Stereociliary bundles of rat inner hair cells at the cochlear apex consist of about 70 stereocilia, most of which are ~0.5 μm in diameter and are arranged in three rows of heights 4 μm, 2 μm and 1.5 μm 12,13
that will be denoted as row 1 (R1), row 2 (R2) and row 3 (R3) respectively. To measure the calcium transients occurring during transduction, whole-cell recordings were made from hair cells in isolated coils of neonatal rat cochleas and calcium indicator dyes of the Fluo4 family were introduced via the patch pipette. When the bundle was deflected with a water jet, a rapid increase in stereociliary calcium could be monitored using a fast swept-field confocal microscope that allowed acquisition at 500 frames per second. This technique enabled full images of the bundle to be taken every 2 ms and showed that an increase in fluorescence could be clearly discerned in individual stereocilia (; Supplementary video 1
). The stereociliary fluorescence changes were eliminated by treatment with MT channel blockers, including the aminoglycoside antibiotic, streptomycin (1 mM, N = 4) and curare (0.2 mM, N = 2) which largely abolished the MT currents at −80 mV (Supplementary Fig. 1
). The fluorescent signal indicative of calcium influx through the MT channels also depended on membrane potential and was abolished by depolarization from the normal holding potential (−80 mV) to +100 mV near the calcium equilibrium potential. To circumvent contamination by motion artifacts, an experimental protocol was adopted5
in which bundle displacement was combined with a depolarizing voltage step, so no calcium influx would occur until the cell was repolarized to its normal holding potential (). Upon repolarization, with the bundle still held in a displaced position, a burst of fluorescence was observed in the second row (R2) stereocilia with little change in the first row (R1). In this experiment, using Fluo-4FF dye, the bundle was viewed vertically and the microscope focused halfway down the bundle just below the top of R2. The responses were quantal in that the fluorescence change in each of the bright stereocilia was similar (). This uniformity in size implies the responses in all active stereocilia were similar and justified averaging the signals. A smaller signal was seen in the two dark (inactive) R2 stereocilia with amplitude and time course similar to the R1 response. The average change in R2 fluorescence developed with a time constant of 25 ms. By comparison the change in the signal in R1 was seven-fold smaller and slower with a time constant of 77 ms. For R3 the onset time constant was 15 ms, more similar to R2 than R1. Although in some cells a double exponential was needed to fit the response onsets, the fast component was dominant and had a mean value of 18 ± 4 ms (R2, N = 6), 15 ± 7 ms, (R3, N = 5) and 40 ± 30 ms, (R1, N= 6). R2 and R3 were thus similar and both faster than R1.
Figure 1 Amplitude and time course of stereociliary calcium signals. (a) Images of an inner hair cell stereociliary bundle in fluorescence at +100 mV and −80 mV holding potential and in brightfield illumination (bottom) with the focus at the top of the (more ...)
Measurements on other cells yielded a ratio of peak fluorescence in R2 to that in R1, FR2/ FR1, of 10.2 ± 0.8 in 15 inner hair cells using the low affinity dye Fluo-4FF which has a calcium dissociation constant, KD, of 10 μM. In comparison, when the dye Fluo-4 with a KD of 0.3 μM was used as the indicator, the fluorescence ratio, FR2/ FR1, was 6.5 ± 0.8 in 12 other inner hair cells. The results suggest that use of a high affinity indicator may underestimate the ratio, probably because the R2 signals are saturated. Fluorescent signals were also visible in R3 stereocilia on focusing down towards the cell body or when the bundle was slightly tilted (). Analysis of such images showed the R3 signals were also large and fast (). A noteworthy feature of the images is that some R3 stereocilia were active even though the adjacent R2 stereocilium was not. This suggests that tension in a given tip link can open channels at its lower attachment point (on R3) without evoking a signal at its upper attachment point (on R2). The signal in R3 with an associated active R2 (black) was brighter than in R3 with an inactive R2 (green) probably because the confocal volume included some of the signal from the shaft of the underlying active R2 stereocilium. Thus the signal measured in R3 coupled to an active R2 is an over-estimate and a comparison of R2 (red trace) with R3, inactive R2 (purple trace) is likely to be a more accurate measure of the relative calcium signals in the two rows. This shows that the signal in R3 was comparable to that in R2. In nine cells, the mean ratio FR2/ FR3 was 1.47 ± 0.5. R2 and R3 therefore behaved similarly with large fast changes while R1 responded more slowly and weakly.
Figure 2 Amplitude and time course of stereociliary calcium signals of an inner hair cell bundle tilted to show all three stereociliary rows. (a) Average fluorescence and Nomarski images. The bright R3 stereocilia (black) have neighboring bright R2 stereocilia (more ...)
It might be argued that no large stimulus-related calcium signal was ever seen in R1 because the tip links from the first to second rows were absent or were damaged during isolation. Tip links between R2 and R1 stereocilia have been reported in inner hair cells of neonatal (postnatal day 3) mice14
and are present in rats of the age used experimentally in both inner (Fig 6A of ref. 12
) and outer hair cells (Fig 4 of ref. 15
). In recordings from more than 50 inner hair cells with MT currents up to 1 nA, no convincing evidence of signals in the first row was ever seen. An MT current of 1 nA probably represents two-thirds of the theoretical maximum, assuming a single channel of 15 pA and 50 tip links (see ref. 12
). It seems unlikely that any damage to the bundle would be restricted to R1, and the simplest conclusion from the small R1 compared to R2 signal is that there are MT channels in the second row of stereocilia but not the first row of inner hair cell bundles.
In some experiments, to visualize all stereociliary rows including those with low calcium signals, the cells were also filled with 50 μM AlexaFluor 488. This addition facilitated clear quantification of the changes in fluorescence at different bundle heights (), at the top, at the middle and at the base of the bundle just above the cuticular plate. At the top, only the tallest row was visible whereas with the focus at the middle or base, R1, R2, and sometimes R3 could be seen. Large fluorescence signals were seen at both levels in R2, though those at the base were smaller. In contrast in R1, small signals were measured at all three positions but paradoxically the amplitude increased towards the base (). These trends were observed in five cells, in which the mean fluorescence changes (in arbitrary units) in R1 were 26 ± 7 (top), 33 ± 5 (middle) and 64 ±18 (base) and in R2 were 210 ± 24 (middle) and 167 ± 21 (base). The mean signal in R3 in these cells was 200 ± 32 (base). One explanation for these trends is that they signify the direction of the calcium gradient. Thus for R2, calcium diffuses from top to bottom of the stereocilia but for R1 it diffuses from bottom to top. Consistent with this notion, FR2/ FR1 was smaller at the base. Such a result would occur if calcium enters through MT channels at the top of R2 and diffuses down into the cell apex and then diffuses back up R1.
Figure 3 Variation in calcium signal along the stereocilia. (a) Transverse images of an inner hair cell bundle at the top, middle and base at +100 mV (left) and −80 mV (right). Each image is the average of ten frames taken at times indicated by black bars (more ...)
If the fluorescent signal mirrors calcium influx through the MT channels, the numbers of active cilia should be proportional to the amplitude of the MT current. Here only bright cilia were counted, so the behavior is independent of dye saturation and results with dyes of different affinities were pooled. To ensure R3 was also counted, images were examined at slightly lower focal planes and in some experiments the bundle were tilted to visualize the entire array ( and ). The number of active cilia was proportional to the MT current amplitude for currents up to 1 nA () with a mean slope of 35.4 ± 1.3 pA per stereocilium at a holding potential of −80 mV. This slope represents the MT current per stereocilium and may be compared with the single-channel current inferred from measurements of unitary events which in apical inner hair cells has a mean amplitude of 15.0 ± 1.6 pA at −80 mV 12
. The ratio of the slope to the single channel amplitude is 2.36 ± 0.34, implying that there are approximately two channels per tip link as previously concluded5,12
Figure 4 Relationship between number of active stereocilia and MT current amplitude. (a) Images of three cells showing different numbers of bright fluorescent (active) stereocilia during mechanical stimulation that evoked different MT current amplitudes which (more ...)
A few measurements were made on outer hair cells which have thinner stereocilia (~0.25 μm in diameter) than the inner hair cells. Although this size is similar to the pixel dimensions, it was sometimes possible to distinguish the different rows (; Supplementary video 2
) and therefore compare the average fluorescence intensities in R1, R2 and R3. The fluorescence change in R2 was about a four-fold larger than R1 (FR2
= 3.8 ± 0.5 for a mean MT current of 580 pA; N=3). One possible reason for the smaller ratio compared to the inner hair cells is that during channel opening the calcium concentration in the stereocilia may be higher due to their smaller diameter and volume producing greater dye saturation. This would lead to an underestimate in FR2
. The calcium response in R2 was also much faster with an onset time constant of 19 ms compared to 116 ms for R1. As with the inner hair cell measurements the intensity in R1 increased as the focus was moved towards the base of the bundle whereas in R2 it showed a small decrease (), consistent with opposite directions of calcium diffusion. These results support the idea that both inner and outer hair cells have only a small calcium signal in the first row stereocilia.
Figure 5 Amplitude and time course of stereociliary calcium signals in an outer hair cell. (a) Transverse images of an outer hair cell bundle at the middle and base of the bundle, each image being the average of ten frames taken at times indicated by black bars (more ...)