Guinea Pig Cochlear Hair Cells Research Articles

Investigation of cochlear hair cells and the perception of ultrasound signals in guinea pigs

We established a specific ultrasound frequency-dependent model of cochlear injury using bone conduction ultrasounds in the inner ear of guinea pigs at 50 kHz and 83 kHz, to explore the effects of bone conduction ultrasound in the cochlea. To establish a unilateral cochlear damage model, the unilateral cochlea was destroyed. The control group consisted of 50 kHz and 83 kHz bone conduction ultrasounds in unaltered guinea pigs. In each group, cerebral blood oxygenation level dependent (BOLD) effects were determined by functional magnetic resonance imaging (fMRI). The cochlear outer hair cell motor protein, Prestin, and the microfilament protein, F-Actin, were detected. We found that bone conduction ultrasound irradiation at 50 kHz and 83 kHz on the guinea pig inner ear for six hours leads to hair cell damage. Furthermore, low frequency bone conduction ultrasound induces major damage to outer hair cells, while high frequency ultrasound damages both internal and external hair cells. fMRI analysis of cerebral BLOD effects revealed an affected cerebral cortex region of interest (ROI) of 4 and 2, respectively, for the normal control group at 50 kHz or 83 kHz, and 2 for the 83 kHz bone conduction ultrasound cochlear injury group, while 50 kHz bone conduction ultrasound failed to induce the cortical ROI within injury model. Results reveal that the spatial location of guinea pig cochlear hair cells determines coding function for lower ultrasound frequencies, and high frequency bone conduction ultrasound may affect the cochlear spiral ganglion or cranial nerve nucleus in bone conduction ultrasound periphery perception.

Changes in Guinea Pig Cochlear Hair Cells after Sound Conditioning and Noise Exposure

Sound conditioning has reduced noise-induced hearing loss in experimental mammalian animals and in clinical observation. Forty guinea pigs were grouped as: A, control; B, conditioning noise exposure group; C, high level noise exposure group; and D, conditioning noise exposure followed by a high level noise exposure group. Auditory brainstem response thresholds were measured. The cochlear sensory epithelia surface was observed microscopically. Calmodulin, F-actin and heat shock protein 70 (HSP70) in hair cells were immunohistochemistrically stained. The intracellular free calcium was stained for confocal microscopy. The ABR threshold shift after noise exposure was higher in group C than D, and showed a quicker and better recovery in group D than C. Stereocilia loss and the disarrangement of outer hair cells were observed, with the greatest changes seen in group C, followed by groups D and B. The most intensive immunohistochemical intracellular expressions of calmodulin, F-actin, and HSP70 were found in group D, followed by groups C, B and A. The highest intensity of the fluorescent intracellular free Ca2+ staining in the isolated outer hair cells was observed in group C. The ABR and morphological studies confirmed the protective effect from noise trauma of sound conditioning. The protective mechanism of hair cells during sound conditioning was enforced through the increase of cellular cytoskeleton proteins and through the relieving of intracellular calcium overloading caused by the traumatic noise.

The dimensions and structural attachments of tip links in mammalian cochlear hair cells and the effects of exposure to different levels of extracellular calcium

The tip links between stereocilia of acousticolateral hair cells have been suggested to contain cadherin 23 (CDH23) comprising an upper branched portion that is bound to a lower portion composed of protocadherin 15 (PCDH15). The molecular conformation of CDH23, its binding to PCDH15, the tip links, and mechanoelectrical transduction have all been shown previously to be sensitive to exposure to low levels of calcium. The aim of this study was to compare the characteristics of tip links in guinea-pig cochlear hair cells with reported features of the CDH23–PCDH15 complex. Tip links were examined using field emission scanning electron microscopy and transmission electron microscopy in conventional preparations and after treatment with the detergent Triton-X-100 or varying calcium concentrations in the extracellular solution. The results showed that tip links have a twisted double-stranded appearance with a branched upper region. They survived demembranation of the stereocilia by detergent suggesting that they have transmembrane domains at both ends. Their lengths, when fixed in the presence of 2 mM extracellular calcium, were ∼150 nm. With prior exposure to 1 mM calcium their lengths were ∼164 nm. The lengths in 50 μM calcium are similar (∼185 nm) to those reported for CDH23–PCDH15 complexes in 100 μM calcium (∼180 nm). Exposure to 1 μM calcium caused loss of tip links and an increased distance between the residual attachment sites. The data indicate that extracellular calcium concentration affects tip-link length. One model compatible with the recently proposed tip-link structure is that the CDH23 double strand undergoes calcium-dependent unfolding, changing the length of the links. The bundle may also tilt in the direction of the tallest row of stereocilia as the tip link lengthens and then is lost. Overall, our data are consistent with a tip link composed of complexes of CDH23 and PCDH15 but do not rule out other possibilities.

An immunogold investigation of the distribution of calmodulin in the apex of cochlear hair cells

Calmodulin is found in the mechanosensitive stereociliary bundle of hair cells where it plays a role in various calcium-sensitive events associated with mechanoelectrical transduction. In this study, we have investigated the ultrastructural distribution of calmodulin in the apex of guinea-pig cochlear hair cells, using post-embedding immunogold labelling, in order to determine in more detail where calmodulin-dependent processes may be occurring. Labelling was found in the cuticular plate as well as the hair bundle, the rootlets of the stereocilia being more densely labelled than the surrounding filamentous matrix. In the bundle, labelling was found almost exclusively at the periphery rather than over the centre of the actin core of the stereocilia, and was clearly associated with the attachments of the lateral links that connect them to their nearest neighbours. It was also found to be denser towards the tips of stereocilia compared to other stereociliary regions and occurred consistently at either end of the tip link that connects stereocilia of adjacent rows. The contact region between stereocilia that is found just below the tip link was also clearly labelled. These concentrations of labelling in the bundle are likely to indicate sites where calmodulin is associated with calcium/calmodulin-sensitive proteins such as the various myosin isoforms and the plasma membrane ATPase (PMCA2a) that are known to occur there, and possibly with the transduction channels themselves. At least one of the myosin isoforms, myosin 1c, is thought to be associated with slow adaptation, and PMCA2a with control of calcium levels in the bundle. The concentration of calmodulin in the contact region further supports the suggestion that this is a functionally distinct region rather than a simple geometrical association between adjacent stereocilia.

Apoptosis of guinea pig cochlear hair cells following chronic aminoglycoside treatment.

Although aminoglycosides have been investigated for their cochleotoxicity, it has still not been determined whether apoptosis or necrosis results in cochlear hair cell death following aminoglycoside treatment. To study possible mechanisms of cell death, we used in situ DNA break-labeling to examine guinea pig cochleae affected by Kanamycin ototoxicity. Chronic kanamycin treatment induced DNA fragmentation that was detectable in both outer and inner hair cells, suggesting the occurrence of apoptosis. These findings suggest that apoptosis achieves deletion of affected hair cells without disrupting tissue architecture in the organ of Corti.

Effect of Furosemide Upon Morphology of Hair Bundles in Guinea Pig Cochlear Hair Cells

Furosemide was administered by intraperitoneal injection and intracochlear perfusion. The peak-to-peak amplitude of the cochlear microphonic at high sound intensities was reduced however the drug was administered. After a period of time ranging from 1-4 h following drug application, cochleae were fixed and prepared for scanning electron microscopy to examine the hair cells. Changes in the hair bundles from basal turns tended to be more extensive than those of more apical turns. Initially there was an increase in the granularity of the surface of the stereociliary membrane and a tendency for cross-links to swell, stretch and break. Later, the surface texture of stereocilia became smoother than normal, and hair cells in the basal turns showed extensive erosion and fracture of cross-links. Tip links could survive even when extensively stretched. It is possible that the morphological changes in stereocilia reported here do not arise from direct actions of furosemide, but indirectly, from perturbations of the ionic composition of cochlear fluids induced by effects of the drug upon the stria vascularis.

Cross-links between stereocilia in the guinea pig cochlea

Cross-links between stereocilia on guinea pig cochlear hair cells have been examined using high resolution scanning (SEM) and transmission electron microscopy (TEM), confirming recent descriptions of these structures. Links from the tips of shorter stereocilia to the sides of the adjacent taller stereocilia (upward-pointing links), between stereocilia of the same row (side-to-side links) and between adjacent rows (row-to-row links), have been observed on inner and outer hair cells. These links have been seen in material fixed using (1) glutaraldehyde only, (2) glutaraldehyde/osmium and (3) glutaraldehyde/osmium/thiocarbohydrazide (a technique which makes gold coating unnecessary).Upward-pointing links were seen less frequently, and the surfaces of stereocilia and microvilli were smoother after fixation (3) compared with fixations (1) and (2) in which they were usually roughened in appearance.In TEM, side-to-side and row-to-row links form a regular lattice between stereocilia, and consist of a number of strands. Upward-pointing links consist of a single strand, the ends of which are associated with electron-dense material. This lies between the stereociliary membrane and the actin filament bundle, at the tip of the shorter stereocilium and the side of the taller stereocilium.

Guinea pig cochlear hair cell density; its relation to frequency discrimination

The density of guinea pig cochlear hair cells fluctuates within a narrow range in a regular and species-specific manner. Two density maxima may be identified within the range. This pattern of density changes resembles the curve of frequency discrimination thresholds reported for guinea pigs in the literature.

Unspecific ion channels in guinea pig cochlear hair cells

It has been proposed that the cochlear microphonic (CM) is produced by a modulated potassium-current through channels in the apical membrane of the hair cells. In support of this concept, Salt and Konishi [J. Acoust. Soc. Am. 66, S47 (1979)] have found that electrophoretically applied tetraethylammonium (TEA; a blocker of K channels of excitable membranes) reduces the CM while raising the endocochlear potential. However, two lines of evidence suggest that these pathways might not be K-selective: (1) Cory and Hudspeth [Nature (in press)] have shown in an in vitro lower-vertebrate hair cell system that the microprohie-producing ion channels are not K selective, since numerous ions can be accommodated. (2) We have observed that injections into scala media of about 50 nl each of phentolamine (1 mM), serotoxin (5 mM), or EGTA (1 mM) in artificial endolymph produce effects similar to those reported for TEA. Control injections produced no effect. The diversity of the known primary actions of each of these agents (alpha-adrenergic blocker, neurotransmitter, and calcium chelating agent, respectively) suggests that TEA may not be acting on a strictly K-selective channel. [Supported by NSF and NIH.]