Cilia pp 203-214 | Cite as

Methods for Visualization of Neuronal Cilia

  • Tamara CasparyEmail author
  • Daniela Marazziti
  • Nicolas F. Berbari
Part of the Methods in Molecular Biology book series (MIMB, volume 1454)


Neuroscientists have been captivated by cilia ever since these slender, microtubule-based projections on the cell body were found to play critical roles in neuronal specification, maintenance, and function. In mammals, the most common cilia marker, acetylated α-tubulin, is extremely difficult to detect in neuronal cilia. Here, we describe methods to detect neuronal cilia in culture, in fixed sections, and in vivo, taking advantage of transgenic mice carrying fluorescently tagged cilia proteins.

Key words

Acetylated α-tubulin Arl13b ACIII Cilia-GFP 



Special thanks to Cheryl Timms Strauss for editing.


  1. 1.
    Askwith CC, Wemmie JA, Price MP, Rokhlina T, Welsh MJ (2004) Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H + -activated currents in hippocampal neurons. J Biol Chem 279(18):18296–18305CrossRefPubMedGoogle Scholar
  2. 2.
    Berbari NF, Bishop GA, Askwith CC, Lewis JS, Mykytyn K (2007) Hippocampal neurons possess primary cilia in culture. J Neurosci Res 85(5):1095–1100CrossRefPubMedGoogle Scholar
  3. 3.
    Brewer GJ, Torricelli JR, Evege EK, Price PJ (1993) Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination. J Neurosci Res 35(5):567–576CrossRefPubMedGoogle Scholar
  4. 4.
    Domire JS, Green JA, Lee KG, Johnson AD, Askwith CC, Mykytyn K (2011) Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet-Biedl syndrome proteins. Cell Mol Life Sci 68(17):2951–2960CrossRefPubMedGoogle Scholar
  5. 5.
    Wemmie JA, Chen J, Askwith CC, Hruska-Hageman AM, Price MP, Nolan BC, Yoder PG, Lamani E, Hoshi T, Freeman JH Jr, Welsh MJ (2002) The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory. Neuron 34(3):463–477CrossRefPubMedGoogle Scholar
  6. 6.
    Berbari NF, Johnson AD, Lewis JS, Askwith CC, Mykytyn K (2008) Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors. Mol Biol Cell 19(4):1540–1547CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bishop GA, Berbari NF, Lewis J, Mykytyn K (2007) Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. J Comp Neurol 505(5):562–571CrossRefPubMedGoogle Scholar
  8. 8.
    Brailov I, Bancila M, Brisorgueil MJ, Miquel MC, Hamon M, Verge D (2000) Localization of 5-HT(6) receptors at the plasma membrane of neuronal cilia in the rat brain. Brain Res 872(1-2):271–275CrossRefPubMedGoogle Scholar
  9. 9.
    Caspary T, Larkins CE, Anderson KV (2007) The graded response to Sonic Hedgehog depends on cilia architecture. Dev Cell 12(5):767–778CrossRefPubMedGoogle Scholar
  10. 10.
    Handel M, Schulz S, Stanarius A, Schreff M, Erdtmann-Vourliotis M, Schmidt H, Wolf G, Hollt V (1999) Selective targeting of somatostatin receptor 3 to neuronal cilia. Neuroscience 89(3):909–926CrossRefPubMedGoogle Scholar
  11. 11.
    Koemeter-Cox AI, Sherwood TW, Green JA, Steiner RA, Berbari NF, Yoder BK, Kauffman AS, Monsma PC, Brown A, Askwith CC, Mykytyn K (2014) Primary cilia enhance kisspeptin receptor signaling on gonadotropin-releasing hormone neurons. Proc Natl Acad Sci U S A 111(28):10335–10340CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Domire JS, Mykytyn K (2009) Markers for neuronal cilia. Methods Cell Biol 91:111–121CrossRefPubMedGoogle Scholar
  13. 13.
    Carson FL, Cappellano CH (2015) Histotechnology a self instructional text, 4th edn. ASCP, Chicago, IL, p 368Google Scholar
  14. 14.
    Berbari NF, Malarkey EB, Yazdi SM, McNair AD, Kippe JM, Croyle MJ, Kraft TW, Yoder BK (2014) Hippocampal and cortical primary cilia are required for aversive memory in mice. PLoS One 9(9), e106576CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Berbari NF, Pasek RC, Malarkey EB, Yazdi SM, McNair AD, Lewis WR, Nagy TR, Kesterson RA, Yoder BK (2013) Leptin resistance is a secondary consequence of the obesity in ciliopathy mutant mice. Proc Natl Acad Sci U S A 110(19):7796–7801CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    O'Connor AK, Malarkey EB, Berbari NF, Croyle MJ, Haycraft CJ, Bell PD, Hohenstein P, Kesterson RA, Yoder BK (2013) An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue. Cilia 2(1):8CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Delling M, DeCaen PG, Doerner JF, Febvay S, Clapham DE (2013) Primary cilia are specialized calcium signalling organelles. Nature 504(7479):311–314CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Bangs FK, Schrode N, Hadjantonakis AK, Anderson KV (2015) Lineage specificity of primary cilia in the mouse embryo. Nat Cell Biol 17(2):113–122CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Berman SA, Wilson NF, Haas NA, Lefebvre PA (2003) A novel MAP kinase regulates flagellar length in Chlamydomonas. Curr Biol 13(13):1145–1149CrossRefPubMedGoogle Scholar
  20. 20.
    Nguyen RL, Tam LW, Lefebvre PA (2005) The LF1 gene of Chlamydomonas reinhardtii encodes a novel protein required for flagellar length control. Genetics 169(3):1415–1424CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Tam LW, Ranum PT, Lefebvre PA (2013) CDKL5 regulates flagellar length and localizes to the base of the flagella in Chlamydomonas. Mol Biol Cell 24(5):588–600CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Tam LW, Wilson NF, Lefebvre PA (2007) A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas. J Cell Biol 176(6):819–829CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Falcon-Urrutia P, Carrasco CM, Lois P, Palma V, Roth AD (2015) Shh signaling through the primary cilium modulates rat oligodendrocyte differentiation. PLoS One 10(7), e0133567CrossRefPubMedCentralGoogle Scholar
  24. 24.
    Menzl I, Lebeau L, Pandey R, Hassounah NB, Li FW, Nagle R, Weihs K, McDermott KM (2014) Loss of primary cilia occurs early in breast cancer development. Cilia 3:7CrossRefPubMedCentralGoogle Scholar
  25. 25.
    Piotrowska-Nitsche K, Caspary T (2012) Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and sonic hedgehog response. Cilia 1:1CrossRefGoogle Scholar
  26. 26.
    Mukhopadhyay S, Wen X, Ratti N, Loktev A, Rangell L, Scales SJ, Jackson PK (2013) The ciliary G-protein-coupled receptor Gpr161 negatively regulates the sonic hedgehog pathway via cAMP signaling. Cell 152(1-2):210–223CrossRefGoogle Scholar
  27. 27.
    Seo S, Zhang Q, Bugge K, Breslow DK, Searby CC, Nachury MV, Sheffield VC (2011) A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and smoothened. PLoS Genet 7(11), e1002358CrossRefPubMedCentralGoogle Scholar
  28. 28.
    Stratigopoulos G, Martin Carli JF, O'Day DR, Wang L, Leduc CA, Lanzano P, Chung WK, Rosenbaum M, Egli D, Doherty DA, Leibel RL (2014) Hypomorphism for RPGRIP1L, a ciliary gene vicinal to the FTO locus, causes increased adiposity in mice. Cell Metab 19(5):767–779CrossRefPubMedCentralGoogle Scholar
  29. 29.
    Berbari NF, Lewis JS, Bishop GA, Askwith CC, Mykytyn K (2008) Bardet-Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia. Proc Natl Acad Sci U S A 105(11):4242–4246CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Tamara Caspary
    • 1
    Email author
  • Daniela Marazziti
    • 2
  • Nicolas F. Berbari
    • 3
  1. 1.Department of Human GeneticsEmory University School of MedicineAtlantaUSA
  2. 2.Institute of Cell Biology and NeurobiologyItalian National Research Council (CNR)RomeItaly
  3. 3.Department of BiologyIndiana University-Purdue University IndianapolisIndianapolisUSA

Personalised recommendations