Calcium Microdomains And Modulation In Mechanosensory Hair Cells

Hair cells from the cochlea and vestibular system contain highly ordered rows of actin-based stereocilia that translate nanoscale physical force from sound, head movement or gravity, into an electrical neural code, through mechanoelectrical transduction (MET). Here we report on calcium signaling in hair cells and neurons of the inner ear using a transgenic mouse line Gad2-Cre::GCaMP5G-tdTomato. Swept field confocal microscopy was used to image [Ca2+] modulation in the inner ear of transgenic mice, in response to physiological hair bundle deflection from either micromechanical movement or chemical stimuli. Images were collected through the intact membranous labyrinth or, in some cases, through a fenestration in the membrane. In semicircular canals, spatially localized [Ca2+]i transients were observed and quantified at the extreme tips of stereocilia, the apex of hair cells, in microdomains at the base of hair cells, and in postsynaptic terminals. Hair bundles were either stimulated with a fluid jet directly attached to a picospritzer or by piezoelectric micromechanical indentation applied to mimic physiological head rotation. Fluorescence were imaged using a custom upright Scientifica microscope configured with Olympus optics and a fast swept field confocal imaging system. In the semicircular canals, ∼1µm deflection of the intact cupula revealed punctate increases in GCaMP5G fluorescence (ΔF/F) at the tips of stereocilia. [Ca2+]i transients were restricted to the tips of stereocilia where MET channels are located and were not present in kinocilia. Cupula deflection also evoked large [Ca2+]i transients just below the cuticular plate, putatively colocalizing with the striated organelle. Depolarization with application of KCl revealed [Ca2+] transients in microdomains at the base of hair cells, likely reflecting voltage gated calcium channels modulating synaptic release. Preliminary results using adult vestibular organs of Gad2-Cre::GCaMP5G-tdTomato transgenic mice reveal highly localized time-resolved [Ca2+] modulation at the tips of stereocilia, in microdomains of hair cells, and at synaptic terminals.