Biology session

Impairment of hearing is the most common sensory deficit in the human population. Sensorineural hearing losses (SNHLs) are the incurable forms of this impairment at present. In this case the lesion affects the inner ear (cochlea) or the neural conductivity or processing. Invention of effective drug therapy needs deepening of our patophysiological knowlege at the cellular level. Our aim was to set up and optimize the method of loading of Ca2+ indicators by electroporation, which makes easier and more reliable the funtional imaging of cells in the organ of Corti.

Majority of SNHLs are developed after the oneset of hearing, but investigations at the cellular level in mice are usually performed before that in the literature. The hemicochlea preparation, we use is suitable for investigations in mice with mature hearing (> P15), i.e., developmental changes do not disturb drawing conclusions. The preparation was made by cutting in half the cochlea of 15-27 days old mice. Cells of interest were identified in an epiflourescent microscope. The fluorescent indicator was injected by a current impulse after touching the cell with the dye loaded micropipette driven by a micromanipulator. The electoporation opens poruses in the cell membrane and the indicator is forced to enter the cell. We used the two Ca2+ indicators, Oregon Green BAPTA-1 and Fura2. By testing them, we have defined their optimal concentration in the pipette (1mM), the intensity (10μA) and the duration (10ms) of the electroporation current and the parameters of the pipette (4-6MΩ). With this method we can load both the sensory- and the suppurting cells of the organ of Corti (4-6 cell per preparation). Due to the selective filling the background staining remains low and the signal-to-noise ratio is much better than in the case of bulk loading we used before. To validate this new method, we stimulated the loaded cells with 50 and 100 μM ATP and measured intracellular Ca2+ transient with functional imaging techique. All cells of the organ of Corti have purinerg receptors (P2X and P2Y) using Ca2+ as a second messenger.

We have set up the method of Ca2+ indicator loading by electroporation of cells of the mature organ of Corti. Using this technique we will have the opportunity to investigate the patomechanism of SNHLs at cellular level in both wild type and genetically modified mice serving as a model of inhereted human deafnesses.