Novel PMCA2 Mutation and Hearing Loss in Mouse Figure 7. Dissipation of Ca2+ transients in utricular hair cells co-loaded with Fluo-4 and caged Ca2+. (A) Immunolocalization of the PMCA2 pump in the stereocilia of Obl/Obl utricular hair cells (details in Materials and Methods). Localisation in wildtype hair cells appears identical to that of mutants. (B) Confocal image of baseline Fluo-4 fluorescence in an organotypic culture of mouse utricular macula. The diagonal dashed line represents the scan line during subsequent data acquisition. The hair bundle comprises the top and middle horizontal dotted lines (the latter crosses the cell cuticular plate). The cell soma comprises middle and lower lines. (C) In this line-scan image, ordinates are pixel positions along the scan line, abscissa is time and fluorescence transients, DF/F0, evoked by a 4 ns UV pulse (arrow), are color-coded according to the color scale-bar at right. Signals from pixels below the lowermost horizontal dotted line arise from the cell adjacent to the hair cell in (B). (D) Fluorescence traces obtained by spatially averaging line-scan pixel signals within the stereociliary compartment of wild type (wt, blue solid line), heterozygous (Obl/+, green solid line), and homozygous Oblivion mice (Obl/Obl, black solid line). Each trace is the population average of n = 6 cells (for wt and Obl/Obl) or n = 5 cells (for Obl/+). The region within the dash-dotted box is shown on a faster time scale in (E). Also shown in (E) are confidence intervals (dash-dotted lines) for the average DF/F0 responses (solid lines). doi:10.1371/journal.pgen.1000238.g007 middle turn (55–65% from the base) at 20 (P20), 72–75 and 121 days old. Between 3 and 7 Obl/+ and +/+ mice were examined at each age. Hair cells with intact stereocilia bundles were counted over a stretch of at least 200–300 mm of the cochlear duct. Hair bundles that were damaged or showed fusion were still counted as being present. A two tailed T-Test was performed on hair cell counts for each hair cell row in each region at 95% confidence intervals, with the number of cases and standard deviations included in the analysis. A two tailed T-test was carried out on the weights of Obl/Obl mutants compared to littermate (Obl/+) controls in the same way. Preyer Reflex and Auditory Brainstem Response (ABR) The Preyer reflex (pinna flick) was detected using a custom-built click box to deliver a calibrated 20 kHz sound burst at 90 dB SPL. Up to 88 mice from Obl/+6+/+ matings were tested weekly from 3 to 8 weeks of age, although the numbers of mice at each time- point varied. For ABR recordings, a separate cohort of mice were PLoS Genetics | www.plosgenetics.org 8 October 2008 | Volume 4 | Issue 10 | e1000238