Skip to main content
PMC home page
JARO: Journal of the Association for Research in Otolaryngology logoLink to JARO: Journal of the Association for Research in Otolaryngology
. 2001 Aug 1;3(1):1–15. doi: 10.1007/s101620010071

Topological and Developmental Gradients of Calbindin Expression in the Chick's Inner Ear

Hakim Hiel 1,✉, Dasakumar S Navaratnam 2, John C Oberholtzer 2, Paul A Fuchs 1
PMCID: PMC3202366  PMID: 12083720

Abstract

Mobile intracellular calcium buffers play an important role in regulating calcium flux into mechanosensory hair cells and calbindin D-28k is expressed at high levels in the chick's basilar papilla. We have used RT-PCR, in situ hybridization, and immunohistology to demonstrate that calbindin expression varies systematically according to hair cell position and developmental age. RT-PCR using microdissected quarters of the posthatch basilar papilla showed that mRNA levels were lowest in the (low frequency) apex and higher in basal quadrants. In situ hybridization revealed calbindin mRNA in posthatch hair cells and supporting cells, with more intense labeling of hair cells from basal (high frequency) positions. A similar topology was obtained with calbindin antibodies. Neither calbindin riboprobe nor calbindin antibody labeled cochlear neurons. In contrast, a subset of large vestibular neurons and their calyciform endings onto Type I vestibular hair cells were strongly labeled by the calbindin antibody, while vestibular hair cells were negative for calbindin immunoreactivity. Likewise, calbindin in situ hybridization was negative for vestibular hair cells but positive in a subset of larger vestibular neurons. Calbindin mRNA was detected in hair cells of the basal half of the papilla at embryonic day 10 (E10) and calbindin immunoreactivity was detected at E12. Hair cells in the apical half of the papilla had equivalent calbindin expression two days later. Immunoreactivity appeared in abneural supporting cells days later than in hair cells, and not until E20 in neurally located supporting cells. These results demonstrate that calbindin message and protein levels are greater in high-frequency hair cells. This "tonotopic" gradient may result from the stabilization of a basal-to-apical developmental gradient and could be related at least in part to calcium channel expression along this axis.

Keywords: cochlea, hair cells, vestibule, mechanotransduction, embryonic, calcium buffering

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Acknowledgements

This work was supported by NIDCD DC02755 to J. Oberholtzer and DC01508 to P. Fuchs.

References

  • 1.Arnold DB, Heintz N. A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells. Proc. Natl. Acad. Sci. USA. 1997;94:8842–8847. doi: 10.1073/pnas.94.16.8842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Art JJ, Fettiplace R. Variation of membrane properties in hair cells isolated from the turtle's cochlea. J. Physiol. 1987;385:207–242. doi: 10.1113/jphysiol.1987.sp016492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Baird RA, Desmadryl G, Fernandez C, Goldberg JM. The vestibular nerve of the chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals. J. Neurophysiol. 1988b;60:182–203. doi: 10.1152/jn.1988.60.1.182. [DOI] [PubMed] [Google Scholar]
  • 4.Baurle J, Vogten H, Grusser–Cornehls U. Course and targets of the calbindin D-28k subpopulation of primary vestibular afferents. J. Comp. Neurol. 1998;402:111–128. doi: 10.1002/(SICI)1096-9861(19981207)402:1<111::AID-CNE8>3.3.CO;2-T. [DOI] [PubMed] [Google Scholar]
  • 5.Chen L, Salvi R, Shero M. Cochlear frequency-place map in adult chickens: intracellular biocytin labeling. Hear. Res. 1994;81:130–136. doi: 10.1016/0378-5955(94)90160-0. [DOI] [PubMed] [Google Scholar]
  • 6.Dechesne CJ, Lavigne–Rebillard M, Brehier Thomasset M, Sans A. Appearance and distribution of neuron-specific enolase and calbindin (CaBP 28 kDa) in the developing human inner ear. Brain Res. 1988a;469:221–230. doi: 10.1016/0165-3806(88)90184-8. [DOI] [PubMed] [Google Scholar]
  • 7.Dechesne CJ, Thomasset M, Brehier A, Sans A. Calbindin (CaBP 28 kDa) localization in the peripheral vestibular system of various vertebrates. Hear. Res. 1988b;33:273–278. doi: 10.1016/0378-5955(88)90157-8. [DOI] [PubMed] [Google Scholar]
  • 8.Edmonds B, Reyes R, Schwaller B, Roberts WM. Calretinin modifies presynaptic calcium signaling in frog saccular hair cells. Nat. Neurosci. 2000;3:786–790. doi: 10.1038/77687. [DOI] [PubMed] [Google Scholar]
  • 9.Fermin CD, Cohen GM. Developmental gradients in the embryonic chick's basilar papilla. Acta Otolaryngol. (Stockh.) 1984;97:39–51. doi: 10.3109/00016488409130963. [DOI] [PubMed] [Google Scholar]
  • 10.Ferrari S, Battini R, Drusiani E. Tissue-specific regulation of the concentration of calbindin D28k mRNA in the developing chicken. Life Sci. 1989;45:1247–1253. doi: 10.1016/0024-3205(89)90126-4. [DOI] [PubMed] [Google Scholar]
  • 11.Firbas W, Muller G. The efferent innervation of the avian cochlea. Hear. Res. 1983;10:109–116. doi: 10.1016/0378-5955(83)90021-7. [DOI] [PubMed] [Google Scholar]
  • 12.Fischer FP. Quantitative analysis of the innervation pattern of the chicken basilar papilla. Hear. Res. 1992;61:167–178. doi: 10.1016/0378-5955(92)90048-R. [DOI] [PubMed] [Google Scholar]
  • 13.Fischer FP, Eisensamer B, Manley GA. Cochlear and lagenar ganglia of the chicken. J. Morphol. 1994;220:71–83. doi: 10.1002/jmor.1052200107. [DOI] [PubMed] [Google Scholar]
  • 14.Fuchs PA, Evans MG. Potassium currents in hair cells isolated from the cochlea of the chick. J. Physiol. 1990;429:529–551. doi: 10.1113/jphysiol.1990.sp018271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Fuchs PA, Murrow BW. Cholinergic inhibition of short (outer) hair cells of the chick's cochlea. J. Neurosci. 1992;12:800–809. doi: 10.1523/JNEUROSCI.12-03-00800.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Fuchs PA, Nagai T, Evans MG. Electrical tuning in hair cells isolated from the cochlea of the chick. J. Neurosci. 1988;8:2460–2467. doi: 10.1523/JNEUROSCI.08-07-02460.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Fuchs PA, Sokolowski BHAS. The acquisition during development of Ca-activated potassium current by cochlear hair cells of the chick. Proc. R. Soc. Lond. B. Biol. Sci. 1990;241:122–126. doi: 10.1098/rspb.1990.0075. [DOI] [PubMed] [Google Scholar]
  • 18.Gray L, Rubel EW. The development of absolute thresholds in chickens. J. Acoust. Soc. Am. 1985;77:1162–1172. doi: 10.1121/1.392180. [DOI] [PubMed] [Google Scholar]
  • 19.Hackney CM, Fettiplace R, Furness DN. The functional morphology of stereociliary bundles on turtle cochlear hair cells. Hear. Res. 1993;69:163–175. doi: 10.1016/0378-5955(93)90104-9. [DOI] [PubMed] [Google Scholar]
  • 20.Hall AK, Norman AW. Vitamin D-independent expression of chick brain calbindin-D28k. Brain Res. Mol. Brain Res. 1991;9:9–14. doi: 10.1016/0169-328X(91)90124-G. [DOI] [PubMed] [Google Scholar]
  • 21.Hirokawa N. Synaptogenesis in the basilar papilla of the chick. J. Neurocytol. 1978a;7:283–300. doi: 10.1007/BF01176994. [DOI] [PubMed] [Google Scholar]
  • 22.Hirokawa N. The ultrastructure of the basilar papilla of the chick. J. Comp. Neurol. 1978b;181:361–374. doi: 10.1002/cne.901810208. [DOI] [PubMed] [Google Scholar]
  • 23.Hudspeth AJ. Extracellular current flow and the site of transduction by vertebrate hair cells. J. Neurosci. 1982;2:1–10. doi: 10.1523/JNEUROSCI.02-01-00001.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hunziker W. The 28-kDa vitamin D-dependent calcium-binding protein has a six-domain structure. Proc. Natl. Acad. Sci. USA. 1986;83:7578–7582. doi: 10.1073/pnas.83.20.7578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Issa NP, Hudspeth AJ. Clustering of Ca2+ and Ca2+-activated K+ channels at fluorescently labeled presynaptic active zones of hair cells. Proc. Natl. Acad. Sci. USA. 1994;91:7578–7582. doi: 10.1073/pnas.91.16.7578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Katayama A, Corwin JT. Cell production in the chicken cochlea. J. Comp. Neurol. 1989;28:129–135. doi: 10.1002/cne.902810110. [DOI] [PubMed] [Google Scholar]
  • 27.Kelley MW, Xu XM, Wagner MA, Warchol ME, Corwin JT. The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture. Development. 1993;119:1041–1053. doi: 10.1242/dev.119.4.1041. [DOI] [PubMed] [Google Scholar]
  • 28.King MW, Norman AW. Analysis of the mRNA coding for the chick vitamin D-induced calbindin and its regulation by 1,25-dihydroxyvitamin D3. Arch. Biochem. Biophys. 1986;248:612–619. doi: 10.1016/0003-9861(86)90515-1. [DOI] [PubMed] [Google Scholar]
  • 29.Martin AR, Fuchs PA. The dependence of calcium-activated potassium currents on membrane potential. Proc. R. Soc. Lond. B Biol. Sci. 1992;250:71–76. doi: 10.1098/rspb.1992.0132. [DOI] [PubMed] [Google Scholar]
  • 30.Martinez-Dunst CM, Michaels RL, Fuchs PA. Release sites and calcium channels in hair cells of the chick's cochlea. J. Neurosci. 1997;17:9133–9144. doi: 10.1523/JNEUROSCI.17-23-09133.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.McNiven AI, Yuhas WA, Fuchs PA. The ionic dependence and agonist preference of an acetylcholine receptor in hair cells. Aud. Neurosci. 1996;2:63–77. [Google Scholar]
  • 32.Murrow BW. Position-dependent expression of potassium currents by chick cochlear hair cells. J. Physiol. 1994;480:247–259. doi: 10.1113/jphysiol.1994.sp020357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Navaratnam DS, Escobar L, Covarrubias M, Oberholtzer JC. Permeation properties and differential expression across the auditory receptor epithelium of an inward rectifier K+ channel cloned from the chick inner ear. J. Biol. Chem. 1995;18:19238–19245. doi: 10.1074/jbc.270.33.19238. [DOI] [PubMed] [Google Scholar]
  • 34.Oberholtzer JC, Buettger C, Summers MC, Matschinsky FM. The 28-kDa calbindin-D is a major calcium-binding protein in the basilar papilla of the chick. Proc. Natl. Acad. Sci. USA. 1988;85:3387–3390. doi: 10.1073/pnas.85.10.3387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Oberholtzer JC, Cohen EL, Davis JG. Molecular cloning of a chick cochlea cDNA encoding a subunit of DNA replication factor C/activator 1. DNA Cell Biol. 1994;8:857–863. doi: 10.1089/dna.1994.13.857. [DOI] [PubMed] [Google Scholar]
  • 36.Oberholtzer JC, Schneider ME, Summers MC, Saunders JC, Matschinsky FM. The developmental appearance of a major basilar papilla-specific protein in the chick. Hear. Res. 1986;23:161–168. doi: 10.1016/0378-5955(86)90013-4. [DOI] [PubMed] [Google Scholar]
  • 37.Oesterle EC, Cunningham DE, Rubel EW. Ultrastructure of hyaline, border and vacuole cells in chick inner ear. J. Comp. Neurol. 1992;318:64–82. doi: 10.1002/cne.903180105. [DOI] [PubMed] [Google Scholar]
  • 38.Ofsie MS, Cotanche DA. Distribution of nerve fibers in the basilar papilla of normal and sound-damaged chick cochleae. J. Comp. Neurol. 1996;370:281–294. doi: 10.1002/(SICI)1096-9861(19960701)370:3<281::AID-CNE1>3.3.CO;2-9. [DOI] [PubMed] [Google Scholar]
  • 39.Pack AK, Slepecky NB. Cytoskeletal and calcium-binding proteins in the mammalian organ of Corti: cell type-specific proteins displaying longitudinal and radial gradients. Hear. Res. 1995;91:119–135. doi: 10.1016/0378-5955(95)00173-5. [DOI] [PubMed] [Google Scholar]
  • 40.Papakostas K, Hackney CM, Furness DN. The distribution of the calcium buffer calbindin in the cochlea of the guinea-pig. Clin. Otolaryngol. 2000;25:570–576. doi: 10.1046/j.1365-2273.2000.00422-9.x. [DOI] [PubMed] [Google Scholar]
  • 41.Rabie A, Thomasset M, Legrand Ch. Immunocytochemical detection of calcium-binding protein in the cochlear and vestibular hair cells of the rat. Cell Tissue Res. 1983;232:691–696. doi: 10.1007/BF00216440. [DOI] [PubMed] [Google Scholar]
  • 42.Rebillard M, Pujol R. Innervation of the chicken basilar papilla during its development. Acta Otolaryngol. 1983;96:379–388. doi: 10.3109/00016488309132723. [DOI] [PubMed] [Google Scholar]
  • 43.Retzius G. Das Gehororgan der Wilbeltiere. II. Das Gehororgan der Reptilien, der Vogel und Saugetiere. Stockholm: Samson and Wallin; 1884. [Google Scholar]
  • 44.Ricci AJ, Fettiplace R. The effects of calcium buffering and cyclic AMP on mechano-electrical transduction in turtle auditory hair cells. J. Physiol. 1997;501:111–124. doi: 10.1111/j.1469-7793.1997.111bo.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Roberts WM, Jacobs R, Hudspeth A. Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J. Neurosci. 1990;10:3664–3684. doi: 10.1523/JNEUROSCI.10-11-03664.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Sans A, Brehier A, Moniot B, Thomasset M. Immuno-electronmicroscopic localization of 'vitamin D-dependent' calcium-binding protein (CaBP-28k) in the vestibular hair cells of the cat. Brain Res. 1987;435:293–304. doi: 10.1016/0006-8993(87)91612-X. [DOI] [PubMed] [Google Scholar]
  • 47.Sans A, Etchecopar B, Brehier A, Thomasset M. Immunocytochemical detection of vitamin D-dependent calcium-binding protein (CaBP-28k) in vestibular sensory hair cells and vestibular ganglion neurons of the cat. Brain Res. 1986;364:190–194. doi: 10.1016/0006-8993(86)91003-6. [DOI] [PubMed] [Google Scholar]
  • 48.Senarita M, Thalmann I, Shibasaki O, Thalmann R. Calcium-binding proteins in the organ of Corti and basilar papilla: CBP-15, an unidentified calcium binding protein of the inner ear. Hear Res. 1995;90:169–175. doi: 10.1016/0378-5955(95)00161-4. [DOI] [PubMed] [Google Scholar]
  • 49.Sokolowski BHA, Csus J, Hafez OI, Haggerty HS. Neurotrophic factors modulate hair cells and their potassium currents in chick otocyst explants. Eur. J. Neurosci. 1999;11:682–690. doi: 10.1046/j.1460-9568.1999.00469.x. [DOI] [PubMed] [Google Scholar]
  • 50.Tanaka K, Smith CA. Structure of the chicken's inner ear: SEM and TEM study. Am. J. Anat. 1978;153:251–272. doi: 10.1002/aja.1001530206. [DOI] [PubMed] [Google Scholar]
  • 51.Tilney LG, Saunders JC. Actin filaments, stereocilia and hair cells of the bird cochlea I. Length, number, width and distribution of stereocilia of each hair cell are related to position of the hair cell on the cochlea. J. Cell Biol. 1983;96:807–821. doi: 10.1083/jcb.96.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Tilney LG, Tilney MS. Functional organization of the cytoskeleton. Hear. Res. 1986;22:55–77. doi: 10.1016/0378-5955(86)90077-8. [DOI] [PubMed] [Google Scholar]
  • 53.Tilney LG, Tilney MS, Saunders JS, DeRosier DJ. Actin filaments, stereocilia, and hair cells of the bird cochlea III. The development and differentiation of hair cells and stereocilia. Dev. Biol. 1986;116:100–118. doi: 10.1016/0012-1606(86)90047-3. [DOI] [PubMed] [Google Scholar]
  • 54.Tucker T, Fettiplace R. Confocal imaging of calcium microdomains and calcium extrusion in turtle hair cells. Neuron. 1995;15:1323–1335. doi: 10.1016/0896-6273(95)90011-x. [DOI] [PubMed] [Google Scholar]
  • 55.Tucker T, Fettiplace R. Monitoring calcium in turtle hair cells with a calcium-activated potassium channel. J. Physiol. 1996;494:613–626. doi: 10.1113/jphysiol.1996.sp021519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Wang YZ, Christakos S. Retinoic acid regulates the expression of the calcium binding protein, calbindin-D28k. Mol. Endocrinol. 1995;11:1510–1521. doi: 10.1210/mend.9.11.8584029. [DOI] [PubMed] [Google Scholar]
  • 57.Whitehead MC, Morest DK. The development of innervation patterns in the avian cochlea. Neuroscience. 1985a;14:255–276. doi: 10.1016/0306-4522(85)90177-0. [DOI] [PubMed] [Google Scholar]
  • 58.Whitehead MC, Morest DK. The growth of cochlear fibers and the formation of their synaptic endings in the avian inner ear: a study with the electron microscope. Neuroscience. 1985b;14:277–300. doi: 10.1016/0306-4522(85)90178-2. [DOI] [PubMed] [Google Scholar]
  • 59.Wu YC, Fettiplace R. A developmental model for generating frequency maps in the reptilian and avian cochleas. Biophys. J. 1996;70:2557–2570. doi: 10.1016/S0006-3495(96)79827-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Wu YC, Tucker T, Fettiplace R. A theoretical study of calcium microdomains in turtle hair cells. Biophys. J. 1996;71:2256–2275. doi: 10.1016/S0006-3495(96)79429-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Zidanic M, Fuchs PA. Synapsin-like immunoreactivity in the chick cochlea: specific labeling of efferent nerve terminals. Aud. Neurosci. 1996;2:347–362. [Google Scholar]

Articles from JARO: Journal of the Association for Research in Otolaryngology are provided here courtesy of Association for Research in Otolaryngology

RESOURCES