Cytoplasm Of Outer Hair Cells Research Articles

The experimental study on endoplasmic reticulum stress-participated outer hair cell apoptosis in cadherin 23 gene mutant mice

Objective: To test the mechanism and upstream pathway of outer hair cell apoptosis in Cadherin 23 (Cdh23) gene mutant mice. Method: The mutant Cdh23(erl/erl)(erl) mice were collected as the study group, while the C57BL/6J (B6) mice were chosen as the control group. A total of 70 mice per group were used in this study. The study group and control group underwent auditory-evoked brainstem response (ABR) tests at the same age. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect outer hair cell(OHC) apoptosis. The qRT-PCR was conducted to test the expression of ER stress markers immunoglobulin-binding protein (BiP) and C/EBP homologous protein (CHOP) mRNA. The expression and location of BiP and CHOP protein in OHC were detected by immunostaining. The expression of BiP protein in cochleae was identified by Western blot. The expression and location of CDH23 protein in OHC were discovered by immunostaining. Results: The ABR thresholds in erl mice were significantly higher than those in B6 mice at the age of 1 and 3 months (both P<0.05). The surface preparation with TUNEL staining confirmed OHC apoptosis in erl mouse cochleae which showed a higher TUNEL positive cell ratio than B6 mouse(t=11.291, P<0.01). The ER stress marker Bip and Chop mRNA were upregulated in the erl mouse inner ear, when compared with those in the B6 mouse(both P<0.05). The BiP protein extracted from the erl mouse cochleae was significantly higher than that of B6 mouse measured by Western blot (t=3.66, P=0.02). Immunostaining showed that BiP and CHOP were highly detected in the OHC in erl mouse cochleae, and was mainly detected in the perinuclear region of OHC. However, a bare BiP and CHOP signal were shown in B6 mouse cochleae. The CDH23 protein was specifically localized at the top of the OHC in B6 mice, indicating the localization of the tip links in hair bundle stereocilia. On the contrary, the CDH23(erl) protein was found to be localized from the top to the nuclei of the OHC in erl mice. Portions of the CDH23(erl) proteins failed to reach the top of the hair bundles and remained in the OHC cytoplasm. Conclusion: As the downstream response of the Cdh23 gene mutation, portions of the mutant CDH23(erl) protein was accumulated in ER lumen resulting in the increase of ER loading and ultimately triggered ER stress and hair cell apoptosis in erl mouse cochleae.

Localization of prestin and expression in the early period after radiation in mice

This study aimed to examine expression and microstructural distribution of prestin in outer hair cells, and the effect of dose and time of radiation on prestin expression in the BALB/c mouse. We also investigated the molecular biological characteristics of prestin and possible mechanisms of sensorineural hearing loss caused by radiation. Seventy 4-week-old mice were randomly divided into four groups, including one control group and three experimental groups. Each experimental group was randomly divided into two groups, which were killed to collect specimens of the cochlea on the 3rd and 7th days after exposure to different doses of 8, 12, and 16 Gy radiation. These cochleas were embedded in paraffin, and then cut into sections. The sections were immunostained with anti-prestin antibodies. The distribution of prestin was observed under optical microscopy and the density of prestin-positive expression was quantitatively calculated by Image-Pro Plus. Prestin had high expression in the lateral membrane and low expression in the cytoplasm of outer hair cells above the nucleus. The density of prestin protein expression of the basal turn was not significantly different after exposure to the different doses of radiation compared with the control group, but up-regulation occurred after radiation in the apex turn. We conclude that prestin protein is mainly expressed in the lateral membrane above the nucleus. Prestin protein may be responsible for the mechanism of injury to the inner ear caused by radiation.

Plasmalemmal ATPase calcium pump localizes to inner and outer hair bundles

Recent studies demonstrate calcium ion influx at the tips of hair cell stereocilia during mechano-transduction. These ions must be either pumped from the cytosol into the extracellular space or endoplasmic envelope, or else sequestered by binding to specific proteins. A plasma membrane calcium pump (ATPase-type) was analysed in whole-mounts of rat organ of Corti using a monoclonal antibody to a large cytoplasmic loop of this protein. The reactivity was particularly high on the tips of longer stereocilia and was found along the shafts. Inner hair cell stereocilia had much less reactivity than outer hair cells. The reactivity lined the plasma membrane of inner hair cell bodies while a higher reactivity appeared in the cytoplasm of outer hair cells. Supporting cells were unreactive. Ultrastructural examination confirmed the plasma membrane calcium pump location on stereocilia and along the endolymph surface of receptor cells. Reaction product lined the plasma membrane of stereocilia as intense puncta. More reactive puncta occurred near the distal ends of stereocilia and the number decreased toward the ciliary base. The endolymph plasma membrane over the cuticular notch was especially reactive. The finding of more intense pump reactivity at the tips of stereocilia than the base is consistent with the hypothesis that during transduction, calcium ions enter stereocilia, distally, and the ATPase plasma membrane calcium pump rapidly extrudes these ions to the extracellular space.

Immunoelectron-microscopic localization of synaptophysin in the organ of Corti of the guinea pig.

Synaptophysin has been localized previously in the mammalian cochlea at the light-microscopic level and in few reports by electron microscopy using either a preincubation procedure or the avidin-biotin reaction. Here we present results of the electron-microscopic analysis for postembedding immunoreactivity of synaptophysin in the cochlea of the guinea pig of LR-White-embedded samples. Strong synaptophysin immunoreactivity is located in the cytoplasm of the efferent nerve endings at the base of inner and outer hair cells. Besides this, some antibodies to synaptophysin were also identified in the cytoplasm of outer hair cells. To get more information about the cellular content of synaptophysin, the density of the immunoreaction per square micrometer was determined for the outer hair cells of the second turn of the cochlea counting the gold particles in numerous ultrathin sections. The role of synaptophysin identified in the cytoplasm of outer hair cells is discussed.

Localization of nitric oxide synthase I and III in the cochlea.

Nitric oxide synthase (NOS) isoforms I and III were localized in the guinea pig cochlea by indirect immunohistochemistry using frozen sections and paraffin sections. NOS I staining was observed in the cytoplasm of outer hair cells, in nerve cell somata and fibers of the spiral ganglion, and in axonal profiles of the spiral lamina next to the base of inner hair cells. In addition, lining cells of the inner sulcus and limbus, and cells of the spiral ligament stained for NOS I but vascular walls remained unstained. NOS III reactivity was seen in the cytoplasm of outer and inner hair cell, in lining cells of the limbus, and on the endolymphatic surface of marginal cells. Staining for NOS III of spiral ganglion perikarya showed varying intensity. Endothelial cells of cochlear glomeruli reacted for NOS III. NOS III in vascular endothelial cells implies regulatory effects of nitric oxide (NO) on vascular wall tonus and cochlear blood supply. NOS I in cochlear neurons indicates these cells as possible sources for NO during neuronal activity. Activated neurons may provide NO that adjusts cochlear perfusion to neuronal activity. Finally, NO that is liberated from hair cells or afferent synaptic terminals may act as an inhibitor on N-methyl-D-aspartate (NMDA) receptors (negative feed-back inhibition).

Microtubule-associated proteins in adult human sensory organs.

The distribution of microtubule-associated proteins MAP-1 and MAP-2 was analysed with immunomorphological techniques in the serially sectioned adult human membranous labyrinth. In the organ of Corti, monoclonal antibodies to MAP-1 did not stain. Positivity for MAP-2 occurred in the entire outer hair cell cytoplasm, in the inner hair cells (?), in the nerve fibres and in the cytoplasm of epithelial cells of the spiral prominence. In addition, staining for MAP-2 was identified in many (but not all) cells or Reissner's membrane. Immunofluorescence for MAP-1 occurred in the supporting cells of the cristae and maculae interpreted to be localized in the apical region adjacent to the sensory cells. Thus, the distribution of MAP-1 and MAP-2 in the adult human membranous labyrinth was the same as described for several animal species with regard to the cochlea. In contrast to such a pattern, both MAP-1 and MAP-2 were identified in the human vestibular organs, thus identifying a subpopulation of centrally located nerve calyces and possibly also the apical portion of vestibular hair cells.

Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti

Calcium is thought to play a major signaling role in outer hair cells to control metabolism, cytoskeletal integrity, cell shape and cell excitability. For this to happen, in resting cells the concentration of free calcium ions must be maintained at low levels so that focal increases can trigger specific events. In this paper, the localization of calcium, calcium-binding and calcium-dependent regulatory proteins in sensory cells from the guinea pig inner ear was demonstrated using immunocytochemical and histochemical techniques. We found the calcium buffer and/or calcium sensor proteins calmodulin, calbindin and calsequestrin predominantly in sensory cells and that when present, these proteins can be enriched in the outer hair cells. Calmodulin is found in the stereocilia, in the cuticular plate and in the cytoplasm and calbindin is found only in the cuticular plate and cytoplasm of both the inner and outer hair cells. The staining for these proteins in the outer hair cells is homogeneous, with no apparent compartmentalization along the lateral wall. Calsequestrin, thought to store and release calcium from membrane bound intracellular storage sites is found only in the cytoplasm of outer hair cells. There, it has a more punctuate staining pattern than does calmodulin or calbindin suggesting that it may be present in calciosomes rather than soluble in the cytoplasm. We did not detect caldesmon and S-100. Using the potassium pyroantimonate technique, we found precipitates containing calcium ions distributed throughout the cytoplasm of outer hair cells, with no evidence that the subsurface cisterns along the lateral wall act as calcium storage sites. Thus, calcium in resting cells is found in the cytoplasm along with calbindin and calmodulin and appears to have a punctate distribution consistent with a co-localization with calsequestrin. The implications of this distribution with respect to the slow shortening and elongation seen in outer hair cells are discussed.

Morphological Changes of Cochlea in a Strain of New-mutant Mice

The hearing ability and histological characteristics of the cochlea of a strain of new-mutant mice were analyzed. This new mutant arose as a spontaneous mutation in the C3H/He stock. The genetic mode is autosomal recessive and the animals show abnormal behavior such as circling, head-tossing and hyperactivity. The audiological findings exhibited no recordable auditory brain stem response (ABR) in any homozygotes at ages ranging from 11 days to 117 days. For morphological examination, we used 36 homozygote with ages ranging from 10 days to 18 months. The primary morphological abnormalities were observed in the organ of Corti. The stereocilia of the outer hair cells showed disarray throughout the whole cochlea, although outer hair cell cytoplasm became fully developed, including the nerve terminals. Age-dependent degeneration of the outer hair cells subsequently occurred from the basal to the apical part of the cochlea. The earliest change demonstrated in the outer hair cells was cuticular degeneration. Although the abnormalities of the inner hair cells occurred late, a complete loss of inner and outer hair cells was demonstrated. The stria vascularis was well preserved at a later age as were spiral ganglion cells. These histological findings confirm that this mouse is classified as a neuroepithelial-type mutant. As this animal was expected to have a single gene abnormality, molecular genetic studies on this animal can provide important information on the nature of histological changes of the hair cell from a mode of gene action.

Actin in cochlear outer hair cells: A structural basis for cell shape and motility

Threshold sensitivity and frequency selectivity in the cochlea depend on intact outer hair cells and models to explain these phenomena suggest that active mechanical processes, residing in outer hair cells, alter micromechanical properties of the basilar membrane. Auditory sensory hair cells isolated from the inner ear change their shape in response to stimulation in vitro. Three areas of the cell have been suggested to be involved in this form of cell motility: (1) the infracuticular network of actin filaments that is seen as a central core; (2) the cortical layer of actin filaments along the lateral wall; and (3) actin in the outer hair cell cytoplasm. The actin filaments in the stereocilia, the cuticular plate, and the infracuticular network are not involved, since mechanically disrupted cells lacking these structures still shorten. Actin filaments along the lateral wall are not involved in shortening since outer hair cell membrane ghosts do not shorten, however, they retain the ability to constrict so...

Effects of caffeine and tetracaine on outer hair cell shortening suggest intracellular calcium involvement

Outer hair cell (OHC) shortening has previously been induced in vitro by the application of solutions containing high potassium (a depolarizing agent), acetylcholine (a suggested efferent transmitter) and cationized ferritin (a positively charged macromolecule), as well as by electrical current. The application of caffeine, which causes contractures in skeletal and smooth muscle by releasing calcium from intracellular stores to activate actin and myosin interaction, also causes shortening of OHCs. Tetracaine, which interferes with calcium movement in muscle and non-muscle cells, blocks potassium-induced and caffeine-induced shortening of OHCs, but does not block electrically-induced shortening. Sodium dantrolene which is an inhibitor of intracellular calcium release in skeletal muscle does not block potassium-induced OHC shortening. Immunocytochemical studies using antibodies to muscle-like contractile and regulatory proteins on unfixed, freeze-dried OHCs demonstrate the co-localization of calmodulin with actin throughout the OHC cytoplasm. These results support the ideas that in OHCs, intracellular calcium release is involved in the activation of shortening and that an actin-mediated cell shape change may be regulated by calmodulin in a manner similar to that which occurs in contraction of smooth muscle.

Elemental composition of individual cells and tissues in the cochlea.

Localization of elements at the cellular and sub-cellular levels was performed with the energy dispersive X-ray microanalysis technique, using shock-frozen, freeze-dried and araldite-embedded mouse (CBA/CBA) cochleae sectioned dry. Anatomical identification occurred in the STEM (scanning transmission electron microscopy) mode. In inner hair cell stereocilia the K/Na ratio was 70:1 but only 20:1 in the cytoplasm. In outer hair cell cytoplasm the K/Na ratio was 11:1 while the ratio in stereocilia was similar to that in inner hair cells. Ca was identified in stereocilia and the upper part of the cytoplasm of both outer and inner hair cells. The elemental composition in the subtectorial space is endolymph-like and that in the inner tunnel of the organ of Corti is similar to extracellular fluid. Considerable regional differences in elemental composition occur in the tectorial membrane with regard to P, K and Ca. The highest concentration of Ca occurs in the basal part of the tectorial membrane towards the sensory hairs. The highest concentration of K occurs in the basal and outer parts whereas the middle part of the tectorial membrane contains low levels of both K and Ca. The elemental composition changes in two main directions: 1) from the limbal (growing) region to the tip of the tectorial membrane, and, 2) from upper to lower surfaces. The three cell types of the stria vascularis differ considerably in elemental composition. The highest concentration of K occurs in marginal cells. The basal cells contain more K than do the intermediate cells. A significantly higher concentration of Ca, Cl and Na occurs in marginal cell cytoplasm than in any other cell type in the stria vascularis.

Receptor synapses without synaptic ribbons in the cochlea of the cat.

This report describes recepto-neural junctions thought to be chemical synapses without synaptic ribbons or vesicle aggregates. Such synapses occur in the cat between the outer hair cells of the cochlea and auditory nerve fibers. These synapses are formed by flat or indented junctions having symmetric membrane complexes associated with smooth endoplasmic reticulum and coated vesicles in the outer hair cell cytoplasm. Inner hair cells have receptoneural junctions with asymmetric membrane complexes; synaptic ribbons and vesicles gather at one type of junction, while the other type associates with endoplasmic reticulum. Gap junctions are not seen at cochlear synapses but do link supporting cells. The results suggest that inner and outer hair cell synapses have different properties and raise questions about the role of synaptic vesicles and endoplasmic reticulum in the storage and release of transmitters.