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Neuroanatomical Tracing Techniques in the Ear: History, State of the Art, and Future Developments

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1427)

Abstract

The inner ear has long been at the cutting edge of tract tracing techniques that have shaped and reshaped our understanding of the ear’s innervation patterns. This review provides a historical framework to understand the importance of these techniques for ear innervation and for development of tracing techniques in general; it is hoped that lessons learned will help to quickly adopt transformative novel techniques and their information and correct past beliefs based on technical limitations. The technical part of the review presents details of our protocol as developed over the last 30 years. We also include arguments as to why these recommendations work best to generate the desired outcome of distinct fiber and cell labeling, and generate reliable data for any investigation. We specifically focus on two tracing techniques, in part developed and/or championed for ear innervation analysis: the low molecular multicolor dextran amine tract tracing technique and the multicolor tract tracing technique with lipophilic dyes.

Key words

Neuronal tracing Lipophilic dyes Dextran amines Ear Innervation Efferents Afferents 
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Notes

Acknowledgements

Confocal images were obtained at the University of Iowa Carver Center for Imaging. We thank the Office of the Vice President for Research (OVPR), University of Iowa College of Liberal Arts and Sciences (CLAS), and the P30 core grant for support (DC 010362). This work was in part supported by a NASA base grant (Bernd Fritzsch) and 1R43GM108470-01 (Gray, Fritzsch).

References

  1. 1.
    Retzius G (1881) Uber die peripherische Endigungsweise des Gehornerven. Biologische Untersuchungen 3:1–51Google Scholar
  2. 2.
    Ramón y Cajal S (1906) In: Nobel lectures, physiology or medicine 1901–1921Google Scholar
  3. 3.
    Golgi C (1906) The neuron doctrine: theory and facts. Nobel Lecture 1921:190–217Google Scholar
  4. 4.
    Retzius G (1881) Das Gehörorgan der Fische und Amphibien. Samson & Wallin, StockholmGoogle Scholar
  5. 5.
    Kersigo J, Fritzsch B (2015) Inner ear hair cells deteriorate in mice engineered to have no or diminished innervation. Front Aging Neurosci 7:33CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Fritzsch B, Wake M (1988) The inner ear of gymnophione amphibians and its nerve supply: a comparative study of regressive events in a complex sensory system (Amphibia, Gymnophiona). Zoomorphol 108(4):201–217CrossRefGoogle Scholar
  7. 7.
    Sarasin P, Sarasin F (1888) Zur Entwicklungsgeschichte und Anatomie der ceylonesischen Blindwühle Ichthyophis glutinosus. Das Gehörorgan. Ergebnisse Naturwiss Forsch auf Ceylon, Bd 2. Kreidels Verlag, WiesbadenGoogle Scholar
  8. 8.
    Sarasin P, Sarasin F (1892) Über das Gehörorgan der Caeciliiden. Anat Anz 7:812–815Google Scholar
  9. 9.
    Retzius G (1891) Das Gehörorgan von caecilia annulata. Anat Anz 6:82–86Google Scholar
  10. 10.
    Ramón y Cajal S (1995) Histology of the nervous system of man and vertebrates, vol 1. Oxford University Press, New York, USAGoogle Scholar
  11. 11.
    Lorent De No R (1981) The primary acoustic nuclei. Raven Press, New YorkGoogle Scholar
  12. 12.
    Lorente de No R (1937) The sensory endings in the cochlea. Laryngoscope 47:373–377Google Scholar
  13. 13.
    Rasmussen GL (1946) The olivary peduncle and other fiber projections of the superior olivary complex. J Comp Neurol 84(2):141–219CrossRefPubMedGoogle Scholar
  14. 14.
    Rasmussen GL (1953) Further observations of the efferent cochlear bundle. J Comp Neurol 99(1):61–74CrossRefPubMedGoogle Scholar
  15. 15.
    Brown M, Pierce S, Berglund A (1991) Cochlear‐nucleus branches of thick (medial) olivocochlear fibers in the mouse: a cochaleotopic projection. J Comp Neurol 303(2):300–315CrossRefPubMedGoogle Scholar
  16. 16.
    Hillman DE (1969) Light and electron microscopical study of the relationships between the cerebellum and the vestibular organ of the frog. Exp Brain Res 9(1):1–15CrossRefPubMedGoogle Scholar
  17. 17.
    Strutz J, Schmidt CL, Stürmer C (1980) Origin of efferent fibers of the vestibular apparatus in goldfish. A horseradish peroxidase study. Neurosci Letts 18(1):5–9CrossRefGoogle Scholar
  18. 18.
    Roberts BL, Meredith GE (1992) The efferent innervation of the ear: variations on an enigma. In: DB Webster, RR Fay, AN Popper. The evolutionary biology of hearing. Springer, New York, pp 185–121Google Scholar
  19. 19.
    Fritzsch B, Wahnschaffe U (1987) Electron microscopical evidence for common inner ear and lateral line efferents in urodeles. Neurosci Letts 81(1):48–52CrossRefGoogle Scholar
  20. 20.
    Sienknecht UJ, Köppl C, Fritzsch B (2014) Evolution and development of hair cell polarity and efferent function in the inner ear. Brain Beh Evol 83(2):150–161Google Scholar
  21. 21.
    Fritzsch B (1993) Fast axonal diffusion of 3000 molecular weight dextran amines. J Neurosci Meth 50(1):95–103Google Scholar
  22. 22.
    Hellmann B, Fritzsch B (1996) Neuroanatomical and histochemical evidence for the presence of common lateral line and inner ear efferents and of efferents to the basilar papilla in a frog, Xenopus laevis. Brain Beh Evol 47(4):185–194CrossRefGoogle Scholar
  23. 23.
    Simmons D, Duncan J, de Caprona DC, Fritzsch B (2011) Development of the inner ear efferent system. In: DK Ryugo, RR Fay, AN Popper. Auditory and vestibular efferents. Springer, New York, pp 187–216Google Scholar
  24. 24.
    Yang T, Bassuk AG, Stricker S, Fritzsch B (2014) Prickle1 is necessary for the caudal migration of murine facial branchiomotor neurons. Cell Tissue Res 357(3):549–561CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Poljak S (1927) Über den allgemeinen Bauplan des Gehörsystems und über seine Bedeutung für die Physiologie, für die Klinik und für die Psychologie. Zeitschrift für die gesamte Neurologie und Psychiatrie 110(1):1–49CrossRefGoogle Scholar
  26. 26.
    Echteler SM (1992) Developmental segregation in the afferent projections to mammalian auditory hair cells. Proc Natl Acad Sci U S A 89(14):6324–6327CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Bulankina A, Moser T (2012) Neural circuit development in the mammalian cochlea. Physiology 27(2):100–112CrossRefPubMedGoogle Scholar
  28. 28.
    Sobkowicz HM, August BK, Slapnick SM (2004) Synaptic arrangements between inner hair cells and tunnel fibers in the mouse cochlea. Synapse 52(4):299–315CrossRefPubMedGoogle Scholar
  29. 29.
    Fritzsch B, Dillard M, Lavado A, Harvey NL, Jahan I (2010) Canal cristae growth and fiber extension to the outer hair cells of the mouse ear require Prox1 activity. PLoS One 5:e9377CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Koundakjian EJ, Appler JL, Goodrich LV (2007) Auditory neurons make stereotyped wiring decisions before maturation of their targets. J Neurosci 27(51):14078–14088CrossRefPubMedGoogle Scholar
  31. 31.
    Cowan CA, Yokoyama N, Bianchi LM, Henkemeyer M, Fritzsch B (2000) EphB2 guides axons at the midline and is necessary for normal vestibular function. Neuron 26(2):417–430CrossRefPubMedGoogle Scholar
  32. 32.
    Fritzsch B, Christensen M, Nichols D (1993) Fiber pathways and positional changes in efferent perikarya of 2.5‐to 7‐day chick embryos as revealed with dil and dextran amiens. J Neurobiol 24(11):1481–1499CrossRefPubMedGoogle Scholar
  33. 33.
    Ayers H, Worthington J (1908) The finer anatomy of the brain of Bdellostoma dombeyi. I. The acustico‐lateral system. Am J Anat 8(1):1–16CrossRefGoogle Scholar
  34. 34.
    Fritzsch B (1981) The pattern of lateral-line afferents in urodeles. Cell Tissue Res 218(3):581–594CrossRefPubMedGoogle Scholar
  35. 35.
    Fritzsch B, Gregory D, Rosa-Molinar E (2005) The development of the hindbrain afferent projections in the axolotl: evidence for timing as a specific mechanism of afferent fiber sorting. Zoology 108(4):297–306CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Fritzsch B (1981) Electroreceptors and direction specific arrangement in the lateral line system of salamanders? Zeitschrift für Naturforschung C 36(5–6):493–496Google Scholar
  37. 37.
    Maklad A, Kamel S, Wong E, Fritzsch B (2010) Development and organization of polarity-specific segregation of primary vestibular afferent fibers in mice. Cell Tissue Res 340(2):303–321CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Fritzsch B, López-Schier H (2014) Evolution of polarized hair cells in aquatic vertebrates and their connection to directionally sensitive neurons. In: H Bleckmann, J Mogdans, SL Coombs. Flow sensing in air and water. Springer, New York, pp 271–294Google Scholar
  39. 39.
    Glover J (1995) Retrograde and anterograde axonal tracing with fluorescent dextran-amines in the embryonic nervous system. Neurosci Prot 30:1–13Google Scholar
  40. 40.
    Fritzsch B, Wilm C (1990) Dextran amines in neuronal tracing. Trends Neurosci 13(1):14CrossRefPubMedGoogle Scholar
  41. 41.
    Tonniges J, Hansen M, Duncan J, Bassett MJ, Fritzsch B, Gray BD, Easwaran A, Nichols MG (2010) Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing. J Microsc 239(2):117–134PubMedGoogle Scholar
  42. 42.
    Godement P, Vanselow J, Thanos S, Bonhoeffer F (1987) A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue. Development 101(4):697–713PubMedGoogle Scholar
  43. 43.
    Wilm C, Fritzsch B (1992) Ipsilateral retinal projections into the tectum during regeneration of the optic nerve in the cichlid fish Haplochromis burtoni: a dil study in fixed tissue. JNeurobiol 23(6):692–707CrossRefGoogle Scholar
  44. 44.
    Huang L-C, Thorne PR, Housley GD, Montgomery JM (2007) Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea. Development 134(16):2925–2933CrossRefPubMedGoogle Scholar
  45. 45.
    Bleckmann H, Niemann U, Fritzsch B (1991) Peripheral and central aspects of the acoustic and lateral line system of a bottom dwelling catfish, Ancistrus sp. J Comp Neurol 314(3):452–466CrossRefPubMedGoogle Scholar
  46. 46.
    Elliott KL, Houston DW, DeCook R, Fritzsch B (2015) Ear manipulations reveal a critical period for survival and dendritic development at the single-cell level in Mauthner neurons. Dev Neurobiol. doi: 10.1002/dneu.22287 PubMedGoogle Scholar
  47. 47.
    Fritzsch B, Northcutt R (1992) A plastic embedding technique for analyzing fluorescent dextran-amine labelled neuronal profiles. Biotech Histochem 67(3):153–157CrossRefPubMedGoogle Scholar
  48. 48.
    Kopecky BJ, Duncan JS, Elliott KL, Fritzsch B (2012) Three-dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy. J Microsc 248(3):292–298Google Scholar
  49. 49.
    Thorne RG, Nicholson C (2006) In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space. Proc Natl Acad Sci U S A 103(14):5567–5572CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Fritzsch B (1979) Observations on degenerative changes of Purkinje cells during early development in mice and in normal and otocyst-deprived chickens. Anat Embryol 158(1):95–102CrossRefPubMedGoogle Scholar
  51. 51.
    Cragg B (1980) Preservation of extracellular space during fixation of the brain for electron microscopy. Tissue Cell 12(1):63–72CrossRefPubMedGoogle Scholar
  52. 52.
    Lu CC, Cao X-J, Wright S, Ma L, Oertel D, Goodrich LV (2014) Mutation of Npr2 leads to blurred tonotopic organization of central auditory circuits in mice. PLoS Genetics 10(12), e1004823CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Jahan I, Kersigo J, Pan N, Fritzsch B (2010) Neurod1 regulates survival and formation of connections in mouse ear and brain. Cell Tissue Res 341(1):95–110CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Maklad A, Fritzsch B (2003) Partial segregation of posterior crista and saccular fibers to the nodulus and uvula of the cerebellum in mice, and its development. Dev Brain Res 140(2):223–236CrossRefGoogle Scholar
  55. 55.
    Costantini I, Ghobril J-P, Di Giovanna AP et al (2015) A versatile clearing agent for multi-modal brain imaging. Sci Rep 5:9808CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Tsuriel S, Gudes S, Draft RW, Binshtok AM, Lichtman JW (2015) Multispectral labeling technique to map many neighboring axonal projections in the same tissue. Nat Methods 12(6):547–552CrossRefPubMedGoogle Scholar
  57. 57.
    Manns M, Fritzsch B (1991) The eye in the brain: retinoic acid effects morphogenesis of the eye and pathway selection of axons but not the differentiation of the retina in Xenopus laevis. Neurosci Letts 127(2):150–154CrossRefGoogle Scholar
  58. 58.
    Gehler S, Gallo G, Veien E, Letourneau PC (2004) p75 neurotrophin receptor signaling regulates growth cone filopodial dynamics through modulating RhoA activity. J Neurosci 24(18):4363–4372CrossRefPubMedGoogle Scholar
  59. 59.
    Pauley S, Lai E, Fritzsch B (2006) Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear. Dev Dyn 235(9):2470–2482CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Cai D, Cohen KB, Luo T, Lichtman JW, Sanes JR (2013) Improved tools for the Brainbow toolbox. Nat Methods 10(6):540–547CrossRefPubMedCentralGoogle Scholar
  61. 61.
    Abe T, Fujimori T (2013) Reporter mouse lines for fluorescence imaging. Dev Growth Differ 55(4):390–405CrossRefPubMedGoogle Scholar
  62. 62.
    Viswanathan S, Williams ME, Bloss EB et al (2015) High-performance probes for light and electron microscopy. Nat Methods 12:568–576CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Mikula S, Denk W (2015) High-resolution whole-brain staining for electron microscopic circuit reconstruction. Nat Methods 12:541–546CrossRefPubMedGoogle Scholar
  64. 64.
    de Boer P, Hoogenboom JP, Giepmans BN (2015) Correlated light and electron microscopy: ultrastructure lights up! Nat Methods 12(6):503–513CrossRefPubMedGoogle Scholar
  65. 65.
    Fritzsch B (2003) Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia. Brain Res Bull 60(5):423–433CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Fritzsch B, Pan N, Jahan I, Elliott KL (2015) Inner ear development: building a spiral ganglion and an organ of Corti out of unspecified ectoderm. Cell Tissue Res 361:7–24CrossRefPubMedGoogle Scholar
  67. 67.
    Maricich SM, Xia A, Mathes EL, Wang VY, Oghalai JS, Fritzsch B, Zoghbi HY (2009) Atoh1-lineal neurons are required for hearing and for the survival of neurons in the spiral ganglion and brainstem accessory auditory nuclei. J Neurosci 29(36):11123–11133CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biology, CLASUniversity of IowaIowa CityUSA
  2. 2.Division of OtolaryngologyUniversity of UtahSalt Lake CityUSA
  3. 3.Molecular Targeting Technologies, Inc.West ChesterUSA

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