Summary
Separated cochlear outer hair cells and isolated strips of organ of Corti containing hair cells and supporting cells have been rapidly frozen before freeze-fracture and deep-etching by immersion of samples sandwiched between two copper plates into liquid nitrogen-cooled propane: isopentane. Assessment of this procedure has shown that no significant freezing damage occurs. The ultrastructure of the hair cells revealed by freeze-fracture of these non-chemically fixed preparations was generally very similar to that seen in fixed material. This indicates that the processing of cochlear tissue normally used for electron microscopy produces few obvious structural artefacts. It also demonstrated that procedures for isolating cochlear hair cells generally do not affect cell structure significantly. However, some isolated hair cells did show abnormalities within the membranes of the lateral cisternae. Such membrane alterations, which would not be identified by light microscopy, occurred to a variable extent but were more commonly present after prolonged periods in maintenance medium. Deep-etching of the preparations to examine extracellular features around stereocilia revealed clearly lateral cross-links between stereocilia. However, tip-links could not be positively identified in either unfixed or prefixed preparations.
Similar content being viewed by others
References
Arima, T., Uemara, T. &Yamamoto, T. (1986) Cytoskeletal organisation in the supporting cell of the guinea pig organ of Corti.Hearing Research 24, 169–75.
Arima, T., Uemara, T. &Yamamoto, T. (1987) Three-dimensional visualisations of the inner ear hair cell of the guinea pig. A rapid-freeze, deep-etch study of filamentous and membranous organelles.Hearing Research 25, 61–8.
Ashmore, J. (1987) A fast motile response in guinea pig outer hair cells: the cellular basis of the cochlear amplifier.Journal of Physiology 388, 323–47.
Billet, T. E., Thorne, P. R. &Gavin, J. B. (1989) The nature and progression of injury in the organ of Corti during ischaemia.Hearing Research 41, 189–98.
Brownell, W. E., Bader, C. R., Bertrand, D. &Ribeaupierre, Y. De (1984) Evoked mechanical responses of isolated cochlear outer hair cells.Science 227, 194–6.
Costello, M. J., Fetter, R. &Hochli, M. (1982) Simple procedures for evaluating the cryofixation of biological samples.Journal of Microscopy 125, 125–36.
Costello, M. J., Fetter, R. &Corless, J. M. (1984) Optimum conditions for the plunge freezing of sandwiched samples. InScience of Biological Specimen Preparation (edited byRevel, J.-P., Barnard, T. &Haggis, G. H.) pp. 105–15. Chicago: SEM Inc.
Davies, S. &Forge, A. (1987) Preparation of the mammalian organ of Corti for scanning electron microscopy.Journal of Microscopy 147, 89–101.
De Leeuw, F. E., Chen, H.-C., Colenbrander, B. &Verkleij, A. J. (1990) Cold-induced ultrastructural changes in bull and boar sperm plasma membranes.Cryobiology 27, 171–83.
Dulon, D., Aran, J.-M. &Schacht, J. (1987) Osmotically induced motility of outer hair cells: implications for Meniere's disease.Archives of Oto-Rhino-Laryngology 244, 104–7.
Dulon, D., Zajic, G. &Schacht, J. (1988) K+ depolarisation induces motility in outer hair cells by an osmotic mechanism.Hearing Research 32, 123–30.
Dulon, D., Zajic, G. &Schacht, J. (1990) Increasing intracellular free calcium induces circumferential contractions in isolated cochlear outer hair cells.Journal of Neuroscience 10, 1388–97.
Evans, B. N. (1990) Fatal contractions: ultrastructural and electromechanical changes in outer hair cells following transmembraneous electrical stimulation.Hearing Research 45, 265–82.
Fetter, R. &Costello, M. J. (1986) A procedure for obtaining complementary replicas of ultra-rapidly frozen sandwiched samples.Journal of Microscopy 141, 277–90.
Flock, A. &Cheung, H. C. (1977) Actin filaments in sensory hairs of inner ear receptor cells.Journal of Cell Biology 75, 339–43.
Flock, A., Flock, B. &Ulfendahl, M. (1986) Mechanisms of movement in outer hair cells and a possible structural basis.Archives of Oto-Rhino-Laryngology 243, 83–96.
Forge, A. (1986) The morphology of the normal and pathological cell membrane and junctional complexes of the cochlea. InBasic and Applied Aspects of Noise-Induced Hearing Loss (edited bySalvi, R. J., Henderson, D., Hamernik, R. J. &Colletti, V.) pp. 55–68. New York: Plenum Publishing Corporation.
Forge, A. (1987) Specialisations of the lateral membrane of inner hair cells.Hearing Research 31, 99–110.
Forge, A. (1989) The lateral walls of inner and outer hair cells. InCochlear Mechanisms (edited byWilson, J. P. &Kemp, D. T.) pp. 29–35. New York: Plenum Publishing Corporation.
Forge, A., Davies, S. &Zajic, G. (1988) Characteristics of the membrane of the stereocilia and cell apex in cochlear hair cells.Journal of Neurocytology 17, 325–34.
Furness, D. N. &Hackney, C. M. (1985) Cross-links between stereocilia in the guinea pig cochlea.Hearing Research 18, 177–88.
Furness, D. N., Richardson, G. P. &Russell, I. J. (1989) Stereociliary bundle morphology in organotypic cultures of the mouse cochlea.Hearing Research 38, 95–110.
Gilkey, J. C. &Staehelin, L. A. (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure.Journal of Electron Microscopy Technique 3, 177–210.
Haggis, G. H. (1985) Towards more precise definition of conditions for satisfactory deep etching.Journal of Microscopy 139, 49–55.
Heuser, J. E. &Salpeter, S. R. (1979) Organisation of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicatedTorpedo postsynaptic membrane.Journal of Cell Biology 82, 150–73.
Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y., Jan, L. &Evans, L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.Journal of Cell Biology 81, 275–300.
Hirokawa, N. &Tilney, L. G. (1982) Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear.Journal of Cell Biology 95, 249–61.
Holley, M. C. (1988) Purification of mammalian cochlear hair cells using small volume Percoll density gradients.Journal of Neuroscience Methods 27, 219–24.
Hudspeth, A. J. (1989) How the ear's works work.Nature 341, 397–404.
Kemp, D. T. (1978) Stimulated acoustic emission from within the human auditory system.Journal of the Acoustical Society of America 64, 1386–91.
McDowell, B., Davies, S. &Forge, A. (1989) The effect of gentamicin-induced hair cell loss on the tight junctions of the reticular lamina.Hearing Research 40, 221–32.
Menco, B.Ph.M. (1986) A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fracturing, freeze-etching, and freeze-substitution.Journal of Electron Microscopy Technique 4, 177–240.
Miller, K. R., Prescott, C. S., Jacobs, T. L. &Lassignal, N. L. (1983) Artefacts associated with quick-freezing and freeze-drying.Journal of Ultrastructure Research 82, 123–33.
Newman, T. M. &Duce, I. R. (1986) Preparation-dependent distribution of intramembrane particles in freeze-fracture replicas of the neuromuscular junction of the locustSchistocerca gregaria.Cell & Tissue Research 243, 323–7.
Osborne, M. P. &Comis, S. D. (1990) High resolution scanning electron microscopy of stereocilia in the cochlea of normal, postmortem, and drug-treated guinea pigs.Journal of Electron Microscopy Technique 15, 254–60.
Phillips, T. E. &Boyne, A. F. (1984) Liquid nitrogen-based quick freezing: experiences with bounce-free delivery of cholinergic nerve terminals to a metal surface.Journal of Electron Microscopy Technique 1, 9–29.
Pickles, J. O., Comis, S. D. &Osborne, M. P. (1984) Cross-links between stereocilia in the guinea pig organ of Corti, and their possible relation to sensory transduction.Hearing Research 15, 103–12.
Pinto Da Silva, P. (1972) Translational mobility of the membrane intercalated particles of human erythrocyte ghosts, pH dependent, reversible aggregation.Journal of Cell Biology 53, 777–87.
Russell, I. J., Richardson, G. P. &Cody, A. R. (1986) Mechanosensitivity of mammalian auditory hair cellsin vitro.Nature 321, 517–19.
Santi, P. A. &Anderson, C. B. (1987) A newly identified cell coat on cochlear hair cells.Hearing Research 27, 47–66.
Severs, N. &Robenek, H. (1983) Detection of microdomains in biomembranes. An appraisal of recent developments in freeze-fracture cytochemistry.Biochemica et Biophysica Acta 737, 373–408.
Shirane, M. &Harrison, R. V. (1987) The effects of deferoxamine mesylate and hypoxia on the cochlea,Acta Otolarygologica 104, 99–107.
Zajic, G. &Schacht, J. (1987) Comparison of isolated outer hair cells from five mammalian species.Hearing Research 26, 249–56.
Zenner, H. P., Zimmermann, U. &Schmitt, U. (1985) Reversible contraction of isolated cochlear hair cells.Hearing Research 18, 127–33.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Forge, A., Davies, S. & Zajic, G. Assessment of ultrastructure in isolated cochlear hair cells using a procedure for rapid freezing before freeze-fracture and deep-etching. J Neurocytol 20, 471–484 (1991). https://doi.org/10.1007/BF01252275
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01252275