Olivocochlear suppression of outer hair cells in vivo: evidence for combined action of BK and SK2 channels throughout the cochlea

Abstract

Cholinergic inhibition of cochlear hair cells via olivocochlear (OC)-efferent feedback is mediated by Ca(2+) entry through α9-/α10-nicotinic receptors, but the nature of the K(+) channels activated by this Ca(2+) entry has been debated (Yoshida N, Hequembourg SJ, Atencio CA, Rosowski JJ, Liberman MC. J Neurophysiol 85: 84-88, 2001). A recent in vitro study (Wersinger E, McLean WJ, Fuchs PA, Pyott SJ. PLoS One 5: e13836, 2010) suggests that small-conductance (SK2) channels mediate cholinergic effects in the apical turn, whereas large-conductance (BK) channels mediate basal turn effects. Here, we measure, as a function of cochlear frequency, the magnitude of BK and SK2 expression in outer hair cells and the strength of in vivo OC suppression in BK(+/+) mice vs. BK(-/-) lacking the obligatory α-subunit (Meredith AL, Thorneloe KS, Werner ME, Nelson MT, Aldrich RW. J Biol Chem 279: 36746-36752, 2004). Except at the extreme apical tip, we see immunostaining for both BK and SK2 in BK(+/+). Correspondingly, at all testable frequencies (8-45 kHz), we see evidence for both SK2 and BK contributions to OC effects evoked by electrically stimulating the OC bundle: OC-mediated suppression was reduced, but not eliminated, at all frequencies in the BK(-/-) ears. The suppression remaining in BK nulls was blocked by strychnine, suggesting involvement of α9-/α10-cholinergic receptors, coupled to activation of the remaining SK2 channels.

Fig. 1.

Cochlear immunostaining shows that inner hair-cell (IHC) synaptic counts in large-conductance BK^−/− are similar to BK^+/+. A and B: immunostaining for presynaptic ribbons (CtBP2; red) and postsynaptic AMPA-receptor patches (GluA2; green) was used to count synaptic elements. Both images are maximum projections from confocal z-stacks in the 16-kHz region. Rough outlines of a pair of adjacent IHCs are shown. C: counts of synaptic elements (means ± SE) at 5 cochlear locations. Samples include data from 4 ears of each genotype: at each frequency region, in each ear, 2 adjacent image stacks were acquired, from ∼80 IHCs total in each cochlear region, in each genotype. GluR, glutamate receptor.

Fig. 2.

Immunostaining for a cholinergic marker shows slightly reduced volume of outer hair-cell (OHC)-efferent terminals in BK^−/−. A and B: maximum projections of confocal z-stacks through the OHC region immunostained with vesicular acetylcholine transporter (VAT; green). Medial olivocochlear (MOC) terminals are normally found in clusters of 2–3 under each OHC, whereas they are often in singlets in BK^−/− (closed arrows). The tiny VAT-positive puncta (open arrows) are located well below the OHC bases, within the outer spiral bundles. C: mean innervation density (±SE) was quantified by measuring the volume of VAT-positive terminals at 6 locations along the cochlear spiral from BK^+/+ (n = 3) and BK^−/− (n = 3) ears: in each location, in each ear, 2 image stacks were obtained from contiguous regions, which together spanned 20 OHCs in each row. D: data from C replotted as the percentage deviation from BK^+/+ data.

Fig. 3.

BK immunolabeling is seen in IHC (A and B) and OHC (C and D) areas. A and B: BK immunostains the IHC near the cuticular plate (red arrowheads), far from the synaptic zone where synaptophysin (Syn)-positive lateral olivocochlear (LOC) terminals (e.g., green-fill arrow) and Na⁺-K⁺-ATPase-positive afferent terminals (e.g., blue-fill arrowhead in A) are located. LOC terminals are seen in the inner spiral bundle (ISB; arrowhead no. 1 in B) and close to afferent terminals contacting the IHC (arrowhead no. 2 in B). MOC fibers to the OHCs are also immunopositive for Na⁺-K⁺-ATPase (blue arrowhead in B). A and B are x-y and y-z projections, respectively, of a confocal z-stack from the 8-kHz region in a BK^+/+ ear: outline of an IHC (dotted lines) is shown for scale. NKAα3, sodium/potassium-transporting ATPase subunit α3. C and D: in BK^+/+ (C), BK (red) and small-conductance (SK2; green) colocalize in puncta at the basal poles of OHCs: each paired panel shows the red and green channels in a confocal image (single optical section) from the 16-kHz region. The outline of 1 OHC base is shown (dashed line). In BK^−/− ear (D), BK immunoreactivity is absent. E: quantification of the mean volumes of BK- and SK2-positive immunopuncta at 5 cochlear regions suggests more robust BK expression in the basal half of the cochlea. No BK-positive puncta were observed at the 4-kHz region. For the other frequencies, data are mean volumes (±SE) from 111 to 181 puncta from all 3 OHC rows from 3 to 5 ears per frequency. F: schematic of the IHC and OHC areas showing the LOC- and MOC-efferent terminals and the type-I and type-II afferent terminals on IHCs and OHCs, respectively. G: BK immunolabeling in the OHC area is postsynaptic. A membrane marker (CellMask; blue-green) labels the OHCs, the Deiters cell cups, and the vesicle-rich efferent terminals. This image of 1st-row OHCs is a maximum projection of 4 consecutive 0.25-μm z-slices from the 22-kHz region.

Fig. 4.

Cochlear thresholds are normal in BK^−/−, at both 6 and 15 wk of age, and for both distortion product otoacoustic emissions (DPOAEs; A) and auditory brain-stem responses (ABRs; B). Means (±SE) are shown; numbers of ears in each DPOAE group were: at 6 wk, 33 BK^+/+ and 24 BK^−/−; at 15 wk, 8 BK^+/+ and 8 BK^−/−. Key in B applies to both panels. SPL, sound-pressure level.

Fig. 5.

Suprathreshold DPOAE responses (A and B) are normal in BK^−/−; however, ABR wave-1 amplitudes (C and D) and the summed responses of the cochlear nerve fibers are reduced, and their latencies (E and F) are prolonged. Mean data (±SE) are shown; group sizes are the same as in Fig. 4. Key in A applies to all panels. Data were obtained at age 6 wk.

Fig. 6.

Efferent effects on OHCs are reduced at all test frequencies in BK^−/− ears. A: in a sample run of the efferent MOC assay, DPOAEs are measured once per second, before, during, and after a 70-s train of shocks to the OC bundle (gray box). Response amplitudes are normalized to the average of the 25 measurements taken before shock-train onset. The size of MOC suppression is the average normalized DPOAE for the 1st 3 points after shock-train onset (circled). B: MOC suppression in BK^−/− vs. BK^+/+ littermates, as a function of stimulus frequency (f₂ is plotted here). For all test frequencies, primary levels are set to evoke a DPOAE 10 dB above the noise floor: typical f₂ levels were ∼30 dB at 16 kHz, ∼40 dB at 32 kHz, and ∼60 dB at 8 kHz. Means from 5 BK^+/+ and 11 BK^−/− ears are shown: SE are smaller than the symbol size.

Fig. 7.

Strychnine blockade of MOC-elicited effects in BK^−/− vs. BK^+/+ suggests BK channels are key contributors to fast and slow suppressive effects as well as the strychnine-insensitive enhancement. A: average effects of MOC shocks on DPOAE amplitudes evoked at 3 f₂ frequencies (8, 16, or 32 kHz, as indicated) from 11 BK^−/− and 5 BK^+/+ ears, as measured either with (thin trace) or without (thick trace) strychnine blockade of effects mediated by the α₉-/α₁₀-ACh receptors. B: contributions of SK2 channels and BK channels to (α₉-/α₁₀-mediated) suppression and (non-α₉-/α₁₀-mediated) enhancement are derived by mathematically combining traces from column A: SK2-mediated suppression (blue) = BK^−/− − BK^−/− with strychnine; BK-mediated enhancement (green) = BK^+/+ with strychnine − BK^−/− with strychnine; BK-mediated suppression (red) = BK^+/+ − BK^−/− − BK^+/+ with strychnine − BK^−/− with strychnine. Gray box in each panel shows duration of shock train. DPOAE amplitudes are normalized before averaging as described in Fig. 6.

Fig. 8.

Strychnine has no effect on the slow enhancement of cochlear responses evoked by shocking the OC bundle in α₁₀-null mice. These data are mean responses from 3 runs performed before strychnine injection (black) and 5 runs performed after (w/) strychnine injection (gray) in 1 animal.

Fig. 9.

BK^−/− mice are more vulnerable to temporary noise-induced hearing loss (A and B) but not to permanent noise-induced hearing loss (C and D), as measured by both DPOAEs (A and C) and ABRs (B and D). For temporary threshold shift (TTS), mice were exposed to the 8- to 16-kHz octave band at 94 dB for 15 min and tested 6 h later. Means (±SE) are shown; numbers of ears in each group were: DPOAE, 12 BK^+/+ and 12 BK^−/−; ABR, 6 BK^+/+ and 6 BK^−/−. For permanent threshold shift (PTS), mice were exposed to the 8- to 16-kHz octave band at 100-dB SPL for 2 h and tested 1 wk later. Means (±SE) are shown; numbers of ears in each group were: DPOAE, 10 BK^+/+ and 10 BK^−/−; ABR, 5 BK^+/+ and 5 BK^−/−.