Experience-dependent flexibility in a molecularly diverse central-to-peripheral auditory feedback system.

Brainstem olivocochlear neurons (OCNs) modulate the earliest stages of auditory processing through feedback projections to the cochlea and have been shown to influence hearing and protect the ear from sound-induced damage. Here, we used single-nucleus sequencing, anatomical reconstructions, and electrophysiology to characterize murine OCNs during postnatal development, in mature animals, and after sound exposure. We identified markers for known medial (MOC) and lateral (LOC) OCN subtypes, and show that they express distinct cohorts of physiologically relevant genes that change over development. In addition, we discovered a neuropeptide-enriched LOC subtype that produces Neuropeptide Y along with other neurotransmitters. Throughout the cochlea, both LOC subtypes extend arborizations over wide frequency domains. Moreover, LOC neuropeptide expression is strongly upregulated days after acoustic trauma, potentially providing a sustained protective signal to the cochlea. OCNs are therefore poised to have diffuse, dynamic effects on early auditory processing over timescales ranging from milliseconds to days.

Just as our pupils dilate or shrink depending on the amount of light available to our eyes, our ears adjust their sensitivity based on the sound environment we encounter. Evidence suggests that a group of cells known as olivocochlear neurons (OCNs for short) may be involved in this process. These cells are located in the brainstem but project into the cochlea, the inner ear structure that converts sound waves into the electrical impulses relayed to the brain. OCNs may mediate how sounds are detected and encoded "at the source." Historically, OCNs have been divided into two groups (medial or lateral OCNs) based on different morphologies and roles in hearing. For instance, medial OCNs are thought to protect our ears against loud sounds by sending molecular signals to the inner ear cells that amplify certain auditory signals. However, it remains difficult to disentangle the precise function of the different types of OCNs, in part because scientists still lack markers that would allow them to distinguish between medial and lateral cells simply based on genetic activity. Frank et al. aimed to eliminate this bottleneck by identifying which genes were switched on and to what degree in individual mouse medial and lateral OCNs; this was done throughout development and after exposure to loud noises. The experiments uncovered a range of genetic markers for medial and lateral OCNs, showing that these cells switch on different sets of genes relevant to their role over development. This gene expression data also revealed that two distinct groups of lateral OCNs exist, one of which is characterised by the production of large amounts of neuropeptides, a type of chemical messenger that can modulate neural circuit activity. Further work in both developing and adult mice showed that this production is shaped by the activity of the cells, with the neuropeptide levels increasing when the animals are exposed to damaging levels of noise. This change lasts for several days, suggesting that such an experience can have long-lasting effects on how the brain provides feedback to the ear. Overall, the results by Frank et al. will help to better identify and characterize the different types of OCNs and the role that they have in hearing. By uncovering the chemical messengers that mediate the response to loud noises, this research may contribute to a better understanding of how to prevent or reduce hearing loss.

Noise Masking in Cochlear Synaptopathy: Auditory Brainstem Response vs. Auditory Nerve Response in Mouse.

After acoustic overexposure, many auditory-nerve fiber (ANF) synapses permanently retract from surviving cochlear hair cells. This synaptopathy is hard to diagnose, since it does not elevate audiometric thresholds until almost no synapses remain, nevertheless it may degrade discrimination of complex stimuli especially in noisy environments. Here, we study an assay based on masking the auditory brainstem responses (ABRs) to a moderate-level probe tone with continuous noise of varied sound levels, and we investigate the underlying ANF responses at the single-fiber level. Synaptopathy was induced by overexposure to octave-band noise, resulting in a permanent synaptic loss of ~50%, without permanent threshold elevation except at the highest frequencies. The normal progressive delay of ABR peaks with increasing masker level is diminished in synaptopathic ears; however, the single-fiber analysis suggests that this normal latency shift does not arise because contributing ANFs shift from low-threshold fibers (with high spontaneous rates) to high-threshold fibers (with low spontaneous rates). Rather, it may arise because of a shift in the cochlear region dominating the response. Surprisingly, the dynamic range of masking, i.e. the difference between the lowest masker level that attenuates the ABR to a fixed-level probe and the lowest masker level that eliminates the ABR, is enhanced in the synaptopathic ears. This ABR behavior mirrors the single-fiber data showing a paradoxical enhancement of onset-response synchrony and resistance to masking in responses of ANFs in the synaptopathic regions. An assay based on the dynamic range of masking could be useful in diagnosing synaptic damage in human populations.

Auditory-nerve responses in mice with noise-induced cochlear synaptopathy.

Cochlear synaptopathy is the noise-induced or age-related loss of ribbon synapses between inner hair cells (IHCs) and auditory-nerve fibers (ANFs), first reported in CBA/CaJ mice. Recordings from single ANFs in anesthetized, noise-exposed guinea pigs suggested that neurons with low spontaneous rates (SRs) and high thresholds are more vulnerable than low-threshold, high-SR fibers. However, there is extensive postexposure regeneration of ANFs in guinea pigs but not in mice. Here, we exposed CBA/CaJ mice to octave-band noise and recorded sound-evoked and spontaneous activity from single ANFs at least 2 wk later. Confocal analysis of cochleae immunostained for pre- and postsynaptic markers confirmed the expected loss of 40%-50% of ANF synapses in the basal half of the cochlea; however, our data were not consistent with a selective loss of low-SR fibers. Rather they suggested a loss of both SR groups in synaptopathic regions. Single-fiber thresholds and frequency tuning recovered to pre-exposure levels; however, response to tone bursts showed increased peak and steady-state firing rates, as well as decreased jitter in first-spike latencies. This apparent gain-of-function increased the robustness of tone-burst responses in the presence of continuous masking noise. This study suggests that the nature of noise-induced synaptic damage varies between different species and that, in mouse, the noise-induced hyperexcitability seen in central auditory circuits is also observed at the level of the auditory nerve.NEW & NOTEWORTHY Noise-induced damage to synapses between inner hair cells and auditory-nerve fibers (ANFs) can occur without permanent hair cell damage, resulting in pathophysiology that "hides" behind normal thresholds. Prior single-fiber neurophysiology in guinea pig suggested that noise selectively targets high-threshold ANFs. Here, we show that the lingering pathophysiology differs in mouse, with both ANF groups affected and a paradoxical gain-of-function in surviving low-threshold fibers, including increased onset rate, decreased onset jitter, and reduced maskability.

Projections from the ventral nucleus of the lateral lemniscus to the cochlea in the mouse.

Auditory efferents originate in the central auditory system and project to the cochlea. Although the specific anatomy of the olivocochlear (OC) efferents can vary between species, two types of auditory efferents have been identified based upon the general location of their cell bodies and their distinctly different axon terminations in the organ of Corti. In the mouse, the relatively small somata of the lateral (LOC) efferents reside in the lateral superior olive (LSO), have unmyelinated axons, and terminate around ipsilateral inner hair cells (IHCs), primarily against the afferent processes of type I auditory nerve fibers. In contrast, the larger somata of the medial (MOC) efferents are distributed in the ventral nucleus of the trapezoid body (VNTB), have myelinated axons, and terminate bilaterally against the base of multiple outer hair cells (OHCs). Using in vivo retrograde cell body marking, anterograde axon tracing, immunohistochemistry, and electron microscopy, we have identified a group of efferent neurons in mouse, whose cell bodies reside in the ventral nucleus of the lateral lemniscus (VNLL). By virtue of their location, we call them dorsal efferent (DE) neurons. Labeled DE cells were immuno-negative for tyrosine hydroxylase, glycine, and GABA, but immuno-positive for choline acetyltransferase. Morphologically, DEs resembled LOC efferents by their small somata, unmyelinated axons, and ipsilateral projection to IHCs. These three classes of efferent neurons all project axons directly to the cochlea and exhibit cholinergic staining characteristics. The challenge is to discover the contributions of this new population of neurons to auditory efferent function.

A simple algorithm for objective threshold determination of auditory brainstem responses.

The auditory brainstem response (ABR) is a sound-evoked neural response commonly used to assess auditory function in humans and laboratory animals. ABR thresholds are typically chosen by visual inspection, leaving the procedure susceptible to user bias. We sought to develop an algorithm to automate determination of ABR thresholds to eliminate such biases and to standardize approaches across investigators and laboratories. Two datasets of mouse ABR waveforms obtained from previously published studies of normal ears as well as ears with varying degrees of cochlear-based threshold elevations (Maison et al., 2013; Sergeyenko et al., 2013) were reanalyzed using an algorithm based on normalized cross-covariation of adjacent level presentations. Correlation-coefficient vs. level data for each ABR level series were fit with both a sigmoidal and two-term power function. From these fits, threshold was interpolated at different criterion values of correlation-coefficient ranging from 0 to 0.5. The criterion value of 0.35 was selected by comparing visual thresholds to computed thresholds across all frequencies tested. With such a criterion, the mean algorithm-computed thresholds were comparable to the visual thresholds noted by two independent observers for each data set. The success of the algorithm was also qualitatively assessed by comparing averaged waveforms at the thresholds determined by the two methods, and quantitatively assessed by comparing peak 1 amplitude growth functions expressed as dB re each of the two threshold measures. Application of a cross-covariance analysis to ABR waveforms can emulate visual thresholding decisions made by highly trained observers. Unlike previous applications of similar methodologies using template matching, our algorithm performs only intrinsic comparisons within ABR sets, and therefore is more robust to equipment and investigator differences in assessing waveforms, as evidenced by similar results across the two datasets.

Descending projections from the inferior colliculus to medial olivocochlear efferents: Mice with normal hearing, early onset hearing loss, and congenital deafness.

Auditory efferent neurons reside in the brain and innervate the sensory hair cells of the cochlea to modulate incoming acoustic signals. Two groups of efferents have been described in mouse and this report will focus on the medial olivocochlear (MOC) system. Electrophysiological data suggest the MOC efferents function in selective listening by differentially attenuating auditory nerve fiber activity in quiet and noisy conditions. Because speech understanding in noise is impaired in age-related hearing loss, we asked whether pathologic changes in input to MOC neurons from higher centers could be involved. The present study investigated the anatomical nature of descending projections from the inferior colliculus (IC) to MOCs in 3-month old mice with normal hearing, and in 6-month old mice with normal hearing (CBA/CaH), early onset progressive hearing loss (DBA/2), and congenital deafness (homozygous Shaker-2). Anterograde tracers were injected into the IC and retrograde tracers into the cochlea. Electron microscopic analysis of double-labelled tissue confirmed direct synaptic contact from the IC onto MOCs in all cohorts. These labelled terminals are indicative of excitatory neurotransmission because they contain round synaptic vesicles, exhibit asymmetric membrane specializations, and are co-labelled with antibodies against VGlut2, a glutamate transporter. 3D reconstructions of the terminal fields indicate that in normal hearing mice, descending projections from the IC are arranged tonotopically with low frequencies projecting laterally and progressively higher frequencies projecting more medially. Along the mediolateral axis, the projections of DBA/2 mice with acquired high frequency hearing loss were shifted medially towards expected higher frequency projecting regions. Shaker-2 mice with congenital deafness had a much broader spatial projection, revealing abnormalities in the topography of connections. These data suggest that loss in precision of IC directed MOC activation could contribute to impaired signal detection in noise.