Stepwise morphological and functional maturation of mechanotransduction in rat outer hair cells

Abstract

Inner ear mechanosensory hair cells convert mechanical vibrations into electrical signals via the coordinated interaction of multiple proteins precisely positioned within the sensory hair bundle. Present work identifies the time course for the acquisition and maturation of mechanoelectric transduction (MET) in rat cochlea outer hair cells maintained in organotypic cultures. A spatiotemporal developmental progression was observed morphologically and functionally with basal cochlea maturation preceding apical cochlea by 2-3 d in all measured properties. The fraction of mechanosensitive cells increased rapidly, with a midpoint at postnatal day 0 for basal cells, and correlated with myosin IIIa immunoreactivity. MET current magnitude increased over several days. Adaptation lagged the onset of transduction by a day and matured more slowly, overlapping but preceding the rise in myosin Ic immunoreactivity. Less than approximately 25% of myosin Ic expression was required for the mature adaptation response, suggesting multiple roles for this protein in hair bundle function. Directional sensitivity, lacking in immature responses, developed rapidly and correlated with the pruning of radial links and an increase in tenting of stereociliary tips. Morphological and electrophysiological data support a hypothesis in which key elements arrive independently at the site of MET, with a mature response occurring as membrane tension increases, likely by the increased tensioning of the tip link with the onset of adaptation. Organotypic cultures developed normal, tonotopically specific, MET response properties, suggesting that maturation was not influenced significantly by external factors such as innervation, endolymph, normal mechanical stimulation, or an intact organ of Corti.

Figure 1.

Cultured hair bundles are morphologically similar to acutely prepared tissue. A, Scanning electron micrograph of the cultured organ of Corti. Solid lines represent where the tissue was cut before placement in culture chamber. The boxes represent the regions recorded from electrophysiology (black being apical and gray being basal). B, Differential interference contrast images of hair bundles from preparations just before electrical recordings from acutely prepared and organotypic cochlea cultures. Bundles appear morphologically similar at each developmental age. Apical hair bundles mature later than basal bundles. Scale bar, 10 μm for all panels. C, Percentage of hair bundles at each age that appear to have normal morphological features. The organotypic hair bundles deteriorate over time so that by P10, 30–50% of the hair bundles are abnormally shaped. Recordings were made exclusively from hair bundles with normal appearance.

Figure 2.

A, Myosin IIIa localizes to the stereocilia in both culture and acute preparations. Both spatial and temporal progression of myosin IIIa staining were similar in vivo and in vitro. Myosin IIIa (green) appears in basal hair bundles before it appears in apical hair bundles both in cultured (top) and acute (bottom) preparations. At P1, myosin IIIa was observed in basally located bundles, only slightly in bundles from midregions, and not in apical hair bundles, a pattern matched in the acute preparations. Red staining is actin labeled with phalloidin. Scale bar, 10 μm for all panels. Misformed hair bundles also show the myosin IIIa localization as depicted in the basal hair cells in P4 organotypic cultures. B, Mechanically elicited currents from apical hair cells at P6 or 6 d in culture. Cells were voltage clamped at −80 mV and mechanically stimulated between −100 and 1000 nm (a single stimulus trace is shown above currents for timing). No differences were observed in activation, adaptation, or current amplitudes between acute and cultured cells (see text for details). C, Averaged activation curve for acute (n = 6) and cultured (n = 5) mechanically activated currents. These curves were not statistically different (see text for details).

Figure 3.

Morphological maturation of rat OHC hair bundles. Scanning electron micrographs of developing OHC bundles from three positions along the cochlea at ages ranging from P1 to P9. The border color reflects the onset of mechanosensitivity. Yellow means no response, and red means mature response. Both temporal and spatial gradients were observed in the shape of the hair bundles, height of the stereocilia, and number of stereocilia. Scale bar, 4 μm for all panels.

Figure 4.

Hair bundles undergo pruning of both stereocilia and interstereociliary links. A, Higher-magnification electron micrographs of an immature (left), middle-aged, and mature hair bundle (right) depicting the change in stereocilium number, size, and interciliary connections. Scale bar, 0.35 μm for all panels. B, Quantification of the number of stereocilia and interciliary links counted in a uniform midregion of apical hair bundles from P0 through P9 (n = 10 for each data point). Larger hair bundle regions than shown in A were used for these measurements (see text). Sigmoidal functions were fit to the data with a resulting slope of 1.6 ± 0.5 for the pruning of stereocilia and 0.6 ± 0.4 number/d for the pruning of interciliary links. The midpoint of these plots was 2.5 ± 0.3 d for stereocilium number and 2.2 ± 1.2 d for interciliary links. Correlation coefficients were 0.99 and 0.98 for stereocilia and links, respectively. C, The ratio of links to stereocilia demonstrates that the links are pruned at a more rapid rate, although over a similar time course, than stereocilia. This plot was also fit with a sigmoidal function having a slope of 0.4 ± 0.6 (links/stereocilium)/d and a midpoint of 3 ± 2 d (r² = 0.99).

Figure 5.

The onset of transduction occurs with a temporal and spatial gradient from base to apex in rat cochlea cultures. A, B, Transduction responses from hair cells voltage clamped at −80 mV from ages P1, P2, P4, and P6 basal (A) and P1, P2, P3, and P6 for apical hair cells (B). The time course of the stimulus is shown above the current records. The stimulus amplitudes varied to encompass the entire activation range for a given cell; for the basal P1, stimuli were −221, 508, 766, 1515, and 1631 nm; for P2, −157, 162, 320, 478, and 803 nm; for P4, −220, 262, 482, 728, and 987 nm; for P6, −90, 150, 290, 350, and 400 nm; for apical recordings at P1 and P2, stimuli were −221, 508, 1515, 1631 nm; for P3, −310, 136, 420, 694, and 1114 nm; and for P6, −265, 286, 450, 603, and 916 nm. C, Time course of the onset of transduction for apical (blue) and basal (red) outer hair cells (n is indicated by each point). The proportion of recorded cells in which transduction was present is plotted against days after birth. Data were fit with sigmoidal functions with slopes of 0.53 ± 0.11 and 0.53 ± 0.17 d⁻¹ and midpoints of 0.04 ± 0.17 and 3.9 ± 0.2 d for basal and apical cells, respectively (r² for fits were 0.96 and 0.98 for base and apex). D, The increase in MET current amplitudes during maturation. Here, too, data were fit with sigmoidal functions. I_max was fixed at 400 pA and 700 pA for apical and basal cells, respectively. Slope values were 0.83 ± 0.06 (r² = 0.9) for basal and 1.02 ± 0.33 pA/d (r² = 0.76) for apical hair cells. Midpoint values were 2.4 ± 0.1 and 3.5 ± 0.4 d for basal and apical cells, respectively. E, Activation kinetics were slower in immature responses than in mature responses. Data were normalized to maximal current and to relative probability of opening. F, Activation kinetics for P1 basal cell recordings. Activation was slower than the stimulus rise time (shown by red line) for each cell. More mature responses activated with kinetics that matched the stimulus rise time.

Figure 6.

The onset of adaptation is delayed compared with the onset of transduction. Adaptation matures over a short time period, first appearing slow and incomplete and then maturing to its final rate. A, Examples of MET currents from basal cells at P1 and P4 voltage clamped at −80 mV in response to 300 and 110 nm stimuli, respectively. Time constants were measured as fits to a simple double-exponential equation, fits shown in red. B, The proportion of transducing cells with adapting responses is plotted against days after birth (n is given near each data point). Data were fit with sigmoidal functions in which the slopes were 0.33 ± 0.13 d⁻¹ and 0.32 ± 0.16 d⁻¹ and the midpoints were 1.3 ± 0.2 and 3.9 ± 0.1 d for basal (r² = 0.95) and apical cells (r² = 0.99), respectively. A plot of the fast adaptation time constant against days in culture (C) demonstrates the development of a tonotopic distribution. Data were fit with exponential curves in which the baseline value represents the mature response and was respectively 0.18 ± 0.01 (r² = 0.9) and 0.35 ± 0.06 ms (r² = 0.89) for basal and apical cells, demonstrating that tonotopy develops in the cultured preparation. The time constant for maturation was 1.2 ± 0.2 and 1.4 ± 0.5 d⁻¹ for basal and apical cells, respectively. D, Examples of maturation of the slow time constant for a P2 and P6 basal cell in response to steps that elicit ∼50% of the maximal current. Double-exponential fits to the current decay are shown in red. E, The extent of adaptation, measured as 1 − (steady-state current)/(peak current), increased during development over a similar time course as the adaptation time constant quickens for both apical and basal cells. Sigmoidal fits to these data yielded slopes that were not significantly different at 0.3 ± 0.1 and 0.5 ± 0.1 d⁻¹ and midpoints of 1.3 ± 0.2 and 4.5 ± 0.2 d for basal and apical cells, respectively (r² values were 0.95 and 0.99 for basal and apical cells). F, The slow adaptation time constant is plotted against days in culture, again demonstrating a temporal delay between apical and basal development. Here, the data were fit with sigmoidal functions with baseline values, representing the mature response, that were not statistically different at 2.4 ± 0.5 and 2.6 ± 0.8 ms. The slopes were not different with values of 0.8 ± 0.8 and 1.1 ± 0.5 d⁻¹, whereas the midpoints were different with values of 4 ± 1 and 8 ± 1 d for basal and apical cells, respectively (r² were 0.76 and 0.95 for basal and apical fits).

Figure 7.

A, Examples of normalized current–displacement (I–x) plots for basal cells recorded at P1 (I_max was 158 pA) and P4 (I_max was 595 pA), demonstrating a leftward shift during maturation. B, C, Data were fit with a single Boltzmann function to obtain information regarding the slope (B) and half-activation properties (C), both plotted against days after birth. No change was seen in the steepness, as demonstrated by the linear fits with slopes not different from 0. The displacement to achieve half-activation shifts leftward over a time course that is similar to the maturation of the MET current response. Data fit with the equation for an exponential had a rate of change of 1.3 ± 0.6 and 2.1 ± 1.1 d for basal (r² = 0.91) and apical (r² = 0.68). D, Correlation between the half-activation displacement and the maximal transducer current. The relationship demonstrates that during the development state, there is a direct relationship that is lost in basal hair cells after P2. The solid line is an exponential with a rate of change of 50 ± 5 pA⁻¹ (r² = 0.9).

Figure 8.

Myosin Ic localization increases over a time course similar but delayed, compared with that of the maturation of fast and slow adaptation. A, B, Staining of actin (red) with phalloidin and myosin Ic (green), in vitro (A) and in vivo (B). No difference in distribution or maturation of myosin Ic was observed between in vivo and in vitro. A spatial and temporal pattern of development was observed in which positive staining was first observed in basal cells, then middle region, and then apical region. Scale bars, 10 μm for all panels.

Figure 9.

A, Data from Figure 8 were quantified by measuring fluorescence intensity (minus background) for bundles at each location and plotted against age. Data were fit with sigmoidal functions with slope values of 1.7 ± 0.5 and 1.9 ± 0.2 (AU/d) and midpoints of 9.8 ± 0.5 and 7.0 ± 0.2 d for apical (r² = 0.99) and basal cells (r² = 0.97), respectively. A, Inset, The initial 12 d for the plot in A to show the separation between apex and base. The n for each point is given near the data point. B, The normalized myosin Ic intensity is plotted against the fast adaptation time constant for apical (blue) and basal cells (red). Single-exponential fits were used in which the rate of change for apical cells was measured as 0.07 ± 0.01 ms compared with 0.04 ± 0.03 ms for basal cells (r² values for fits were 0.99 and 0.96 for apical and basal cells, respectively). C, Similar plots were made for the slow time constant of adaptation resulting in rates of change of 0.17 ± 0.13 and 0.17 ± 0.03 ms for apical (r² = 0.69) and basal cells (r² = 0.95), respectively.

Figure 10.

Immature bundles show a lack of directional sensitivity. A, Example of a probe positioned at the back end of an OHC hair bundle. Scale bar, 10 μm. Positions of directly behind the center of the hair bundle or behind one side of the hair bundle were used with no apparent difference in response properties. B, A response from a P3 basal hair cell stimulated (shown above current) with the probe behind the bundle so that stimuli that would normally close channels opened channels. There was no current on at rest in these cells. Similar responses were obtained in three cells. C, A mixed response, also obtained with the probe behind the hair bundle. Stimuli toward the kinocilium opened channels (a), whereas stimuli away from the kinocilium largely produced off responses as indicated by the overshoot in the tail current (b). However, larger negative stimuli were capable of producing a mixed response that both opened and closed channels (c). This response shows channels opening when the bundle was pushed in the off direction, but the return to baseline elicited a current implicating channel closure, i.e., tail current.

Figure 11.

Summary of stepwise maturation time course of the hair bundle and mechanosensitivity in relation to cochlear development. A, Time line of key events from exiting the cell cycle, to expression of transcription factor math1, to maturation of adaptation. B, Stages of development depicting stereocilium maturation, thickening, elongation, pruning of interciliary links, the appearance of myosin Ic, the onset of mechanosensitivity, and the appearance of directional sensitivity. C, Schematized MET responses. D, Top-down view of stereocilia depicting the pruning of interciliary linkages. Blue represents myosin IIIa, and yellow represents myosin Ic.