Auditory Research publications

In many excitatory synapses, mobile zinc is found within glutamatergic vesicles and is coreleased with glutamate. Ex vivo studies established that synaptically released (synaptic) zinc inhibits excitatory neurotransmission at lower frequencies of synaptic activity but enhances steady state synaptic responses during higher frequencies of activity. However, it remains unknown how synaptic zinc affects neuronal processing in vivo. Here, we imaged the sound-evoked neuronal activity of the primary auditory cortex in awake mice. We discovered that synaptic zinc enhanced the gain of sound-evoked responses in CaMKII-expressing principal neurons, but it reduced the gain of parvalbumin- and somatostatin-expressing interneurons. This modulation was sound intensity-dependent and, in part, NMDA receptor-independent. By establishing a previously unknown link between synaptic zinc and gain control of auditory cortical processing, our findings advance understanding about cortical synaptic mechanisms and create a new framework for approaching and interpreting the role of the auditory cortex in sound processing.

Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.

Cannabinoid receptors (CBRs) are widely distributed in the brain, including the inferior colliculus (IC). Here, we aim to study whether endocannabinoids influence a specific type of neuronal adaptation, namely, stimulus-specific adaptation (SSA) found in some IC neurons. SSA is important because it has been found as early as the level of the midbrain and therefore it may be a neuronal correlate of early indices of deviance detection. Furthermore, recent studies have demonstrated a direct link between SSA and MMN, that is widely used as an outcome measure in a variety of human neurodegenerative disorders. SSA is considered a form of short-term plasticity, and CBRs have been shown to play a role in short-term neural plasticity. Therefore, it is reasonable to hypothesize that endocannabinoids may play a role in the generation or modulation of SSA. We recorded single units in the IC under an oddball paradigm stimulation. The results demonstrate that cannabinoid agonists lead to a reduction in the neuronal adaptation. This change is due to a differential increase of the neuronal firing rate to the standard tone alone. Furthermore, we show that the effect is mediated by the cannabinoid receptor 1 (CBR1). Thus, cannabinoid agonists down-modulate SSA in IC neurons.

Sound stimuli are characterized by their rich spectral and temporal dynamic properties. Individual neurons in auditory cortex (ACX) encode both spectral and temporal aspects of sounds e.g. sound onset and/or offset. While the different fields of the ACX show gradients of frequency selectivity the large-scale organization of sound dynamics is unknown. We used widefield imaging of GCaMP6s in awake mouse ACX combined with a novel unsupervised image segmentation technique to investigate the spatiotemporal representation of sound onset and offset. Using this technique, we identified known auditory fields but also detected novel ACX areas. Furthermore, we found that ACX areas differed in their responses to tone onset and offset. Multiple areas were preferentially activated by tone offset, and on-response areas were more spatially localized than off-response areas. We also found tonotopy in off-responses. Together our results demonstrate a different spatial distribution of neurons across ACX for processing sound onsets versus offsets.

Hair cells of the cochlea are mechanosensors for the perception of sound. Mutations in the LRTOMT gene, which encodes a protein with homology to the catecholamine methyltransferase COMT that is linked to schizophrenia, cause deafness. Here, we show that Tomt/Comt2, the murine ortholog of LRTOMT, has an unexpected function in the regulation of mechanotransduction by hair cells. The role of mTOMT in hair cells is independent of mTOMT methyltransferase function and mCOMT cannot substitute for mTOMT function. Instead, mTOMT binds to putative components of the mechanotransduction channel in hair cells and is essential for the transport of some of these components into the mechanically sensitive stereocilia of hair cells. Our studies thus suggest functional diversification between mCOMT and mTOMT, where mTOMT is critical for the assembly of the mechanotransduction machinery of hair cells. Defects in this process are likely mechanistically linked to deafness caused by mutations in LRTOMT/Tomt.

Nicotine enhances sensory and cognitive processing via actions at nicotinic acetylcholine receptors (nAChRs), yet the precise circuit- and systems-level mechanisms remain unclear. In sensory cortex, nicotinic modulation of receptive fields (RFs) provides a model to probe mechanisms by which nAChRs regulate cortical circuits. Here, we examine RF modulation in mouse primary auditory cortex (A1) using a novel electrophysiological approach: current-source density (CSD) analysis of responses to tone-in-notched-noise (TINN) acoustic stimuli. TINN stimuli consist of a tone at the characteristic frequency (CF) of the recording site embedded within a white noise stimulus filtered to create a spectral “notch” of variable width centered on CF. Systemic nicotine (2.1 mg/kg) enhanced responses to the CF tone and to narrow-notch stimuli, yet reduced the response to wider-notch stimuli, indicating increased response gain within a narrowed RF. Subsequent manipulations showed that modulation of cortical RFs by systemic nicotine reflected effects at several levels in the auditory pathway: nicotine suppressed responses in the auditory midbrain and thalamus, with suppression increasing with spectral distance from CF so that RFs became narrower, and facilitated responses in the thalamocortical pathway, while nicotinic actions within A1 further contributed to both suppression and facilitation. Thus, multiple effects of systemic nicotine integrate along the ascending auditory pathway. These actions at nAChRs in cortical and subcortical circuits, which mimic effects of auditory attention, likely contribute to nicotinic enhancement of sensory and cognitive processing.

Cortical responses to repeated stimuli are highly dynamic and rapidly adaptive. Such rapid changes are prominent in all sensory cortices, across which many aspects of circuitry are conserved. As an example, in the auditory cortex, preceding sounds can powerfully suppress responses to later, spectrally similar sounds – a phenomenon called forward suppression. Whether cortical inhibitory networks shape such suppression, or whether it is wholly regulated by common mechanisms such as synaptic depression or spike-frequency adaptation, is controversial. Here, we show that optogenetically suppressing somatostatin-positive interneurons reveals facilitation in neurons that are normally forward-suppressed. This is accompanied by a weakening of forward suppression, suggesting that these interneurons regulate the strength of forward interactions. In contrast, inactivating parvalbumin-positive interneurons does not change suppression strength, but does alter its frequency-dependence. These results establish a role of cortical inhibition in forward suppression and link specific aspects of rapid sensory adaptation to genetically distinct interneuron types.

Hair cells in the mature cochlea cannot spontaneously regenerate. One potential approach for restoring hair cells is stem cell therapy. However, when cells are transplanted into scala media (SM) of the cochlea, they promptly die due to the high potassium concentration. We previously described a method for conditioning the SM to make it more hospitable to implanted cells and showed that HeLa cells could survive for up to a week using this method. Here, we evaluated the survival of human embryonic stem cells (hESC) constitutively expressing GFP (H9 Cre-LoxP) in deaf guinea pig cochleae that were pre-conditioned to reduce potassium levels. GFP-positive cells could be detected in the cochlea for at least 7 days after the injection. The cells appeared spherical or irregularly shaped, and some were aggregated. Flushing SM with sodium caprate prior to transplantation resulted in a lower proportion of stem cells expressing the pluripotency marker Oct3/4 and increased cell survival. The data demonstrate that conditioning procedures aimed at transiently reducing the concentration of potassium in the SM facilitate survival of hESCs for at least one week. During this time window, additional procedures can be applied to initiate the differentiation of the implanted hESCs into new hair cells.

Repeated noise exposure induces inflammation and cellular adaptations in the peripheral and central auditory system resulting in pathophysiology of hearing loss. In this study, we analyzed the mechanisms by which noise-induced inflammatory-related events in the cochlea activate glial-mediated cellular responses in the cochlear nucleus (CN), the first relay station of the auditory pathway. The auditory function, glial activation, modifications in gene expression and protein levels of inflammatory mediators and ultrastructural changes in glial-neuronal interactions were assessed in rats exposed to broadband noise (0.5-32 kHz, 118 dB SPL) for 4 h/day during 4 consecutive days to induce long-lasting hearing damage. Noise-exposed rats developed a permanent threshold shift which was associated with hair cell loss and reactive glia. Noise-induced microglial activation peaked in the cochlea between 1 and 10D post-lesion; their activation in the CN was more prolonged reaching maximum levels at 30D post-exposure. RT-PCR analyses of inflammatory-related genes expression in the cochlea demonstrated significant increases in the mRNA expression levels of pro- and anti-inflammatory cytokines, inducible nitric oxide synthase, intercellular adhesion molecule and tissue inhibitor of metalloproteinase-1 at 1 and 10D post-exposure. In noise-exposed cochleae, interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) were upregulated by reactive microglia, fibrocytes, and neurons at all time points examined. In the CN, however, neurons were the sole source of these cytokines. These observations suggest that noise exposure causes peripheral and central inflammatory reactions in which TNF-α and IL-1β are implicated in regulating the initiation and progression of noise-induced hearing loss.

Acoustic environments are composed of complex overlapping sounds that the auditory system is required to segregate into discrete perceptual objects. The functions of distinct auditory processing stations in this challenging task are poorly understood. Here we show a direct role for mouse auditory cortex in detection and segregation of acoustic information. We measured the sensitivity of auditory cortical neurons to brief tones embedded in masking noise. By altering spectrotemporal characteristics of the masker, we reveal that sensitivity to pure tone stimuli is strongly enhanced in coherently modulated broadband noise, corresponding to the psychoacoustic phenomenon comodulation masking release. Improvements in detection were largest following priming periods of noise alone, indicating that cortical segregation is enhanced over time. Transient opsin-mediated silencing of auditory cortex during the priming period almost completely abolished these improvements, suggesting that cortical processing may play a direct and significant role in detection of quiet sounds in noisy environments. SIGNIFICANCE STATEMENT Auditory systems are adept at detecting and segregating competing sound sources, but there is little direct evidence of how this process occurs in the mammalian auditory pathway. We demonstrate that coherent broadband noise enhances signal representation in auditory cortex, and that prolonged exposure to noise is necessary to produce this enhancement. Using optogenetic perturbation to selectively silence auditory cortex during early noise processing, we show that cortical processing plays a crucial role in the segregation of competing sounds.

Wide-field optical imaging techniques constitute powerful tools to sample and study mesoscale neuronal activity. The sampled data constitutes a sequence of image frames in which one can perceive the flow of brain activity starting and terminating at source and sink locations respectively. The most common data analyses include qualitative assessment to identify sources and sinks of activity as well as their trajectories. The quantitative analyses is mostly based on computing the temporal variation of the intensity of pixels while a few studies have also reported estimates of wave motion using optical-flow techniques from computer vision. A comprehensive toolbox for the quantitative analyses of mesoscale brain activity data however is still missing. We present a graphical-user-interface based Matlab ® toolbox for investigating the spatiotemporal dynamics of mesoscale brain activity using optical-flow analyses. The toolbox includes the implementation of three optical-flow methods namely Horn-Schunck, Combined Local-Global, and Temporospatial algorithms for estimating velocity vector fields of perceived flow in mesoscale brain activity. From the velocity vector fields we determine the locations of sources and sinks as well as the trajectories and temporal velocities of activity flow. Using our toolbox, we compare the efficacy of the three optical-flow methods for determining spatiotemporal dynamics by using simulated data. We also demonstrate the application of optical-flow methods onto sensory-evoked calcium and voltage imaging data. Our results indicate that the combined local-global method we employ, yields results that correlate with the manual assessment. The automated approach permits rapid and effective quantification of mesoscale brain dynamics and may facilitate the study of brain function in response to new experiences or pathology. Conflicts of Interest none Author contribution statement MHM, MM, NV, and SI designed the study. NA and SI wrote Matlab ® code for the toolbox and designed the simulated data. MHM, and NA performed the experiments. NA and SI analyzed the data. SI, NA, and MHM wrote the manuscript.

Numerous behavioral paradigms have been developed to assess tinnitus-like behavior in animals. Nevertheless, they are often limited by prolonged training requirements, as well as an inability to simultaneously assess onset and lasting tinnitus behavior, tinnitus pitch or duration, or tinnitus presence without grouping data from multiple animals or testing sessions. To enhance behavioral testing of tinnitus, we developed a conditioned licking suppression paradigm to determine the pitch(s) of both onset and lasting tinnitus-like behavior within individual animals. Rats learned to lick water during broadband or narrowband noises, and to suppress licking to avoid footshocks during silence. After noise exposure, rats significantly increased licking during silent trials, suggesting onset tinnitus-like behavior. Lasting tinnitus-behavior, however, was exhibited in about half of noise-exposed rats through 7 weeks post-exposure tested. Licking activity during narrowband sound trials remained unchanged following noise exposure, while ABR hearing thresholds fully recovered and were comparable between tinnitus(+) and tinnitus(-) rats. To assess another tinnitus inducer, rats were injected with sodium salicylate. They demonstrated high pitch tinnitus-like behavior, but later recovered by 5 days post-injection. Further control studies showed that 1): sham noise-exposed rats tested with footshock did not exhibit tinnitus-like behavior, and 2): noise-exposed or sham rats tested without footshocks showed no fundamental changes in behavior compared to those tested with shocks. Together, these results demonstrate that this paradigm can efficiently test the development of noise- and salicylate-induced tinnitus behavior. The ability to assess tinnitus individually, over time, and without averaging data enables us to realistically address tinnitus in a clinically relevant way. Thus, we believe that this optimized behavioral paradigm will facilitate investigations into the mechanisms of tinnitus and development of effective treatments.

Bidirectional manipulations – activation and inactivation – are widely used to identify the functions supported by specific cortical interneuron types. Implicit in much of this work is the notion that tonic activation and inactivation will both produce valid, internally consistent insights into interneurons’ computational roles. Here, using single-unit recordings in auditory cortex of awake mice, we show that this may not generally hold true. Optogenetically manipulating somatostatin-positive (Sst+) or parvalbumin-positive (Pvalb+) interneurons while recording tone-responses showed that Sst+ inactivation increased response gain, while Pvalb+ inactivation weakened tuning and decreased information transfer, implying that these neurons support delineable computational functions. But activating Sst+ and Pvalb+ interneurons revealed no such differences. We used a simple network model to understand this asymmetry, and showed how relatively small changes in key parameters, such as spontaneous activity or strength of the light manipulation, determined whether activation and inactivation would produce consistent or paradoxical conclusions regarding interneurons’ computational functions.

Age-related hearing loss (ARHL) -presbycusis - is the most prevalent neurodegenerative disease and number one communication disorder of our aged population; and affects hundreds of millions of people worldwide. Its prevalence is close to that of cardiovascular disease and arthritis, and can be a precursor to dementia. The auditory perceptual dysfunction is well understood, but knowledge of the biological bases of ARHL is still somewhat lacking. Surprisingly, there are no FDA-approved drugs for treatment. Based on our previous studies of human subjects, where we discovered relations between serum aldosterone levels and the severity of ARHL, we treated middle age mice with aldosterone, which normally declines with age in all mammals. We found that hearing thresholds and suprathreshold responses significantly improved in the aldosterone-treated mice compared to the non-treatment group. In terms of cellular and molecular mechanisms underlying this therapeutic effect, additional experiments revealed that spiral ganglion cell survival was significantly improved, mineralocorticoid receptors were upregulated via post-translational protein modifications, and age-related intrinsic and extrinsic apoptotic pathways were blocked by the aldosterone therapy. Taken together, these novel findings pave the way for translational drug development towards the first medication to prevent the progression of ARHL.

Gap detection or gap pre-pulse inhibition of the acoustic startle (GPIAS) has been successfully used in rat and guinea pig models of tinnitus, yet this system has been proven to have low efficacy in CBA mice, with low basal GPIAS and subtle tinnitus-like effects. Here, we tested five mouse strains (CBA, BalbC, CD-1, C57BL/6 and 129sv) for pre-pulse inhibition (PPI) and gap detection with varying interstimulus intervals (ISI) and found that mice from a CBA genetic background had the poorest capacities of suppressing the startle response in the presence of a pre-pulse or a gap. CD-1 mice displayed variable responses throughout all ISI. Interestingly, C57BL/6, 129sv and BalbC showed efficient suppression with either pre-pulses or gaps with shorter ISI. The glutamate aspartate transporter (GLAST) is expressed in support cells from the cochlea and buffers the excess of glutamate. We hypothesized that loss of GLAST function could sensitize the ear to tinnitus-inducing agents, such as salicylate. Using shorter ISI to obtain a greater dynamic range to assess tinnitus-like effects, we found that disruption of gap detection by salicylate was exacerbated across various intensities of a 32-kHz narrow band noise gap carrier in GLAST knockout (KO) mice when compared to their wild-type (WT) littermates. Auditory brainstem responses (ABR) and distortion-product otoacoustic emission (DPOAE) were performed to evaluate the effects on hearing functions. Salicylate caused greater auditory threshold shifts (near 15 dB) in GLAST KO mice than in WT mice across all tested frequencies, despite similarly reduced DPOAE. Despite these changes, inhibition using broad-band gap carriers and 32 kHz pre-pulses were not affected. Our study suggests that GLAST deficiency could become a useful experimental model to decipher the mechanisms underlying drug-induced tinnitus. Future studies addressing the neurological correlates of tinnitus in this model could provide additional insights into the mechanisms of tinnitus.

In adult animals, the neuropeptide calcitonin gene‐related peptide ( CGRP ) is contained in cochlear efferent fibers projecting out to the cochlea, and contributes to increased suprathreshold sound‐evoked activity in the adult auditory nerve. Similarly, CGRP applied to the lateral‐line organ (hair cell organ) increases afferent nerve activity in adult frogs (post‐metamorphic day 30), yet this increase is developmentally delayed from post‐metamorphic day 4–30. In this study, we discovered that there was also a developmental delay in increased suprathreshold sound‐evoked activity auditory nerve between juvenile and adult mice similar to what had been observed previously in frog. Moreover, juvenile mice with a targeted deletion of the α CGRP gene [ CGRP null (−/−)] did not show a similar developmental increase in nerve activity, suggesting CGRP signaling is involved. This developmental delay is not due to a delay in CGRP expression, but instead is due to a delay in receptor formation. We observed that the increase in sound‐evoked nerve activity is correlated with increased formation of cochlear CGRP receptors, which require three complexed proteins ( CLR , RAMP 1, RCP ) to be functional. CGRP receptor formation in the cochlea was incomplete at 1 month of age (juvenile), but complete by 3 months (adult), which corresponded to the onset of suprathreshold enhancement of sound‐evoked activity in wild‐type animals. Taken together, these data support a model for cochlear function that is enhanced by maturation of CGRP receptor complexes.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this "toughening" effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with "toughening" and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol.

Different forms of plasticity are known to play a critical role in the processing of information about sound. Here, we report a novel neural plastic response in the inferior colliculus, an auditory center in the midbrain of the auditory pathway. A vigorous, long-lasting sound-evoked afterdischarge (LSA) is seen in a subpopulation of both glutamatergic and GABAergic neurons in the central nucleus of the inferior colliculus of normal hearing mice. These neurons were identified with single unit recordings and optogenetics in vivo . The LSA can continue for up to several minutes after the offset of the sound. LSA is induced by long-lasting, or repetitive short-duration, innocuous sounds. Neurons with LSA showed less adaptation than the neurons without LSA. The mechanisms that cause this neural behavior are unknown but may be a function of intrinsic mechanisms or the microcircuitry of the inferior colliculus. Since LSA produces long-lasting firing in the absence of sound, it may be relevant to temporary or chronic tinnitus or to some other aftereffect of long-duration sound.

WBP2 encodes the WW domain-binding protein 2 that acts as a transcriptional coactivator for estrogen receptor α (ESR1) and progesterone receptor (PGR). We reported that the loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene. The earliest abnormality we detect in Wbp2-deficient mice is a primary defect at inner hair cell afferent synapses. This study defines a new gene involved in the molecular pathway linking hearing impairment to hormonal signalling and provides new therapeutic targets.