Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molecularly defined cortical astroglia subpopulation modulates neurons via secretion of Norrin

Nature Neuroscience volume 22, pages 741–752 (2019)Cite this article

Subjects

Abstract

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Generation of GLT1 promoter reporter mice employing increasing lengths of the DNA sequence upstream of the transcription start site.
Fig. 2: 8.3 kb tdTomato expression is static and limited to an astroglia subset in the cerebral motor cortex.
Fig. 3: Astroglial cell populations isolated from 8.3-astroglia mouse CNS and human cortex have unique transcriptomes, as validated with IHC and RNA in situ hybridization.
Fig. 4: Functional and dysfunctional properties of astroglia subpopulation by examination of LGR6 pathways in the cerebral motor cortex.
Fig. 5: Norrin is a neuromodulating protein that affects cortical neuron dendritic morphology and spine density.
Fig. 6: Norrin alters the electrophysiological properties of cortical neurons; Norrin-null mice display neurobehavioral abnormalities.

Similar content being viewed by others

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Zhang, Y. & Barres, B. A. Astrocyte heterogeneity: an underappreciated topic in neurobiology. Curr. Opin. Neurobiol. 20, 588–594 (2010).

    Article  CAS  Google Scholar 

  2. Bruijn, L. I. et al. ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron 18, 327–338 (1997).

    Article  CAS  Google Scholar 

  3. Molofsky, A. V. et al. Astrocyte-encoded positional cues maintain sensorimotor circuit integrity. Nature 509, 189–194 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Tsai, H. H. et al. Regional astrocyte allocation regulates CNS synaptogenesis and repair. Science 337, 358–362 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Oberheim, N. A., Goldman, S. A. & Nedergaard, M. Heterogeneity of astrocytic form and function. Methods Mol. Biol. 814, 23–45 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Chaboub, L. S. & Deneen, B. Developmental origins of astrocyte heterogeneity: the final frontier of CNS development. Dev. Neurosci. 34, 379–388 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Miller, S. J. Astrocyte heterogeneity in the adult central nervous system. Front. Cell. Neurosci. 12, 401 (2018).

    Article  PubMed  Google Scholar 

  8. Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015).

    Article  CAS  Google Scholar 

  9. Rothstein, J. D., Van Kammen, M., Levey, A. I., Martin, L. J. & Kuncl, R. W. Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann. Neurol. 38, 73–84 (1995).

    Article  CAS  Google Scholar 

  10. Rothstein, J. D. et al. Glutamate transporter subtypes: role in excitotoxicity and amyotrophic lateral sclerosis. Ann. Neurol. 36, abstr. 282 (1994).

    Google Scholar 

  11. Regan, M. R. et al. Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS. J. Neurosci. 27, 6607–6619 (2007).

    Article  CAS  Google Scholar 

  12. Tanaka, K. et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702 (1997).

    Article  CAS  Google Scholar 

  13. Higashimori, H. et al. Astroglial FMRP-dependent translational down-regulation of mGluR5 underlies glutamate transporter GLT1 dysregulation in the fragile X mouse. Hum. Mol. Genet. 22, 2041–2054 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Swanson, R. A. et al. Neuronal regulation of glutamate transporter subtype expression in astrocytes. J. Neurosci. 17, 932–940 (1997).

    Article  CAS  Google Scholar 

  15. Ellis, B. L., Hirsch, M. L., Porter, S. N., Samulski, R. J. & Porteus, M. H. Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs. Gene Ther. 20, 35–42 (2013).

    Article  CAS  Google Scholar 

  16. Parr-Brownlie, L. C. et al. Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms. Front. Mol. Neurosci. 8, 14 (2015).

    Article  PubMed  Google Scholar 

  17. Foo, L. C. Purification of astrocytes from transgenic rodents by fluorescence-activated cell sorting. Cold Spring Harb. Protoc. 2013, 551–560 (2013).

    Article  Google Scholar 

  18. Cahoy, J. D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Nieweg, K., Schaller, H. & Pfrieger, F. W. Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats. J. Neurochem. 109, 125–134 (2009).

    Article  CAS  Google Scholar 

  20. Snippert, H. J. et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327, 1385–1389 (2010).

    Article  CAS  Google Scholar 

  21. Jiang, P. et al. hESC-derived Olig2+ progenitors generate a subtype of astroglia with protective effects against ischaemic brain injury. Nat. Commun. 4, 2196 (2013).

    Article  PubMed  Google Scholar 

  22. Tatsumi, K. et al. Olig2-lineage astrocytes: a distinct subtype of astrocytes that differs from GFAP astrocytes. Front. Neuroanat. 12, 8 (2018).

    Article  PubMed  Google Scholar 

  23. Hrvatin, S. et al. Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex. Nat. Neurosci. 21, 120–129 (2018).

    Article  CAS  Google Scholar 

  24. Schaum, N. et al. Transcriptomic characterization of 20 organs and tissues from mouse at single cell resolution creates a Tabula Muris. Nature 562, 367–372 (2018).

    Article  Google Scholar 

  25. Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Li, J. Y. et al. LGR4 and its ligands, R-spondin 1 and R-spondin 3, regulate food intake in the hypothalamus of male rats. Endocrinology 155, 429–440 (2014).

    Article  Google Scholar 

  27. Ye, X., Smallwood, P. & Nathans, J. Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain. Gene Expr. Patterns 11, 151–155 (2011)./5/5/20

    Article  CAS  Google Scholar 

  28. Braunger, B. M. & Tamm, E. R. The different functions of Norrin. Adv. Exp. Med. Biol. 723, 679–683 (2012).

    Article  CAS  Google Scholar 

  29. Seitz, R., Hackl, S., Seibuchner, T., Tamm, E. R. & Ohlmann, A. Norrin mediates neuroprotective effects on retinal ganglion cells via activation of the Wnt/β-catenin signaling pathway and the induction of neuroprotective growth factors in Muller cells. J. Neurosci. 30, 5998–6010 (2010).

    Article  CAS  Google Scholar 

  30. Deng, C. et alMulti-functional norrin is a ligand for the LGR4 receptor. J. Cell Sci. 126, 2060–2068 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Ott, S., Patel, R. J., Appukuttan, B., Wang, X. & Stout, J. T. A novel mutation in the Norrie disease gene. J. AAPOS 4, 125–126 (2000).

    Article  CAS  Google Scholar 

  32. Warburg, M. Norrie’s disease. Birth Defects Orig. Artic. Ser. 7, 117–124 (1971).

    CAS  PubMed  Google Scholar 

  33. Tong, X. et al. Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington’s disease model mice. Nat. Neurosci. 17, 694–703 (2014).

    Article  CAS  PubMed  Google Scholar 

  34. Liao, X. H. & Nguyen, H. Epidermal expression of Lgr6 is dependent on nerve endings and Schwann cells. Exp. Dermatol. 23, 195–198 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Yang, Y. et al. Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1. Neuron 61, 880–894 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Swann, J. W., Al-Noori, S., Jiang, M. & Lee, C. L. Spine loss and other dendritic abnormalities in epilepsy. Hippocampus 10, 617–625 (2000).

    Article  CAS  Google Scholar 

  37. Fogarty, M. J., Noakes, P. G. & Bellingham, M. C. Motor cortex layer V pyramidal neurons exhibit dendritic regression, spine loss, and increased synaptic excitation in the presymptomatic hSOD1G93A mouse model of amyotrophic lateral sclerosis. J. Neurosci. 35, 643–647 (2015).

    Article  Google Scholar 

  38. Fiala, J. C. S. J., Spacek, J. & Harris, K. M. Dendritic spine pathology: cause or consequence of neurological disorders? Brain Res. Brain Res. Rev. 39, 29–54 (2002).

    Article  PubMed  Google Scholar 

  39. Orre, M. et al. Isolation of glia from Alzheimer’s mice reveals inflammation and dysfunction. Neurobiol. Aging 35, 2746–2760 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Blanco-Suarez, E., Liu, T. F., Kopelevich, A. & Allen, N. J. Astrocyte-secreted chordin-like 1 drives synapse maturation and limits plasticity by increasing synaptic GluA2 AMPA receptors. Neuron 100, 1116–1132.e13 (2018).

    Article  CAS  Google Scholar 

  41. Lanjakornsiripan, D. et al. Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers. Nat. Commun. 9, 1623 (2018).

    Article  PubMed  Google Scholar 

  42. Yang, Y. et al. Molecular comparison of GLT1+ and ALDH1L1+ astrocytes in vivo in astroglial reporter mice. Glia 59, 200–207 (2011).

    Article  PubMed  Google Scholar 

  43. Mastorakos, P. et al. Highly PEGylated DNA nanoparticles provide uniform and widespread gene transfer in the brain. Adv. Healthc. Mater. 4, 1023–1033 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Zhang, K. et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56–61 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Li, Y. et al. A comprehensive library of familial human amyotrophic lateral sclerosis induced pluripotent stem cells. PLoS One 10, e0118266 (2015).

    Article  PubMed  Google Scholar 

  46. Donnelly, C. J. et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415–428 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Miller, S. J. & Rothstein, J. D. Astroglia in thick tissue with super resolution and cellular reconstruction. PLoS One 11, e0160391 (2016).

    Article  PubMed  Google Scholar 

  48. Tomer, R., Ye, L., Hsueh, B. & Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat. Protoc. 9, 1682–1697 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Staudt, T., Lang, M. C., Medda, R., Engelhardt, J. & Hell, S. W. 2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Tech. 70, 1–9 (2007).

    Article  CAS  Google Scholar 

  50. Paukert, M. et al. Norepinephrine controls astroglial responsiveness to local circuit activity. Neuron 82, 1263–1270 (2014).

    Article  CAS  PubMed  Google Scholar 

  51. Ferreira, T. A. et al.Neuronal morphometry directly from bitmap images. Nat. Methods 11, 982–984 (2014).

    Article  CAS  PubMed  Google Scholar 

  52. Salinas, E. & Sejnowski, T. J. Correlated neuronal activity and the flow of neural information. Nat. Rev. Neurosci. 2, 539–550 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Poli, D., Pastore, V. P. & Massobrio, P. Functional connectivity in in vitro neuronal assemblies. Front. Neural Circuits 9, 57 (2015).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Johns Hopkins Deep Sequencing and Microarray Core Facility and C. Conover Talbot Jr. for insight on microarray, and analyses using the Partek and Spotfire software suites, H. Zhang for assistance with FACS at the Johns Hopkins University School of Public Health FACS Center, the Johns Hopkins Medicine Microscopy Core for the use of the Zeiss LSM 700 laser scanning confocal microscope, J. Nathans for the Norrin mice, L. Ostrow for postmortem tissue, and members of the Rothstein laboratory for helpful discussions. This work was funded by grants from the National Science Foundation Graduate Fellowship Research Program (S.J.M.), grant no. R01NS092067 (J.D.R., M.B.R.), and NIH grant no. R01NS094239 (J.D.R.).

Author information

Authors and Affiliations

  1. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Sean J. Miller, Thomas Philips, Zhuoxun Chen, Yi-Chun Hsieh, J. Gavin Daigle, Jeannie Chew, Svetlana Vidensky, Jacqueline T. Pham, Rita Sattler & Jeffrey D. Rothstein

  2. Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Sean J. Miller, Thomas Philips, Zhuoxun Chen, Yi-Chun Hsieh, J. Gavin Daigle, Jeannie Chew, Svetlana Vidensky, Jacqueline T. Pham, Rita Sattler & Jeffrey D. Rothstein

  3. Cellular & Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Sean J. Miller, Jacqueline T. Pham & Jeffrey D. Rothstein

  4. The Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Namho Kim, Jung Soo Suk & Justin Hanes

  5. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Namho Kim & Justin Hanes

  6. Department of Neurology, Richard T. Johnson Division of Neuroimmunology and Neurological Infections, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Raha Dastgheyb & Norman Haughey

  7. Department of Biological Sciences, Columbia University, New York, NY, USA

    Malika Datta & Raju Tomer

  8. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Ethan G. Hughes, Dwight E. Bergles & Jeffrey D. Rothstein

  9. Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA

    Michael B. Robinson

  10. Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Michael B. Robinson

  11. Departments of Biomedical Engineering, Environmental and Health Sciences, Oncology, Neurosurgery, and Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD, USA

    Jung Soo Suk & Justin Hanes

  12. Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Norman Haughey & Mikhail Pletnikov

Authors
  1. Sean J. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas Philips
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Namho Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raha Dastgheyb
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhuoxun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yi-Chun Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J. Gavin Daigle
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Malika Datta
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jeannie Chew
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Svetlana Vidensky
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jacqueline T. Pham
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ethan G. Hughes
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Michael B. Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Rita Sattler
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Raju Tomer
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Jung Soo Suk
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Dwight E. Bergles
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Norman Haughey
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Mikhail Pletnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Justin Hanes
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Jeffrey D. Rothstein
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.J.M. designed, performed, and analyzed the experiments, and wrote the manuscript. T.P. performed and E.G.H. assisted with the window surgeries and multiphoton imaging. J.G.D. and S.V. cultured and isolated the primary cortical neurons and maintained the mouse colonies. J.C. helped in Golgi imaging and interpretation; M.P. helped in behavioral assay generation and interpretation. Z.C. and Y-C.H. assisted with tissue dissociation and transferred samples to the Johns Hopkins University FACS and Sequencing/Microarray cores. N.K. generated the nanoparticles and performed the transmission electron microscopy, which was overseen by J.S.S. and J.H. M.D. and R.T. performed the tissue clearing and CLARITY-optimized light-sheet microscopy imaging. J.T.P. assisted with maintenance and differentiation of hiPSC lines. R.D. performed the MEA experimentation, which was overseen by N.H. M.B.R., R.S., and D.E.B. contributed to data interpretation and manuscript review. J.D.R. oversaw project development, experimental design, data interpretation, data representation, and manuscript writing.

Corresponding author

Correspondence to Jeffrey D. Rothstein.

Ethics declarations

Competing interests

S.J.M. and J.D.R. have filed a patent on the use of Norrin. The remaining authors declare no competing interests.

Additional information

Journal peer review information: Nature Neuroscience thanks Matthew Holt, Baljit Khakh, and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Selection and generation of the Glt1 promoter fragment transgenic mice.

Glt1 (human nomenclature EAAT2) shows four highly methylated sites in its hypothesized promoter region, which were selected for the generation of the transgenic mice.

Supplementary Figure 2 Promoter fragments < 8.3 kb result in tdTomato in nonastroglia cells in the cerebral cortex.

(a) 2.5 kb and 7.9 kb appear to label neurons in the cerebral cortex. (b) The fragment sizes 2 kb, 6.7 kb, and 7.9 kb result in no colocalization with astroglia marker Glt1 but colocalize with neuronal marker, NeuN. At least two to three mice were imaged with 3–5 images per mouse.

Supplementary Figure 3 Additional approaches to validate astroglia tdTomato expression and to validate 8.3 promoter construct using nanoparticles.

(a) CLARITY-optimized light-sheet microscopy (COLM) imaging was employed to visualize in 3D the whole brain distribution of the 8.3-astroglia. COLM shows the restricted pattern of expression of the 8.3-astroglia in the cortex as well paucity of expression in deep telencephalon structures, arrows indicate 8.3 astroglia. (b) Glt1-eGFP colocalizes with 8.3 kb-tdTomato. (c) Average cell counts (± SEM) per cortical layer of Glt1-eGFP and 8.3-astroglia in the mouse adult cortex (P60-P90; n = 3). (d, e) Nanoparticles were packaged and injected intracortically. 8.3 kb-tdTomato expression is observed in cortical layers II/III and V, consistent with the transgenic mice. (f, g) Nanoparticles were packaged with CMV-eGFP and the 8.3 kb constructs as an additional tool to evaluate astroglial specific TdTomato expression. Transmission electron microscopy at 80,000x magnification was used to show the diameter of CMV-GFP nanoparticles. Transmission electron microscopy at 80,000x magnification was used to show the diameter of 8.3 kb-tdTomato nanoparticles. At least two mice were used for COLM. For panels C,D,E, at least three mice were used for ex vivo histology with 3–5 images analyzed per mouse, representative images are shown.

Supplementary Figure 4 8.3-astroglia maintain stable tdTomato expression in vitro and have a unique transcriptome.

(a) FACS gating for isolation of CNS cells. FACS was performed on cells isolated from adult dissociated adult cortex of BAC-Glt1-eGFP/8.3-astroglia mice (n = 3) and shows reliably identified three fluorescently-unique cell populations: eGFP-only, TdTomato/eGFP, and negative. (b) 8.3-astroglia maintain stable tdTomato-expression in vitro. (c) GLT1-eGFP-only astroglia do not express tdTomato in vitro. (d) The top Ingenuity canonical pathways are listed for each astroglia population and for pathways shared between both astroglia populations from the microarray data. (e) OLIG2 colocalizes with 8.3-astroglia in the motor cortex. The collected populations were subjected to microarray RNA and qPCR analyses (n = 3 mice). Cells isolated by dissociation into single-cell and maintained in astroglia medium without FBS for up to two weeks before passaging. For histology, at least three mice were used with 3–5 images per mouse analyzed.

Supplementary Figure 5 8.3-astroglia are LGR6- and KCNJ10-positive.

(a) LGR6 colocalizes with ALDH1L1 in adult mouse motor cortex, arrows indicate immunoreactive cells. (b) KCNJ10 is enriched in 8.3-astroglia, red arrows indicate 8.3-astroglia and green arrows indicate Glt1-eGFP only astroglia. (c) KCJN10 (Kir4.1)mean intensity fluorescence (MIF) is increased significantly in 8.3-astroglia vs Glt1-eGFP astroglia (N = 100 cells). At least 3–5 mice were imaged with 3–5 images per mouse analyzed. Values plotted represent the mean with SEM error bars. Statistical analytics were performed by a two-sided Student T-test, ****p < 0.0001.

Supplementary Figure 6 Rspo1 is restricted to a neuronal subset in the lower cortical layers in the motor cortex.

(a) Rspo1-positive mRNA is identified in a cell in the lower cortical layers. (b) Rspo1 is not observed in all cells in lower cortical layers. (c) Rspo1 is restricted to neurons in the lower cortical layers of the motor cortex. (d) Rspo1 is in not observed in all neurons in the lower cortical layers of the motor cortex. At least 3–5 mice were analyzed with 3–5 images per mouse. Black arrows indicate Rspo1 RNA.

Supplementary Figure 7 RSPO1 treatment increases LGR6-positive astroglia proliferation, but in vivo knockdown of LGR6 leads to reduced cortical thickness.

(a) Dose-dependent increase in astroglia post 24-hour RSPO1-treatment. (b) Dose-dependent increase in the overall amount of LGR6-positive astroglia post 24-hour RSPO1-treatment. (c) Lgr6-heterozygote null mice display a significant reduction in cortical thickness. Data shown reflect 5 different primary astroglia isolations with 5 repeats per treatment were used for RSPO1 treatments. At least seven mice were used with 3–5 images per mouse for cortical thickness. Thickness was calculated by measuring the distance from the corpus callosum to the pia of the motor cortex following the Allen Brain Mouse Atlas Reference. Values plotted represent the mean with SEM error bars. Statistical analytics were performed by a two-sided Student’s T-test between experimental and control or PBS, *p < 0.05, **p < 0.01, ***p < 0.001.

Supplementary Figure 8 Lgr6-heterozygote and Norrin-null mice exhibit spine-density deficits in cortical layer V of the motor cortex.

(a) Wild-type mice dendritic spines. (b) Lgr6-heterozygote spine density is reduced compared to wild-type. Five mice were analyzed per genotype for spine density. (c) Norrin-null spine density is reduced compared to wild-type. Representative images are shown from apical dendrites from pyramidal layer V neurons, a minimum of 5 neurons analyzed per mouse.

Supplementary Figure 9 Norrin treatment affects the overall morphology of cortical neurons.

(a) Primary cortical neurons treated with a PBS vehicle control. (b) Primary cortical neurons treated with Norrin for 48 h show increased branching and dendritic length compared to the PBS control. (c) Primary cortical neurons treated for 48 h with truncated protein 1. (d) Primary cortical neurons treated for 48 h with truncated protein 2. At least 5–10 primary cortical isolations with 3–5 repeats per treatment and 15–20 neurons analyzed per treatment. Somas are drawn in, images not to scale.

Supplementary Figure 10 Norrin alters neuronal morphology.

(a) Primary cortical neurons treated with a PBS vehicle control. (b) Primary cortical neurons treated with Norrin for 48 h show increased branching and dendritic length compared to the PBS control. (c) Primary cortical neurons treated for 48 h with truncated protein 1. (d) Primary cortical neurons treated for 48 h with truncated protein 2. At least 5–10 primary cortical isolations with 3–5 repeats per treatment and 15–20 neurons analyzed per treatment.

Supplementary Figure 11 Norrin treatment alters the electrophysiology of cortical neurons.

(a) Rastaplots of the MEA recordings after 24 h of treatment with Norrin. Representative images are shown. MEA analyses were performed 3 times at each time point, each with three different treatments as shown.

Supplementary Figure 12 Norrin treatment alters the electrophysiology of cortical neurons.

(a) Number of spikes is significantly increased post Norrin-treatment. (b) Percent of spikes in a burst on the MEA recordings is significantly increased post Norrin-treatment. (c) Burst total weight is significantly increased in Norrin treated neurons. (d) Degree of bursts is significantly increased post Norrin-treatment. (e) Degree of spikes is significantly increased in Norrin treated neurons. (f) Weight total of the neuronal spikes is significantly increased in Norrin treated neurons. MEA analyses were performed 3 times at each time point, each with three different treatments. Values plotted represent the mean with SEM error bars, along with individual data points. Statistical analytics were performed by a two-sided Student’s T-test between control, denatured and norrin treatments, *p < 0.05, **p < 0.01, ***p < 0.001.

Supplementary Figure 13 Sample voltage traces and spike-time stamps from MEA experiments.

A 30 second sample is shown from a single electrode for each condition. In black are the voltages which have been filtered using a second order butterworth filter with a 200 Hz cutoff frequency. Spike time stamps, identified as time points where voltages exceed a threshold of at least five standard deviations from baseline, are marked as blue dots above the voltages. Representative images are shown. Electrophysiological analyses were performed 3 times at each time point, each with three different treatments as shown.

Supplementary Figure 14 The physiological pathway hypothesized for 8.3-astroglia interacting with corresponding neuronal neighbors.

8.3-astroglia residing the adult motor cortex typically display normal levels of Lgr6 and Norrin, which lead to typical neuronal morphology, electrophysiology, and overall behavior. Deletion or knockdown of LGR6 or Norrin leads to altered dendritic morphology, spine density, and behavior. This drawing was created by the Johns Hopkins Art as Applied to Medicine Department expressly for this article.

Supplementary information

Supplementary Figs. 1–14 and Supplementary Tables 1–5.

Reporting Summary

41593_2019_366_MOESM3_ESM.mov

Supplementary Video 1 CLARITY-optimized light-sheet microscopy (COLM) video of coronal view of mouse cortex post-tissue clearing of 8.3-astroglia mice. This preparation provides a 3D visualization of the whole brain of the 8.3-astroglia mice, illustrating the selective localization of 8.3-astroglia in the cortex.

41593_2019_366_MOESM4_ESM.mov

Supplementary Video 2 Live multiphoton in vivo imaging of motor cortex of adult 8.3-astroglia mice demonstrates visualization of individual 8.3-astroglia. This study provides in vivo evidence that the tdTomato fluorescence is stably expressed in 8.3-astroglia over time.

41593_2019_366_MOESM5_ESM.mov

Supplementary Video 3 MEA activity post 24-h treatment with PBS control. These studies demonstrate that the PBS control treatment does not alter the overall electrophysiological activity of cortical neurons.

41593_2019_366_MOESM6_ESM.mov

Supplementary Video 4 MEA activity after 24-h treatment with Norrin denatured control. These studies demonstrate that the denatured protein control treatment does not alter the overall electrophysiological activity of cortical neurons.

41593_2019_366_MOESM7_ESM.mov

Supplementary Video 5 MEA activity after 24-h treatment with Norrin. These studies demonstrate that treating the cultured neurons with Norrin alters the overall electrophysiological activity of cortical neurons.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Miller, S.J., Philips, T., Kim, N. et al. Molecularly defined cortical astroglia subpopulation modulates neurons via secretion of Norrin. Nat Neurosci 22, 741–752 (2019). https://doi.org/10.1038/s41593-019-0366-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-019-0366-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing