Mécanosensibilité des cellules ciliées de la cochlée du rat en réponse à un micro-jet de fluide

Hearing begins with mechanoelectrical transduction of sound-evoked vibrations by hair cells of the cochlea—the sensory auditory organ of the mammalian ear. Each hair cell is equipped with a hair bundle, a tuft of a few tens to a few hundred stereocilia that protrude from the cell’s apical surface into the surrounding fluid. The hair bundle acts as a mechanosensory antenna. Deflections of the hair bundle modulate tension in extracellular “tip links” that interconnect the stereocilia at their tips. This change in tension directly activates mechanosensitive ion channels, giving rise to an electrical signal. Hair-cell mechanosensitivity depends on the collective response of about 100 transduction channels within the hair bundle. Ideally, channel gating would be concerted so that their contributions add up, maximising the total current. In outer hair cells of the cochlea, however, the stereocilia are organized in a hair bundle with a U- or V-shape and the bundle’s cohesiveness is weak. The functional significance of this peculiar morphological feature and the ability of transduction channels to gate together in a poorly cohesive structure is unclear.My PhD work is organized in two parts. First, I aimed to clarify how the individual contributions from the transduction channels of an outer-hair-cell bundle add up to shape the response of the hair cell. Achieving this goal required precise knowledge of individual stereociliary movements and the orientational field of the tip links that convey force to the transduction channels. To tackle this challenge, I used an excised preparation of the rat cochlea and mechanically stimulated single hair bundles with a fluid jet, which deflects the hair bundle through hydrodynamic interactions. I recorded the hair-bundle response with a high-speed camera and developed an image-analysis software to automatically track the position of individual stereocilia. Using finite-element simulations, I computed the viscous stress field generated by a fluid jet on a hair-bundle-like structure and confronted the measured stereociliary displacements to the estimated forces, investigating the role of interstereociliary links. Finally, I characterized the orientational field of the tip links by monitoring the reorientation of stereocilia movements upon disruption of the tip links and the contraction of the hair bundle upon application of a calcium chelator.My results indicate that the stress and displacement fields are nearly proportional for stimulations from the neural edge, but not for stimulations from the abneural edge, for which the lateral links must provide a significant contribution to the response. While neural stimulation efficiently recruited all the tip links in the bundle, though with maximal stimulation at the bundle’s edges, abneural stimulation excited mostly the transduction elements within the central region of the V-shaped bundle. Based on our estimate of the orientational field of the tip links, we conclude that mechanosensitivity of an outer hair-cell bundle is greater for stimulations from the neural edge than for stimulations from the abneural edge.In the second part of my PhD work, I studied how the ionic environment of a hair cell affects a hair bundle’s mechanical properties. Excised sensory tissues from animal models of the mammalian cochlea generally fail to reconstitute several key in-vivo properties, including the large difference in calcium concentration between the basal and apical aspects of the hair cells. In an effort to better control the ionic environment of the hair cell ex vivo, I developed a micro-perfusion system to tune the calcium concentration in direct proximity of a hair bundle. Transitioning from an artificial extracellular saline with high calcium concentration to a more physiological one, I found that the hair-bundle stiffness decreased by an average of 30%, which is consistent with the softening of the tip links at low calcium concentrations.