otani, S. The presence of background dopamine signal converts long-term synaptic depression to potentiation in rat prefrontal cortex. J. Neurosci. 26, 4803–4810 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Vijayraghavan, S., Wang, M., Birnbaum, S.G., Williams, G.V. & Arnsten, A.F.T. Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat. Neurosci. 10, 376–384 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Yang, C.R.C. & Seamans, J.K.J. Dopamine D1 receptor actions in layers V–VI rat prefrontal cortex neurons in vitro: modulation of dendritic-somatic signal integration. J. Neurosci. 16, 1922–1935 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lapish, C.C., Kroener, S., Durstewitz, D., Lavin, A. & Seamans, J.K. The ability of the mesocortical dopamine system to operate in distinct temporal modes. Psychopharmacology (Berl.) 191, 609–625 (2007).

    Article  CAS  Google Scholar 

  48. Roberts, A.C. et al. 6-Hydroxydopamine lesions of the prefrontal cortex in monkeys enhance performance on an analog of the Wisconsin card sort test: possible interactions with subcortical dopamine. J. Neurosci. 14, 2531–2544 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Crofts, H.S. et al. Differential effects of 6-OHDA lesions of the frontal cortex and caudate nucleus on the ability to acquire an attentional set. Cereb. Cortex 11, 1015–1026 (2001).

    Article  CAS  PubMed  Google Scholar 

  50. Miller, E.K. & Cohen, J.D. An integrative theory of prefrontal cortex function. Annu. Rev. Neurosci. 24, 167–202 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Mo, R. et al. Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development. Development 124, 113–123 (1997).

    CAS  PubMed  Google Scholar 

  52. Corrales, J.D., Blaess, S., Mahoney, E.M. & Joyner, A.L. The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation. Development 133, 1811–1821 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Kimmel, R.A. et al. Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation. Genes Dev. 14, 1377–1389 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Li, J.Y.H., Lao, Z. & Joyner, A.L. Changing requirements for Gbx2 in development of the cerebellum and maintenance of the mid/hindbrain organizer. Neuron 36, 31–43 (2002).

    Article  CAS  PubMed  Google Scholar 

  55. Sudarov, A. & Joyner, A.L. Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers. Neural Dev. 2, 26 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ferri, A.L.M. et al. Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner. Development 134, 2761–2769 (2007).

    Article  CAS  PubMed  Google Scholar 

  57. Pedersen, I.L. et al. Generation and characterization of monoclonal antibodies against the transcription factor Nkx6.1. J. Histochem. Cytochem. 54, 567–574 (2006).

    Article  CAS  PubMed  Google Scholar 

  58. Eisenstat, D.D. et al. DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation. J. Comp. Neurol. 414, 217–237 (1999).

    Article  CAS  PubMed  Google Scholar 

  59. Kilpatrick, I.C., Jones, M.W. & Phillipson, O.T. A semiautomated analysis method for catecholamines, indoleamines, and some prominent metabolites in microdissected regions of the nervous system: an isocratic HPLC technique employing coulometric detection and minimal sample preparation. J. Neurochem. 46, 1865–1876 (1986).

    Article  CAS  PubMed  Google Scholar 

  60. van Loo, K.M.J. et al. Transcriptional regulation of T-type calcium channel CaV3.2: bi-directionality by early growth response 1 (Egr1) and repressor element 1 (RE-1) protein-silencing transcription factor (REST). J. Biol. Chem. 287, 15489–15501 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Garaschuk, O., Milos, R.-I. & Konnerth, A. Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat. Protoc. 1, 380–386 (2006).

    Article  CAS  PubMed  Google Scholar 

  62. Van Eden, C.G. & Uylings, H.B. Cytoarchitectonic development of the prefrontal cortex in the rat. J. Comp. Neurol. 241, 253–267 (1985).

    Article  CAS  PubMed  Google Scholar 

  63. Franklin, K.B.J. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates, Third Edition (Academic Press, 2007).

  64. Steckler, T., Sauvage, M. & Holsboer, F. Glucocorticoid receptor impairment enhances impulsive responding in transgenic mice performing on a simultaneous visual discrimination task. Eur. J. Neurosci. 12, 2559–2569 (2000).

    Article  CAS  PubMed  Google Scholar 

  65. Bevins, R.A. & Besheer, J. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study 'recognition memory'. Nat. Protoc. 1, 1306–1311 (2006).

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank V. Bosch, O. van Ray and M. Reitze for technical assistance, R. Edwards (University of California, San Francisco) for the vGlut2 in situ probe, C. Wotjak (Max Planck Institute of Psychiatry) for lending the automatized chambers used for the attentional task, K. Deisseroth (Stanford University) for the viral construct, and S. Schoch and K. van Loo (University of Bonn) for the AAV preparation. This work was supported by the North-Rhine-Westphalia Repatriation Program (Ministry for Innovation, Science and Research of North Rhine Westphalia) and the Maria von Linden-Program (University of Bonn) (to S.B.), SFB1089 (Project C04) and ERANET Neuron EpiNet (to H.B.), the Mercator Stiftung (to M.S.), and the Backus Foundation and the Centres of Excellence in Neurodegeneration Research (D.A.D.M.). A.R.V. is a recipient of a German Academic Exchange Service doctoral fellowship.

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Author notes

  1. Anne Boehlen

    Present address: Present address: Institute of Cellular Neurosciences, University of Bonn, Bonn, Germany.,

  2. Anna Kabanova and Milan Pabst: These authors contributed equally to this work.

Authors and Affiliations

  1. Neurodevelopmental Genetics, Institute of Reconstructive Neurobiology, Life and Brain Center, University of Bonn, Bonn, Germany

    Anna Kabanova, Ankita R Vaswani & Sandra Blaess

  2. Department of Epileptology, Laboratory of Experimental Epileptology and Cognition Research, University of Bonn, Bonn, Germany

    Milan Pabst, Oliver Braganza, Anne Boehlen, Negar Nikbakht, Leonie Pothmann & Heinz Beck

  3. Functional Architecture of Memory Unit, Mercator Research Group, Faculty of Medicine, Ruhr University Bochum, Bochum, Germany

    Markus Lorkowski & Magdalena Sauvage

  4. German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany

    Ruth Musgrove, Donato A Di Monte & Heinz Beck

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Contributions

A.K. performed in situ hybridization and immunostaining experiments and analyzed the mutant phenotype. M.P. and L.P. performed and analyzed electrophysiological experiments. M.L. and N.N. performed and analyzed behavioral experiments. O.B. performed and analyzed the calcium imaging experiments. A.B. performed retrograde tracing experiments. A.R.V. performed structured illumination and confocal microscopy and analyzed data. R.M. performed and analyzed the HPLC experiments. S.B., H.B., A.K., M.P., M.S. and D.A.D.M. analyzed data and designed the experiments. S.B. and H.B. wrote the manuscript with contributions from all of the authors. S.B. conceived the project.

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Correspondence to Sandra Blaess.

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Integrated supplementary information

Supplementary Figure 1 The size of the lateral MbDN precursor domain is severely reduced in Gli2ΔMb>E9.0 mice.

(a) Quantitative analysis of the size of the MbDN progenitor domain (Lmx1a+, orange plus yellow) and the lateral (Lmx1a+ Corin–, orange) and medial (Lmx1a+ Corin+, yellow) MbDN progenitor domain in E9.5 and E10.5 coronal ventral midbrain sections. n = 3 mice. (b) Quantitative analysis of the size of the Arx+ MbDN progenitor domain in E9.5 and E10.5 coronal sections. n = 3 mice. (c) Area in µm2 of Lmx1+ Corin–, Lmx1+ Corin+, Lmx1a+ and Arx+ domains. Statistical significance was determined using one way ANOVA and Tukey’s multiple comparison test (ANOVA: P < 0.0001 for all comparisons; medial: F(3,8) = 120.5, lateral: F(3,8) = 39.43, Lmx1a: F(3,8) = 252.5, Arx: F(3,8) = 409.7; P-values for pairwise comparisons are listed in (d) Red: statistically significant. Error bars indicate s.e.m.

Supplementary Figure 2 Progenitor proliferation and neurogenesis are not affected in Gli2ΔMb>E9.0 mice.

(a,b) Proliferating progenitors were labeled with a 1–hour BrdU pulse at E10.5. (c,d) RNA in situ hybridization for the precursor marker Hes5 at E11.5. (e,f) Immunofluorescent staining for Neurogenin 2 (Neurog2) and Lmx1a on E11.5 coronal section. (a-f) The Lmx1a+ MbDN progenitor domain is indicated by the dashed lines. Scale bars represent 50 µm (a,b) and 100 µm (c-f). (g) Quantitative analysis of the number of BrdU+ cells within the Lmx1a+ domain at E10.5. n = 3 mice, P = 0.5086, unpaired t test, t(4) = 0.725. (h) Quantitative analysis of the number of Neurog2+ cells within the Lmx1a+ domain at E11.5. n = 3 mice, unpaired t test, P = 0.13, t(4) = 1.87. Error bars indicate s.e.m.

Supplementary Figure 3 Reduced number and altered distribution of MbDNs in adult Gli2ΔMb>E9.0 mice.

(a-c) Quantitative analysis of MbDNs at different rostrocaudal levels in P21 control and Gli2ΔMb>E9.0 brains. (a) TH+ cells (n = 3 mice, unpaired t test: rostral: P < 0.0001, t(10) = 10.8, intermediate: P < 0.0001, t(10) = 35.3 and P = 0.0014, t(5.9) = 5.86 (Welch’s correction), caudal: P = 0.002, t(10) = 4.26). (b) TH+ Calbindin+ cells (n = 3 mice, unpaired t test, rostral: P = 0.0086, t(4) = 4.80, intermediate: P = 0.0016, t(4)=7.59 and P = 0.0008, t(4) = 8.93, caudal: P = 0.0020, t(4) = 7.21). (c) TH+ Girk2+ cells (n = 3 mice, rostral: P = 0.0011, t(4) = 8.36, intermediate: P = 0.0012, t(4) = 8.23 and P = 0.1536, t(4) = 1.76, caudal: P = 0.2513, t(4) = 1.34). (d,e) Mediolateral distribution of TH+ Girk2+ cells in control and Gli2ΔMb>E9.0 brains at P21. (e) The ventral midbrain was divided into 600 μm wide bins starting at the midline. (d) TH+ Girk2+ cells were counted in each bin (bilaterally) and normalized for the total number of counted cells n = 3 mice; unpaired t test: 0–600 μm: P = 0.0020, t(4) = 7.185; 600–1,200 μm: P = 0.0062, t(4) = 5.262; >1,200 μm: P = 0.0011, t(4) = 8.346). Error bars indicate s.e.m.

Supplementary Figure 4 MbDNs are reduced in Gli2ΔMb>E9.0 mice during embryonic development.

Immunostaining for differentiating MbDNs (TH+) in E11.5 (a,b), E12.5 (c,d), E14.5 (e,f) and E18.5 (g,h) control and Gli2ΔMb>E9.0 brains. Red arrowheads indicate areas in which MbDNs are severely reduced in Gli2ΔMb>E9.0 embryos. (i,j) Immunostaining for Nurr1, which is expressed in differentiated MbDNs and in a medial TH– cell population at E18.5. Insets in i, j: Coexpression of Nurr1 and TH. (k,l) Immunostaining for Foxa2, which is expressed in differentiated TH+ cells, in a medial TH– cell population and in the red nucleus (RN) at E18.5. Insets in k, l: Coexpression of Foxa2 and TH. Asterisks indicate midline. Scale bars in (a-f) and g-h represent 100 µm.

Supplementary Figure 5 Projections from MbDNs to forebrain targets and number of TH+ cells in the locus coeruleus.

(a-c) MbDN mesostriatal projections are not significantly reduced in Gli2ΔMb>E9.0 mice. (a) Quantitative analysis of glyco-DAT+ projections to striatum (relative fluorescence intensity normalized for the area). n = 3 mice, unpaired t test: CPu: P = 0.2867, t(2) = 1.43 (Welch’s correction), NAc: P = 0.3361, t(4) = 1.09, OT: P = 0.9048, t(4) = 0.13. (b,c) TH+ and glyco-DAT+ projections in the striatum of control and Gli2ΔMb>E9.0 mice. Scale bar represents 400 µm. (d) The number of TH+ noradrenergic neurons in the locus coeruleus is not significantly changed in Gli2ΔMb>E9.0 compared to control mice. n = 5 mice, unpaired t test, P = 0.6601, t(8) = 0.4566. (e,f) Retrograde tracing of mPFC- and NAc-projecting neurons using cholera toxin subunit B (CTB). Number of TH+ CTB+ neurons (e) and of TH– CTB+ neurons (f) in the VTA shows that only mPFC-projecting MbDNs (TH+ CTB+ in e) are significantly reduced in the mutants. NAc control: n = 6 mice, NAc mutant: n = 5 mice, mPFC control: n = 6 mice, mPFC mutant: n = 7 mice. (e) Unpaired t test: NAc: P = 0.8784, t(9) = 0.16; mPFC: P = 0.0201, t(6.06) = 3.13 (Welch’s correction). (f) unpaired t test: NAc: P = 0.0987, t(5.72) = 1.97 (Welch’s correction); mPFC: P = 0.1737, t(11) = 1.46. Error bars indicate s.e.m.

Supplementary Figure 6 Dopamine content in the PFC and striatum as measured by HPLC.

Dopamine content in the PFC and striatum as measured by HPLC. The levels of dopamine and its metabolite DOPAC are significantly decreased in the PFC and striatum of Gli2ΔMb>E9.0 mice compared with those of control mice. (a) PFC control: n = 32 samples from 18 mice; Gli2ΔMb>E9.0: n = 44 samples from 24 mice. Mann-Whitney test: DA: P < 0.0001, U =109, DOPAC: P = 0.0024, U = 419. (b) Striatum: control: n = 36 samples form 18 mice; Gli2ΔMb>E9.0: n = 47 samples from 24 mice. Mann-Whitney test: DA: P < 0.0001, U = 237.5, DOPAC: P < 0.0001, U = 373. Error bars indicate s.e.m.

Supplementary Figure 7 Properties of TH+ Chr2-eYFP+ VTA neurons.

(a) Representative example of an immunohistochemically identified biocytin-labeled TH+ neuron in the VTA. Scale bar represents 10 μm. (b,c) Electrophysiological properties of the neuron shown in (a). The neuron demonstrated no obvious sag component. With increasing membrane depolarization, this TH+ neuron showed a pronounced slow depolarization until the first action potential, resulting in a prolonged latency for action potential firing (depicted in the lower panel in c).

Supplementary Figure 8 Electrophysiological properties of two distinct groups of mPFC interneurons.

(a,c) Morphology and intrinsic firing properties of type 1 and type 2 mPFC layer V/VI interneurons. (a) Type 1 interneuron with rapid firing, and no hyperpolarizing sag. (b) Type 1 interneurons are characterized by narrow and small amplitude action potentials with large fAHP (fast afterhyperpolarization, see inset). (c) Type 2 interneuron: Slow firing with accommodation and a prominent hyperpolarizing sag. (d) Type 2 interneurons generate wider and larger action potentials, no or small fAHPs and noticeable mAHPs (medium afterhyperpolarization, see inset and Supplementary Table 5). (e) Quantification of the EPSP amplitude elicited by blue-light stimulation of ChR2-eYFP+ mPFC-projecting VTA neurons. Note, that type 1 mPFC interneurons (n = 5) received significantly larger EPSPs compared to type 2 (n = 7) interneurons (unpaired t test, P = 0.01). Error bars indicate s.e.m.

Supplementary Figure 9 Five-choice serial reaction time task to investigate visual attentional processes.

(a-d) Autoshaping. Gli2ΔMb>E9.0 mice and controls learned to associate the light stimulus and the food reward to the same extent. This is evident in an increase in the number of trials completed before automatic pellet delivery (a, session effect: F(2,19) = 6.66; P = 0.005; genotype effect: F(1,9) = 0.16; interaction effect: F(2,19) = 0.37; both P-values > 0.05), a decrease in response latency over sessions (b, session effect: F(4,37) = 5.96; P = 0.001; genotype effect: F(1,9) = 0.55; interaction effect: F(4,37) = 1.55; both P-values > 0.05) and the absence of genotype differences for those parameters. Gli2ΔMb>E9.0 and control mice completed a comparable number of trials, indicating that both groups were equally motivated to perform the task (c, genotype effect: F(1,9) = 1.55; session effect: F(2,25) = 1.07; interaction effect: F(2,25) = 0.62; all P-values > 0.05). In addition, the number of nose-pokes during the inter trial interval (ITI, i.e. impulsivity index) was comparable in Gli2ΔMb>E9.0 and control mice (d, genotype effect: F(1,9) = 0.07 session effect: F(4,37) = 2.01; interaction effect: F(4,37) = 2.06; all P-values > 0.05). (e,f) 5-CSRTT. (e) Gli2ΔMb>E9.0 and control mice completed a comparable number of trials (all P values > 0.05) revealing that Gli2ΔMb>E9.0 and control mice had similar motivation levels. (f) Impulsivity was found to be comparable between groups (all P-values > 0.05). (a-f) Gli2ΔMb>E9.0: n = 5 mice, controls: n = 6 mice. Significance was determined by repeated measures ANOVA. Error bars indicate s.e.m.

Supplementary Figure 10 Motor activity and object recognition.

(a) Horizontal locomotor activity (the distance traveled over 10 min) was not significantly different in Gli2ΔMb>E9.0 and control mice. Unpaired t test, P = 0.369, t(9) = 0.891. (b) Vertical locomotor activity (number of rearings over 10 min) was reduced in Gli2ΔMb>E9.0 mice compared to control mice. Unpaired t test, P = 0.024, t(9) = 3,725. Gli2ΔMb>E9.0: n = 5 mice, controls: n = 6 mice. (c-e) Recognition memory in control and Gli2ΔMb>E9.0 mice. (c) Object recognition test with presentation of two identical objects during an acquisition phase (10 min), a retention period (50 min), and a recognition period (5 min). (d) Total time spent exploring objects during the first (acquisition) trial and the second (recognition) trial was increased in control mice (from 22.6 to 33.6 s, P = 0.009, t(10) = 3.2, paired t test), but decreased in Gli2ΔMb>E9.0 mice (from 31.7 to 22.3 s, P = 0.015, t(11) = 2.9, paired t test). (e) Discrimination ratio (i.e. the ratio of time spent exploring object 1 divided by the time spent exploring object 2) was increased in control and Gli2ΔMb>E9.0 mice (control: from 0.52 ± 0.04 to 0.66 ± 0.07, P = 0.047, t(10) = 2.3; mutants: 0.42 to 0.58, P = 0.004; t(11) = 3.7 paired t test). The amount of increase was not significantly different between control and Gli2ΔMb>E9.0 mice (P = 0.64, unpaired t test). Gli2Δ Mb>E9.0: n = 11 mice, controls: n = 12 mice. Error bars indicate s.e.m.

Supplementary Figure 11 Mesocortical inhibitory circuit.

Mesocortical MbDNs co-releasing glutamate (TH-vGlut2 neurons, light green) are the primary component of a specific circuit that directly targets local interneurons (INs) in the PFC, thereby inhibiting principal neurons (PNs). vGlut2-only neurons might also provide some contribution to this circuit. This mesocortical circuit is essentially lost in Gli2ΔMb>E9.0 mice. Potential direct projections of VTA neurons to PNs or projections of TH-only neurons to INs are not depicted in the schematic.

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Kabanova, A., Pabst, M., Lorkowski, M. et al. Function and developmental origin of a mesocortical inhibitory circuit. Nat Neurosci 18, 872–882 (2015). https://doi.org/10.1038/nn.4020

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