Elsevier

Neuroscience

Volume 194, 27 October 2011, Pages 326-336
Neuroscience

Regeneration, Repair, and Developmental Neuroscience
Research Paper
Spatial and temporal expression patterns of nicotinic acetylcholine alpha 9 and alpha 10 subunits in the embryonic and early postnatal inner ear

https://doi.org/10.1016/j.neuroscience.2011.08.005Get rights and content

Abstract

The expression and function of nicotinic receptor subunits (nAChRs) in the inner ear before the onset of hearing is not well understood. We investigated the mRNA expression of the α9 and α10 nAChR subunits in sensory hair cells of the embryonic and postnatal rat inner ear. We mapped their spatial and temporal expression in cochlear and vestibular hair cells using qPCR, [35S] labeled cRNA in situ hybridization, and α-bungarotoxin (α-Bgt) to label the presumptive membrane-bound receptor on cochlear hair cells. The results suggest that (1) the mRNA expression of the α9 subunit precedes expression of the α10 subunit in both cochlear and vestibular hair cells, (2) the mRNA expression of both the α9 and α10 subunits occurs earlier in the vestibular system than in the cochlea, (3) the mRNA expression of both subunits is required for the assembled receptor complexes, and (4) the presumptive assembled receptor, at least in the cochlea, is associated with synapse formation and the onset of function.

Highlights

▶α9 and α10 nicotinic receptor subunits first appear in vestibular hair cells. ▶α9 and α10 subunits have higher expression at birth and at postnatal day 10. ▶Bungarotoxin labeling is found first on inner hair cells at postnatal day 0. ▶α9/α10 receptors are on hair cells after α10 expression and efferent innervation.

Section snippets

Animals and tissue preparation

Sprague–Dawley albino rats varying in age from embryonic day 16 (E16) to young adult (6 weeks) were anesthetized with near-lethal i.p. injections of sodium pentobarbital (Nembutal, 100 mg/kg) or by hypothermia (only animals less than one postnatal week). The day of birth (E21.5) represented postnatal day 0 (P0). We also bred transgenic mice overexpressing the α9 gene [Tg(Chrna9-EGFP)1Jnz] that were obtained from J. Zuo (Zuo et al., 1999). All procedures used for this study followed NIH

α9 and α10 mRNA expression in the inner ear using RT-PCR

How neuronal nicotinic synapses acquire nAChRs at cholinergic synapses in the inner ear is not well understood. A recent study suggests that a rapsyn-mediated signaling cascade similar to the NMJ may be involved in neuronal nicotinic synapse formation and demonstrated an early expression of rapsyn in the embryonic inner ear as well as punctate localization of rapsyn immunoreactivity in vestibular hair cells before cochlear hair cells (Osman et al., 2008). Our previous studies (Simmons and

Discussion

The present study further demonstrates the differential regulation of α9 and α10 subunits in the inner ear. Although hair cells in vestibular organs express α9 and α10 subunits before cochlear hair cells, cell surface receptors could only be detected on cochlear hair cells following the arrival of cholinergic terminals during the early postnatal period in rats. Importantly, this work supports the hypothesis that functional cell surface expression may require not only the expression of both

Conclusions

Knowledge of the expression and cell surface localization pattern of nAChRs in hair cells at different developmental ages is essential to understanding the role of the receptor subunits during efferent synaptogenesis. α9 and α10 nAChR subunits show differential expression patterns among inner and outer hair cells as well as between cochlear and vestibular hair cells. The data reported in this study are consistent with the following developmental sequence: first α9 subunits are expressed,

Acknowledgments

This publication was made possible by NIH grants DC004086 (D.D.S.), DC006907 (B.J.M.) and NIDCD P30 core center grants DC004665 and DC004662. This research was also supported by the Deafness Research Foundation, National Organization for Hearing Research, the American Hearing Research Foundation, and the State of Nebraska (B.J.M.). The authors thank Ms. Xiaona Huang, Mr. Steve Gum, Ms. Angela Schrader, Ms. Jennifer Spice, and Ms. Aubrey Hornak for their excellent technical assistance.

References (41)

  • A.A. Osman et al.

    Muscle-like nicotinic receptor accessory molecules in sensory hair cells of the inner ear

    Mol Cell Neurosci

    (2008)
  • A. Schrattenholz et al.

    Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit

    J Biol Chem

    (1998)
  • D.D. Simmons et al.

    Differential expression of the alpha9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear hair cells

    Mol Brain Res

    (1998)
  • D.D. Simmons et al.

    Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study

    Dev Brain Res

    (1996)
  • D.E. Vetter et al.

    Role of alpha9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation

    Neuron

    (1999)
  • J. Adler et al.

    Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient

    Cytometry A

    (2010)
  • A.L. Bergeron et al.

    The final stage of cholinergic differentiation occurs below inner hair cells during development of the rodent cochlea

    J Assoc Res Otolaryngol

    (2005)
  • L.L. Bruce et al.

    Postnatal development of efferent synapses in the rat cochlea

    The Journal of comparative neurology

    (2000)
  • C.C. Chang et al.

    Isolation of Neurotoxins from the Venom of Bungarus Multicinctus and Their Modes of Neuromuscular Blocking Action

    Arch Int Pharmacodyn Ther

    (1963)
  • C. Colomer et al.

    Functional characterization of alpha9-containing cholinergic nicotinic receptors in the rat adrenal medulla: implication in stress-induced functional plasticity

    J Neurosci

    (2010)
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