Tonotopic gradient in the developmental acquisition of sensory transduction in outer hair cells of the mouse cochlea

Abstract

Inner ear hair cells are exquisite mechanosensors that transduce nanometer scale deflections of their sensory hair bundles into electrical signals. Several essential elements must be precisely assembled during development to confer the unique structure and function of the mechanotransduction apparatus. Here we investigated the functional development of the transduction complex in outer hair cells along the length of mouse cochlea acutely excised between embryonic day 17 (E17) and postnatal day 8 (P8). We charted development of the stereociliary bundle using scanning electron microscopy; FM1-43 uptake, which permeates hair cell transduction channels, mechanotransduction currents evoked by rapid hair bundle deflections, and mRNA expression of possible components of the transduction complex. We demonstrated that uptake of FM1-43 first occurred in the basal portion of the cochlea at P0 and progressed toward the apex over the subsequent week. Electrophysiological recordings obtained from 234 outer hair cells between E17 and P8 from four cochlear regions revealed a correlation between the pattern of FM1-43 uptake and the acquisition of mechanotransduction. We found a spatiotemporal gradient in the properties of transduction including onset, amplitude, operating range, time course, and extent of adaptation. We used quantitative RT-PCR to examine relative mRNA expression of several hair cell myosins and candidate tip-link molecules. We found spatiotemporal expression patterns for mRNA that encodes cadherin 23, protocadherin 15, myosins 3a, 7a, 15a, and PMCA2 that preceded the acquisition of transduction. The spatiotemporal expression patterns of myosin 1c and PMCA2 mRNA were correlated with developmental changes in several properties of mechanotransduction.

FIG. 1.

A: maturation of mouse outer hair cell bundles along the developing organ of Corti. Scanning electron micrographs (SEMs) were taken at P0 and P6 at 3 positions along the cochlea: base, middle, and apex and in all 3 rows. When hair bundles were viewed from behind the longest stereocilia, changes in its morphology could be observed. Representative images are shown. Bundle height was measured as indicated on basal hair bundles at P0 and P6. Scale bar: 1 μm. B: bundle height estimated from stereopairs of SEM images at calibrated magnifications of ×20,000 and ×10,000. Measures were taken from 3 to 6 stereocilia closest to the kinocilium in each of ≥3 different bundles at each location in 3 different cochleae at P0 and P6 (total number of bundles studied for each location are n = 17 P0BA; n = 15 P6BA; n = 9 P0AP; n = 16 P6AP). Graph shows total number of stereocilia and means ± SE. ** statistically significant differences in bundle height.

FIG. 2.

FM1-43 uptake along the developing cochlea. A: Differential interference contrast microscopy image of a P2 organ of Corti. Bars illustrate the cuts performed to obtain 4 quarters for physiological and PCR experiments. BA: base, MB: mid basal, MA: mid apical, AP: apex. B–E: confocal images of unfixed organ of Corti (base through apex counterclockwise with the most basal side on the left) that have been exposed to 5 μM FM1-43 for 10 s, washed and imaged 5 min later. P0, the arrow indicates the basal end (B), P2 (C), P4 (D), and P6 (E) images obtained from animals of the same litter are shown. F: FM1-43 fluorescence intensity along the organ of Corti for these respective stages.

FIG. 3.

Hair cell response to mechanical stimuli along the developing organ of Corti from E17 to P8. Representative recordings for each stage and each quarter are illustrated. No current was detected in embryonic preparations. The onset of mechanosensitivity in outer hair cells progressed from P0 in the base to P2 in the apex. The largest currents were obtained at P2 in the base. Relaxation of the current during square step stimuli was observed in all of the recordings including at the onset of mechanosensitivity.

FIG. 4.

Onset and development of outer hair cell mechanosensitivity. A: percentage of transducing cells in each cochlea quarters from E17 to P8. Boltzmann fits to the data demonstrated a sharp rise in the number of mechanosensitive cells from E18 to P0 in the base and delayed by 1 and 2 days, respectively, for MA and AP (n = 234). For numerical values and averages, see Supplemental Table S2. B: 3-dimensional (3D) plot illustrating averaged maximal transduction currents elicited in outer hair cells of each quarter at each of the 10 developmental stages. Numerical values, standard errors and number of cells are listed in Supplemental Table S2.

FIG. 5.

Developmental changes in outer hair cell response to mechanical stimuli. A and B: normalized open probability as a function of displacement during hair cell development from P0 to P5 in the base (A) and P2 to P8 in the apex (B). Representative responses are shown. C: averaged 10–90% operating range as a function of development in BA and AP. Fitting functions were used to help visualize the developmental changes occurring along the cochlea from P0 to P8 (exponential decay for BA and Boltzmann for AP).

FIG. 6.

Adaptation in basal and apical outer hair cells (BA and AP) between P0 and P8. A and B: representative half-maximal transduction currents from basal (A) and apical cells (B) from which slow and fast time constants were derived. C and D: averaged fast (C) and slow (D) time constants (± SE) obtained using double-exponential fits to half-maximal transduction current traces. The fit parameters are plotted as a function of development in the 2 extreme quarters of the cochlea. An example of the double-exponential fitting curve is shown in a P2-BA cell in A: Tau fast = 2.16 ms; Tau slow = 17.65 ms. E: extent of adaptation derived from the ratio of peak vs. steady-state current at the end of a 80-ms square displacement step. Averaged values for each stage are plotted (± SE). Developmental changes were fitted with a Boltzmann curve. In both the base and the apex, adaptation became more complete in more mature stages. Its extent reached a steady stage value of ∼92% of the peak current. The same number of cells were analyzed for C–E.

FIG. 7.

Expression of β actin (Actb), prestin (Slc26a5), Myo3a, Myo7a, Myo15a, Myo1c, Cdh23, Pcdh15, and Atp2b2 (PMCA2) along the cochlea from E17 to P8. A: quantitative PCR measurements of house keeping gene β actin in the 28 samples. 3D plot illustrates fold change in expression from E17 AP. B–G, 3D plots represent relative quantitative PCR measurements of the 7 genes normalized to β actin and the most immature stage: E17-AP. H: Pcdh15 expression normalized against the most mature stage (P8-BA) illustrated on an inverse time scale to reveal the gradual decreased expression during development in all four quarters. Corresponding numerical values ± SD are listed in Supplemental Table S3.