Article Figures & Data
Figures
- Figure 1.
Schematic of the cochlear sensory epithelium showing inner and outer hair cells and their afferent innervation as they appear in tissue immunostained for neurofilament (green) and a synaptic ribbon protein (CTBP2: red). The approximate orientations of the confocal z stacks shown in subsequent figures are also indicated (small box for Figs. 4 and 8; larger box for Fig. 7): the viewing angle for the xy projections is noted. Efferent terminals in IHC and OHC areas have few neurofilaments and thus do not stain brightly in the confocal images.
- Figure 2.
The level and duration of an acoustic overexposure were adjusted so that cochlear thresholds were elevated for several days before returning to normal. a–c , A 2 h exposure to an octave-band (8–16 kHz) noise at 100 dB SPL produced ∼40 dB maximum threshold shifts 1 d postexposure that recovered by 2 weeks to normal preexposure values, as assessed via DPOAEs ( a ), ABRs ( b ), and CAPs ( c ). Thresholds are expressed re age-matched unexposed controls. Group means ± SEMs are shown: n = 6–21 ears per group. ABR and DPOAE measurements are from the same animals; CAP thresholds are from a separate group.
- Figure 3.
Despite threshold recovery, suprathreshold neural responses at high frequencies were permanently attenuated, although recovery of otoacoustic emissions suggests cochlear sensory cells are normal. b , d , At 8 weeks postexposure, suprathreshold amplitudes of ABR wave 1, the far-field response of the cochlear nerve, were less than half their preexposure values ( d ) in regions where temporary threshold shift was maximal (Fig. 2: 32 kHz), but recovered more completely ( b ) where initial shifts were less severe (Fig. 2: 12 kHz). a , c , In contrast, mean DPOAE amplitudes returned to normal by 8 weeks postexposure at both 12 kHz ( a ) and 32 kHz ( c ), suggesting complete recovery of OHC function, endolymphatic potentials, and cochlear mechanics. Together, these data suggest a primary loss of afferent innervation in the 32 kHz region. Group means ± SEMs are shown: n = 7–21 ears per group.
- Figure 4.
a–d , Despite reversibility of threshold shift and intact sensory cells, noise-exposed ears show rapid loss of cochlear synaptic terminals ( a , b ) and delayed loss of cochlear ganglion cells ( c , d ). Immunostaining reveals synaptic ribbons (red, anti-CtBP2) and cochlear nerve dendrites (green, anti-neurofilament) in the IHC area of a control ( a ) and an exposed ( b ) ear at 1 d post noise. Outlines of selected IHCs are indicated ( a , b : dashed lines); the position of IHC nuclei is more irregular in the traumatized ears. Each confocal image ( a , b ) is the maximum projection of a z-series spanning the IHC synaptic region in the 32 kHz region: the viewing angle is from the epithelial surface (see Fig. 1). Each image pair (red/merge) shows the same confocal projection without, or with, the green channel, respectively. Merged images show juxtaposed presynaptic ribbons and postsynaptic terminals, in both control and exposed ears ( a , b : filled arrows), and the lack of both in denervated regions ( b : dashed box). Anti-CtBP2 also stains IHC nuclei; anti-neurofilament also stains efferent axons to OHCs ( a , b : unfilled arrowheads). Cochlear sections show normal density of ganglion cells 2 weeks postexposure ( c ) compared with diffuse loss after 64 weeks ( d ): both images are from the 32 kHz region of the cochlea.
- Figure 5.
Double-staining for anti-neurofilament (green) and anti-CtBP2 (red) suggests cochlear nerve terminals have disappeared where there is loss of synaptic ribbons. a–d , Tissues double stained for anti-neurofilament (green) and anti-CtBP2 (red) are shown as confocal projections of the 45 kHz region from a control ( a , b ) and an exposed ( c , d ) ear 3 d after noise; viewed from the surface of the sensory epithelium (xy projections in a , c ) and in cross-section views (xz projection, b , d ) of half the extent in the x dimension (dashed box). The dramatic reduction in cochlear terminals is especially clear in the xz projections. In the xy projections, filled arrows indicate some of the synaptic ribbons paired with nerve terminals; filled arrows ( c ) point to three ribbons that are displaced from the basolateral IHC membrane and appear uncoupled from nerve terminals. Open arrows ( a , c ) point to spiraling efferent axons in the inner spiral bundle and the open arrowheads show efferents to OHCs crossing the tunnel of Corti. Scale bar in a applies to all panels.
- Figure 6.
Immunostaining cochlear-nerve terminal swellings suggests that ribbon counts underestimate the degree of IHC denervation. a , b , These confocal projections of the IHC area in the 45 kHz region of a control ear ( a ) and an ear 3 d postexposure ( b ) are immunostained with anti-parvalbumin (green), which stains terminal swellings, and anti-CtBP2 (red), which stains synaptic ribbons. In the control ear, there is close to a one-for-one relation between ribbons and terminals (e.g., filled arrows). In the exposed ear, almost all terminals are near a ribbon (e.g., filled arrows); however, some ribbons are not paired with terminals (e.g., unfilled arrows): some appear intracellular, i.e., far from the IHC membrane. The vacuolization of terminals in the exposed ear is part of the acute excitotoxic response to overstimulation (Wang et al., 2002).
- Figure 7.
Synaptic ribbon counts in six cochlear regions of control and noise-exposed ears show synaptic loss throughout the basal half of the cochlea. Mean numbers (±SEMs) of synaptic ribbons per IHC were computed from confocal z-stacks such as those in Figure 2 from control ears (n = 11) and exposed ears at 6 cochlear locations and 4 postexposure times: 1 d (n = 6), 3 d (n = 5), and 8 weeks (n = 6).
- Figure 8.
a–d , Normalized functional and histopathological metrics versus postexposure time show a close match between synaptic loss ( c ) and loss of neural amplitudes ( b ); ganglion cell loss ( d ) is significantly delayed and hair cell responses ( a ) return to normal. There is a close match between synaptic loss ( c ) and loss of neural amplitudes ( b ); ganglion cell loss ( d ) is significantly delayed and hair cell responses ( a ) return to normal. Suprathreshold response amplitudes ( a , b ) are for 80 dB SPL; complete growth functions are in Figure 3. Values are expressed as a percentage of control means (± SEMs, n = 7–21 per group). Loss of ribbons was quantified ( c ) by comparing age-matched controls (n = 11) to exposed ears at four postexposure times: 1 d (n = 6), 3 d (n = 5), 2 weeks (n = 4), and 8 weeks (n = 6). Data from two cochlear regions are shown: 12 kHz and 32 kHz (see key). To control for aging, ribbons were counted in unexposed 104 week animals (n = 3: triangles in c ). Loss of ganglion cells ( d ) was quantified at the 32 kHz place in control (n = 7) and exposed ears at 3 postexposure times: 2 weeks (n = 6), 52–64 weeks (n = 7) and 104 weeks (n = 6). To control for aging, cells were counted in unexposed 104 week animals (n = 12: triangles in e ). For all counts ( c , d ), means ± SEMs are shown, and data are expressed as a percentage of values from unexposed 16 week animals.
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