Late-onset loss of cochlear neurons following neuropathic noise.

<p>Density of spiral ganglion neuron somata as a function of post exposure time, as seen in the ~11 kHz (left) and ~32 kHz (right) regions after exposure to neuropathic (97 dB SPL) or non-neuropathic (94 dB SPL) noise. Data are group means (± SD), expressed relative to data from the youngest unexposed ears. Statistical significance of the group differences is indicated by asterisks: *, <i>p < 0</i>.<i>05</i>; **, <i>p < 0</i>.<i>01</i>. N = 3–7 animals per time and noise exposure.</p

Confocal imaging shows a reduction in CtBP2 stained presynaptic ribbons following neuropathic noise.

<p>Short-term (<b>a</b>-<b>c</b>) and long-term (<b>d</b>-<b>f</b>) loss of presynaptic ribbons in inner hair cells (IHCs) after exposure to neuropathic (97 dB SPL) noise compared to age-matched animals exposed to non-neuropathic noise (94 dB SPL) and unexposed controls. Presynaptic ribbons are labeled with anti-CtBP2 antibodies (red). Confocal images are maximal projections of z-stacks of ribbons within 4–5 IHCs in the 32 kHz region. Dashed lines depict IHC outlines. Scale bar: 10 μm (<b>a</b>-<b>f</b>).</p

Cochlear thresholds are either permanently or temporarily shifted following neuropathic noise.

<p>ABR (left column) and DPOAE (right column) thresholds resulting from exposure of 6 week old mice to 8–16 kHz noise for 2 hours at 100 dB SPL (purple), 97 dB SPL (neuropathic exposure, blue) and 94 dB SPL (non-neuropathic exposure, green) compared to unexposed controls (red). Thresholds are shown as group means ± SD. The noise band is depicted as a gray vertical bar. Time post exposure (pe) is specified in hours (h), weeks (w) or months (m) in vertical boxes on the right side of each row. N = 5–9 mice per time point and noise exposure.</p

Ability of transgenic <i>endoA</i> constructs to rescue the development of <i>endoA</i> null mutants to adulthood.

<p>Shown is the proportion of eclosed adult rescuants (genotype <i>elav-GAL4/Y or w</i>; <i>UAS-endoA</i>*<i>/+; endoA^Δ4/endoA^Δ4</i>) relative to the total number of adult progeny resulting from the rescue cross. The <i>UAS-endoA</i>* transgene carried the mutations indicated on the abscissa and in some cases also encoded a hemagglutinin epitope tag (indicated by the suffix “−HA”). Also shown is the proportion of rescuants in which the <i>endoA</i> transgene encoded either wild type EndoA (“wt”), or HA-tagged wild type EndoA (“wt-HA”). Each <i>bar</i> represents one transgenic integration line, specified <i>below the abscissa</i>. The total number of adult progeny resulting from the rescue cross is indicated for each line (<i>numbers above the bars</i>). The lower and upper 95% confidence intervals are given. ^*<i>P</i><0.01. N.s., not significant. ^†Besides <i>UAS-endoA^{A66W 4.1}</i>, the rescue efficiency of two other <i>UAS-endoA^A66W</i> transgenes was evaluated (<i>UAS-endoA^{A66W 41.3}</i> and <i>UAS-endoA^89.1</i>). They both caused lethality of all the progeny from the rescue cross, as detailed in the text.</p

Neural response is reduced following neuropathic noise.

<p>ABR wave I peak-to-peak amplitudes (<b>a</b>) and DPOAE amplitudes (<b>b</b>) in response to 11.3 kHz tones (left column) and 32.0 kHz tones (right column) recorded from 6 hours to 16 months post exposure to noise causing PTS (100 dB SPL, purple), neuropathic noise causing TTS (97 dB SPL, blue), non-neuropathic noise causing TTS (94 dB SPL, green), and unexposed animals (red). ABR amplitudes are in response to 80 dB SPL tones (the maximum level tested) and DPOAE amplitudes are in response to 80 dB SPL tones. Noise parameters are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125160#pone.0125160.g001" target="_blank">Fig 1</a>. Statistical significance of the group differences is indicated by asterisks: *: <i>p < 0</i>.<i>05</i>, **: <i>p < 0</i>.<i>01</i>, ***: <i>p < 0</i>.<i>001</i>. Data are group means ± SD. N = 5–9 mice per time point for noise-exposed and control groups.</p

Cochlear sensory cells are intact after neuropathic noise.

<p>Photomicrographs of the organ of Corti from the ~32 kHz region of mice exposed to 97 dB SPL noise and evaluated either 1 month (<b>a</b>) or 16 months (<b>b</b>) after exposure. Scale bar: 20 μm (<b>a</b>-<b>b</b>).</p

The small-eye trait induced by expression of the <i>UAS-endoA^A66W</i> mutant transgene.

<p>A, eye of a control fly carrying the <i>elav-GAL4</i> driver but no <i>endoA</i> transgene. B, <i>endoA</i> null fly rescued to the pharate adult stage by <i>UAS-endoA^A66W</i> expression, driven by <i>elav-GAL4</i> (<i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA^Δ4/endoA^Δ4</i>). Note that the eye size is reduced and that the lower eye tip is pointy rather than rounded. C, The small-eye trait also appears when <i>UAS-endoA^A66W</i> expression occurs on a wild-type <i>endoA</i> background (<i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA⁺/endoA⁺</i>). D–G, Scanning electron micrographs of <i>elav-GAL4</i> (D, F) and <i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA⁺/endoA⁺</i> (E, G) eyes. In G, some examples of ommatidia that lack bristles are indicated by <i>asterisks</i>, and aberrant dual bristles by <i>arrowheads</i>. Pitting is indicated by an <i>arrow</i>. Scale bars: C, 100 µm (applies to A–C); E, 50 µm (D, E); G, 20 µm (F, G).</p

Numbers of presynaptic ribbons are reduced after neuropathic noise.

<p>Synaptic counts in inner hair cells in the 11.3 kHz (left) and 32.0 kHz (right) regions following exposure to neuropathic (97 dB SPL) or non-neuropathic (94 dB SPL) noise. Statistical significance of the group differences is indicated by asterisks: *: <i>p < 0</i>.<i>05</i>, **: <i>p < 0</i>.<i>01</i>, ***: <i>p < 0</i>.<i>001</i>. Data are group means ± SD. N = 6–7 animals per time and noise exposure.</p

Cochlear-nerve peripheral axons can be counted in tangential sections through the osseous spiral lamina.

<p>Tangential cuts though the osseous spiral lamina in the ~32 kHz region of cochleae 1 month (<b>b</b>), 8 months (<b>d</b>) or 16 months (<b>f</b>) after exposure to neuropathic noise (97 dB SPL) demonstrate loss of peripheral axons compared to age-matched unexposed controls (<b>a</b>, <b>c</b>, <b>e</b>). These sections show fascicles of cochlear-nerve peripheral axons roughly midway between their cell bodies in the spiral ganglion and their peripheral terminals in the organ of Corti. Scale bar: 10 μm (<b>a</b>-<b>f</b>).</p

Degeneration of spiral ganglion neurons is assessed in cochlear sections after neuropathic noise.

<p>Midmodiolar sections through Rosenthal’s canal show permanent loss of spiral ganglion neurons in the apex (<b>b</b>) and base (<b>d</b>) 16 months after exposure to neuropathic noise (97 dB SPL) compared to age-matched unexposed controls (<b>a</b>, <b>c</b>). Scale bar: 50 μm (a-d).</p