Article Figures & Data
Figures
- Figure 1.
A, B, Anti-peripherin (red) immunostains the cell bodies of type II SGNs in Prph+/+ ears (A) but not in Prph−/− (B), confirming the null expression in the knockout. Each image is a maximum projection of a confocal z-stack through the apical turn. Antibodies to β-tubulin (TuJ1; green) stain the cell bodies of type I SGNs, and myosin VIIa is used to stain hair cells (blue). Scale bar: B (for A, B), 100 μm.
- Figure 2.
In the organ of Corti, peripherin weakly stains the peripheral projections of type II SGNs, while it strongly stains a subset of MOC efferents. A, Thin outer spiral fibers (green arrowheads) in the outer spiral bundles are immunopositive for β-tubulin (TuJ1), as are thick MOC fibers running radially across the tunnel of Corti. Some of MOC fibers are also positive for peripherin (green-rimmed red arrowheads). Peripherin also strongly stains MOC fibers in the inner spiral bundle (ISB) and, rarely, a thin type II projection running diagonally across the floor of the tunnel (red arrowhead). B, Antibodies to synaptophysin (blue) and Na+/K+ ATPase (green) identify the thick tunnel-crossing fibers as MOC neurons, a subset of which are peripherin positive (green-rimmed red arrowhead). An occasional thin type II projection is also peripherin positive (red arrowhead). Images are maximum projections from the 11.3 kHz region of two different wild-type animals. Scale bar: A (for A, B), 20 μm.
- Figure 3.
Immunostaining for parvalbumin (which stains type II terminals and outer spiral fibers green) and vesicular acetylcholine transporter (which stains MOC terminals blue) suggests that both afferent and efferent innervations are normal in Prph−/− ears. A, B, Maximum projections of confocal z-stacks from the 16 kHz region of a Prph−/− ear (A) and a Prph+/+ ear (B), shown in the acquisition plane (x–y) and in the orthogonal plane (z–y) showing a cross-sectional view, as schematized in C. The dotted box in C shows the approximate region imaged in A and B. Green-filled and blue-filled arrowheads in A and B highlight the spatially offset clusters of type II and olivocochlear terminals, respectively, underneath the third-row OHCs. White arrowheads in Azy and Bzy point to the three outer spiral bundles (OSBs) running between the Deiter’s cells. Scale bar: Bzy (for all panels), 20 μm.
- Figure 4.
Type II terminals are apposed to presynaptic ribbons in Prph−/− and Prph+/+ ears. A, B, Maximum projections of confocal z-stacks from the 11.3 kHz region of a knock-out and a wild-type ear, shown in the acquisition plane (x–y) and the orthogonal plane (z–y; Fig. 3C ). Green-filled, red-rimmed arrowheads point to appositions between (parvalbumin-positive) type II terminals and (CtBP2-positive) presynaptic ribbons. Red-filled, white-rimmed arrowheads in the z–y projections point to nonsynaptic ribbons. Scale bar and immunostaining keys apply to all panels. Dashed white boxes in the z–y projections show the z-cropping used to generate the x–y projections.
- Figure 5.
A, B, Quantitative analysis shows that the afferent (A) and efferent (B) innervation of OHCs is similar in Prph−/− and Prph+/+ ears. A, Mean ribbon counts and synaptic ribbon counts per OHC (±SEM) as a function of cochlear location. B, Mean silhouette area of MOC terminals per OHC (±SEM) as a function of cochlear location. Both graphs are based on data from four cochleae, two of each genotype. Each point is based on data from four high-power z-stacks, each containing ∼25 OHCs.
- Figure 6.
Cochlear thresholds are similar in Prph−/− and Prph+/+ ears. Data are the means (±SEMs) from eight ears of either genotype.
- Figure 7.
Shock-evoked MOC suppression is absent in Prph−/− ears. A, Schematic cross-section through the brainstem at the level of the lateral superior olive (LSO) and medial superior olive (MSO), showing (1) the cell bodies of MOC neurons projecting to one ear (ipsilateral), (2) the circuit underlying the sound-evoked MOC reflex, and (3) the location of the electrical stimulator at the floor of the fourth ventricle. B, Mean time course of the changes in DPOAE amplitude (compared with mean preshock baseline) evoked by a 70 s shock train to the OC bundle. Data are averaged from four WT and two KO ears. DPOAEs in the ipsilateral ear were evoked with primary tones at 16.0 kHz (f2) and 13.33 kHz (f1), presented at levels evoking a DPOAE 10 dB above the noise floor. C, Mean shock-evoked suppression in DPOAE amplitudes for f2 = 16 or 22.6 kHz for Prph−/− vs. Prph+/+ ears (n = 2 and n = 4, respectively). To collapse traces such as those in B to a single number, we average, for each test iteration, the first three points after shock onset.
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