Opposing gradients of ribbon size and AMPA receptor expression underlie sensitivity differences among cochlear-nerve/hair-cell synapses

The auditory system transduces sound-evoked vibrations over a range of input sound pressure levels spanning six orders of magnitude. An important component of the system mediating this impressive dynamic range is established in the cochlear sensory epithelium, where functional subtypes of cochlear nerve fibers differ in threshold sensitivity, and spontaneous discharge rate (SR), by more than a factor of 1000 (Liberman, 1978), even though, regardless of type, each fiber contacts only a single hair cell via a single ribbon synapse. To study the mechanisms underlying this remarkable heterogeneity in threshold sensitivity among the 5–30 primary sensory fibers innervating a single inner hair cell, we quantified the sizes of presynaptic ribbons and postsynaptic AMPA receptor patches in >1200 synapses, using high-power confocal imaging of mouse cochleas immunostained for CtBP2 (C-terminal binding protein 2, a major ribbon protein) and GluR2/3 (glutamate receptors 2 and 3). We document complementary gradients, most striking in mid-cochlear regions, whereby synapses from the modiolar face and/or basal pole of the inner hair cell have larger ribbons and smaller receptor patches than synapses located in opposite regions of the cell. The AMPA receptor expression gradient likely contributes to the differences in cochlear nerve threshold and SR seen on the two sides of the hair cell in vivo (Liberman, 1982a); the differences in ribbon size may contribute to the heterogeneity of EPSC waveforms seen in vitro (Grant et al., 2010).National Institute on Deafness and Other Communication Disorders (U.S.) (Grants RO1 DC0188)National Institute on Deafness and Other Communication Disorders (U.S.) (P30 DC5029

Chronic Conductive Hearing Loss Leads to Cochlear Degeneration

Synapses between cochlear nerve terminals and hair cells are the most vulnerable elements in the inner ear in both noise-induced and age-related hearing loss, and this neuropathy is exacerbated in the absence of efferent feedback from the olivocochlear bundle. If age-related loss is dominated by a lifetime of exposure to environmental sounds, reduction of acoustic drive to the inner ear might improve cochlear preservation throughout life. To test this, we removed the tympanic membrane unilaterally in one group of young adult mice, removed the olivocochlear bundle in another group and compared their cochlear function and innervation to age-matched controls one year later. Results showed that tympanic membrane removal, and the associated threshold elevation, was counterproductive: cochlear efferent innervation was dramatically reduced, especially the lateral olivocochlear terminals to the inner hair cell area, and there was a corresponding reduction in the number of cochlear nerve synapses. This loss led to a decrease in the amplitude of the suprathreshold cochlear neural responses. Similar results were seen in two cases with conductive hearing loss due to chronic otitis media. Outer hair cell death was increased only in ears lacking medial olivocochlear innervation following olivocochlear bundle cuts. Results suggest the novel ideas that 1) the olivocochlear efferent pathway has a dramatic use-dependent plasticity even in the adult ear and 2) a component of the lingering auditory processing disorder seen in humans after persistent middle-ear infections is cochlear in origin

Type II Cochlear Ganglion Neurons Do Not Drive the Olivocochlear Reflex: Re-Examination of the Cochlear Phenotype in Peripherin Knock-Out Mice

Abstract The cochlear nerve includes a small population of unmyelinated sensory fibers connecting outer hair cells to the brain. The functional role of these type II afferent neurons is controversial, because neurophysiological data are sparse. A recent study (Froud et al., 2015) reported that targeted deletion of peripherin, a type of neurofilament, eliminated type II afferents and inactivated efferent feedback to the outer hair cells, thereby suggesting that type II afferents were the sensory drive to this sound-evoked, negative-feedback reflex, the olivocochlear pathway. Here, we re-evaluated the cochlear phenotype in mice from the peripherin knock-out line and show that (1) type II afferent terminals are present in normal number and (2) olivocochlear suppression of cochlear responses is absent even when this efferent pathway is directly activated by shocks. We conclude that type II neurons are not the sensory drive for the efferent reflex and that peripherin deletion likely causes dysfunction of synaptic transmission between olivocochlear terminals and their peripheral targets

Age-related loss of outer hair cells was increased by OC lesion, but not by chronic conductive hearing loss.

<p>Mean survival (± SEMs) of outer hair cells (A) and inner hair cells (B) as a function of cochlear location. Cochleas were harvested at 64 wks. Groups and group sizes are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g002" target="_blank">Fig 2</a>. Key in A also applies to B.</p

Schematics of the auditory periphery, including the TM, the cochlea and the brainstem locations of the cell bodies of the medial (M) and lateral (L) olivocochlear (OC) neurons.

<p>A: Conductive hearing loss was produced by unilateral resection of the TM (<i>TMx</i>). B: Cochlear de-efferentation was produced by a stereotaxic section of the olivocochlear bundle in the dorsal surface of the brainstem (<i>OCx</i>).</p

Age-related threshold shifts are exacerbated by ipsilateral OC lesion (<i>OCx</i>), TM removal (<i>TMx</i>) or otitis media (<i>OM</i>).

<p>Threshold shift in each group, at each age, was defined <i>re</i> mean values at the same test frequency in 8-wk controls. Each point shows mean threshold shift (±SEM) for either ABRs (A) or DPOAEs (B) for frequencies from 5–45 kHz inclusive. Group sizes are: Controls n = 11, <i>OCx</i> n = 6; <i>TMx</i> n = 10; <i>OM</i> n = 2. <i>OCx</i> cases were exclusively those where the degree of de-efferentation was greater than 75% at all cochlear regions, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g006" target="_blank">Fig 6</a>. Key in A applies to both panels. Downward arrows indicate that some DPOAE thresholds were at the measurement ceiling, thus the shifts represent a minimum estimate.</p

Threshold shifts (A,B) and changes in suprathreshold amplitude (C,D) for ABR (A,C) and DPOAEs (B,D), as measured at 64 wks and normalized to mean age-matched controls.

<p>Means (±SEMs) are shown. Amplitudes are computed by extracting the mean response for each ear at each stimulus frequency for stimulus levels from 60–80 dB SPL, inclusive. Groups and group sizes are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g002" target="_blank">Fig 2</a>. Key in C applies to all panels. Downward arrows indicate that the DPOAE thresholds were at or near the measurement ceiling, thus response changes are a minimum estimate.</p

Effects of chronic conductive hearing loss (E,F,G,H) or OC lesion (I,J,K,L) on efferent and afferent innervation in the inner and outer hair cells areas, as compared to control (A,B,C,D).

<p>Each row of images (e.g. A,B) contains a pair of maximal projections of a confocal z-stack through 10–12 adjacent hair cells, viewed either in the xy (acquisition) plane (left) or the zy (digitally rotated) plane (right). Each afferent synapse in the inner hair cell area (C,G,K) appears as a closely apposed pair of red (anti-CtBP2) and green (anti-GluA2) puncta. GluA2 puncta are not visible in the outer hair cell area (A,E,I). OC terminals in both inner and outer hair cell areas appear in the blue (anti-VAT) channel. Positions of inner hair cell nuclei are shown as dotted white circles—as seen in the red channel by adjusting gamma (not shown). The white lines in D show the modiolar-pillar and habenular-cuticular axes used in the spatial analysis of inner hair cell synapses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g009" target="_blank">Fig 9</a>). Orientation of inner hair cells in the zy plane (D,H,L) is as schematized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g009" target="_blank">Fig 9</a>, as are the outer hair cells (B,F,J). Scale in C applies to all micrographs, which are from the 22 kHz region.</p

The relation between the degree of de-efferentation in the inner hair cell area and loss of afferent synapses (A) or ABR Wave 1 amplitude (B) is similar whether the de-efferentation is caused by OC lesion or by chronic conductive hearing loss.

<p>Data include all frequency regions from 8 to 45 kHz. Data are normalized to the mean value for age-matched controls at the appropriate frequency region. Wave 1 amplitudes (B) are extracted for stimulus levels from 60–80 dB SPL, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g003" target="_blank">Fig 3</a>. Data are not shown for <i>TMx ipsi</i> and <i>OM ipsi</i> groups since conductive hearing loss, <i>per se</i> profoundly decreases ABR amplitudes. Groups and group sizes are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g002" target="_blank">Fig 2</a>. Key in A also applies to B.</p