Auditory Research publications

The investigation of neuronal activity in non-human primate models is of critical importance due to their genetic similarity to human brains. In this study, we tested the feasibility of using photoacoustic imaging for the detection of cortical and subcortical responses due to peripheral electrical stimulation in a squirrel monkey model. Photoacoustic computed tomography and photoacoustic microscopy were applied on squirrel monkeys for real-time deep subcortical imaging and optical-resolution cortical imaging, respectively. The electrically evoked hemodynamic changes in primary somatosensory cortex, premotor cortices, primary motor cortex, and underlying subcortical areas were measured. Hemodynamic responses were observed in both cortical and subcortical brain areas at the cortices during external stimulation, demonstrating the feasibility of photoacoustic technique for functional imaging of non-human primate brain.

Cisplatin-related ototoxicity is a critical side effect of chemotherapy and can lead to irreversible hearing loss. This study aimed to assess the potential effect of the DNA methyltransferase (DNMT) inhibitor RG108 on cisplatin-induced ototoxicity. Immunohistochemistry, apoptosis assay, and auditory brainstem response (ABR) were employed to determine the impacts of RG108 on cisplatin-induced injury in murine hair cells (HCs) and spiral ganglion neurons (SGNs). Rhodamine 123 and TMRM were utilized for mitochondrial membrane potential (MMP) assessment. Reactive oxygen species (ROS) amounts were evaluated by Cellrox green and Mitosox-red probes. Mitochondrial respiratory function evaluation was performed by determining oxygen consumption rates (OCRs). The results showed that RG108 can markedly reduce cisplatin induced damage in HCs and SGNs, and alleviate apoptotic rate by protecting mitochondrial function through preventing ROS accumulation. Furthermore, RG108 upregulated BCL-2 and downregulated APAF1, BAX, and BAD in HEI-OC1 cells, and triggered the PI3K/AKT pathway. Decreased expression of low-density lipoprotein receptor-related protein 1 (LRP1) and high methylation of the LRP1 promoter were observed after cisplatin treatment. RG108 treatment can increase LRP1 expression and decrease LRP1 promoter methylation. In conclusion, RG108 might represent a new potential agent for preventing hearing loss induced by cisplatin via activating the LRP1-PI3K/AKT pathway.

Background The study investigated the cause of varying estimates of frequency difference limens (DLs) in delayed comparison tasks involving pitch retention in auditory working memory (AWM). Using procedures adapted from the method of constant stimuli (MCS) and the single-interval adjustment matrix (SIAM), we sought to determine via 3 experiments whether the disparity in frequency DLs obtained using each procedure was due to the method of measurement (Experiment 1), the response format (Experiment 2), or performance feedback (Experiment 3). Material and methods Five adults (ages 21 to 38 years) with hearing within normal limits participated in Experiments 1 and 2, and seven adults (ages 20 to 30 years) with hearing within normal limits participated in Experiment 3. Delayed comparison tasks were used to evaluate frequency DLs under SIAM and MCS. Results Our preliminary results suggest that DL values for pitch discrimination are more influenced by response format than by the measurement procedure or performance feedback. Regardless of the method used, DL values were greater in the condition containing intervening stimuli compared to the condition lacking intervening stimuli. Conclusions Preliminary findings suggest there is consistency in the listener’s adopted criterion (i.e., judgment rationale) across the psychoacoustic methods investigated. Performance measures suggest that SIAM is as accurate as MCS, but it is noteworthy that two SIAM measurement runs using the "same/different" response format is more efficient than four runs with MCS. Future application of the SIAM procedure for measuring DL values might, with larger sample sizes, identify additional factors that contribute to performance and the listener’s adopted criterion, since data collection time is appreciably shorter with SIAM.

Cisplatin-induced ototoxicity can be partially attributed to excessive reactive oxygen species (ROS) production, and agmatine is well-known for the activation of the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway to inhibit ROS production. Whether agmatine could be used to alleviate cisplatin-induced ototoxicity is investigated. Cisplatin-exposed House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants showed increased ROS production detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining and decreased cell viability detected by Cell Counting Kit-8 (CCK-8) or Myosin 7a staining, which could be reversed by the agmatine pretreatment. Cisplatin intraperitoneally injected C57BL/6 mice demonstrated damaged auditory function as indicated by distortion products otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) assays, and trans-tympanically administrated agmatine in the left ears could partly prevent the auditory function loss. Mechanistically, downregulated B-cell lymphoma 2 (Bcl-2) expression, upregulated Bcl2-associated x (Bax) expression, and diminished p-PI3K and p-AKT expression were detected in cisplatin-exposed HEI-OC1 cells and cochlear explants, which could be prevented by the pretreatment with agmatine. Our investigation demonstrates that agmatine pretreatment could alleviate cisplatin-induced ototoxicity with the activation of PI3K/AKT signaling pathway.

Background The transcription factor Sox2 plays important roles in the developmental processes of multiple organs and tissues. However, whether Sox2 can protect mature or terminally differentiated cells against injury is still unknown. Methods We investigated the roles of Sox2 in cochlear hair cells, which are terminally differentiated cells, using conditional transgenic mice and several hearing loss models. Results Sox2 overexpression dramatically mitigated the degree of cochlear hair cell loss when exposed to ototoxic drugs. Noise-induced apoptosis of cochlear hair cells and hearing loss were also significantly alleviated by Sox2 overexpression. Notably, noise-induced upregulation of pro-inflammatory factors such as TNF-α and IL6 was inhibited by Sox2 overexpression. Then we used lipopolysaccharide to clarify the effect of Sox2 on cochlear inflammation, and Sox2 overexpression significantly inhibited lipopolysaccharide-induced upregulation of pro-inflammatory factors and alleviated inflammation-related cochlear hair cell death. Conclusions These results demonstrate a novel protective role of Sox2 in mature and terminally differentiated cochlear hair cells by inhibiting inflammation.

Tinnitus is often triggered by cochlear damage and has been linked with aberrant patterns of neuronal activity. Acoustic Coordinated Reset (CR®) Neuromodulation is a sound therapy hypothesised to reduce tinnitus symptoms by desynchronising pathological brain activity using a portable acoustic device (the T30 neurostimulator). We report results of a pivotal trial to test the efficacy of this intervention. This two-centre, double-blind randomised controlled trial with long-term open-label extension, was undertaken between February 2012 and February 2014 in the UK. Participants were 100 adults with tinnitus as a primary complaint, recruited through hearing clinics and media advertisements. Intervention was the device programmed either with the proprietary sound sequence or placebo algorithm, fit by one of five trained audiologists. Minimisation software provided group allocation (1:1 randomisation), with groups matched for age, gender, hearing loss and tinnitus severity. Allocation was masked from participants and assessors during the trial. The primary measure of efficacy was change in tinnitus symptom severity between groups, measured using the Tinnitus Handicap Questionnaire at 12 weeks. Secondary outcomes were other measures of tinnitus symptom severity, health-related quality of life, and perceptual characteristics (pitch, loudness, bandwidth) at 12 weeks, and Tinnitus Handicap Questionnaire at 36 weeks (open-label extension). A statistician blinded to the allocation conducted an intention-to-treat analysis that employed linear regressions on minimisation variables, trial centre and intervention group, with multiple imputations for missing data. The study was registered on clinicaltrials.gov (NCT01541969). We screened 391 individuals and assigned interventions to 100 eligible participants. The primary outcome was not statistically significant between groups (mean group = −0.45, 95% CI −5.25 to 4.35; p = 0.85), nor were any of the secondary outcomes. Four adverse events occurred during the trial. Analysis of tinnitus symptom severity data collected across the 24-week open-label extension showed no statistically significant within-group changes after 12, 24, or 36 weeks treatment with the proprietary sound sequence. While individual participants may benefit from sound therapy, Acoustic CR® Neuromodulation did not lead to group-mean reductions on tinnitus symptom severity or other measures compared to placebo, or over time.

Stimulus-specific adaptation (SSA), defined as a decrease in responses to a common stimulus that only partially generalizes to other rare stimuli, is a widespread phenomenon in the brain that is believed to be related to novelty detection. Although cross-modal sensory processing is also a widespread phenomenon, the interaction between the two phenomena is not well understood. In this study, the thalamic reticular nucleus (TRN), which is regarded as a hub of the attentional system that contains multi-modal neurons, was investigated. The results showed that SSA existed in an interactive oddball stimulation, which mimics stimulation changes from one modality to another. In the bimodal integration, SSA to bimodal stimulation was stronger than to visual stimulation alone but similar to auditory stimulation alone, which indicated a limited integrative effect. Collectively, the present results provide evidence for independent cross-modal processing in bimodal TRN neurons.

The transcription factor c-Myb is vital for cell survival, proliferation, differentiation, and apoptosis. We have previously reported that c-Myb knockdown exacerbates neomycin-induced damage to cochlea cells. However, the function and regulation of c-Myb in the mammalian inner ear remains unclear. Here, we first found that the expression of c-Myb in cochlear HCs was downregulated after cisplatin damage in vivo. Next, to investigate the role of c-Myb in HCs treated with cisplatin, the recombinant virus AAV-ie-CAG-Myb-HA (AAV-c-Myb) that overexpresses c-Myb was constructed and transfected into HCs. The protein expression of c-Myb was effectively up-regulated in cultured cochlear HCs after the virus transfection, which increased cochlear HC viability, decreased HC apoptosis and reduced intracellular reactive oxygen species (ROS) levels after cisplatin injury in vitro. The overexpression of c-Myb in HCs after AAV-c-Myb transfection in vivo also promoted HC survival, improved the hearing function of mice and reduced HC apoptosis after cisplatin injury. Furthermore, c-Myb-HC conditional knockout mice (Prestin; c-Myb-cKO) in which c-Myb expression is downregulated only in cochlear OHCs were generated and the cisplatin-induced HCs loss, apoptosis and hearing deficit were all exacerbated in Prestin; c-Myb-cKO mice treated with cisplatin in vivo. Finally, mechanistic studies showed that upregulation of the PI3K/Akt signaling pathway by c-Myb contributed to the increased HC survival after cisplatin exposure in vitro. The findings from this work suggest that c-Myb might serve as a new target for the prevention of cisplatin-induced HC damage and hearing loss.

The molecular mechanisms that regulate the proliferation and differentiation of inner ear spiral ganglion cells (SGCs) remain largely unknown. Shikonin (a naphthoquinone pigment isolated from the traditional Chinese herbal medicine comfrey root) has anti-oxidation, anti-apoptosis and promoting proliferation and differentiation effects on neural progenitor cells. To study the protective effect of shikonin on auditory nerve damage, we isolated spiral ganglion neuron cells (SGNs) and spiral ganglion Schwann cells (SGSs) that provide nutrients in vitro and pretreated them with shikonin. We found that shikonin can reduce ouabain, a drug that can selectively destroy SGNs and induce auditory nerve damage, caused SGNs proliferation decreased, neurite outgrowth inhibition, cells apoptosis and mitochondrial depolarization. In addition, we found that shikonin can increase the expression of Nrf2 and its downstream molecules HO-1 and NQO1, thereby enhancing the antioxidant capacity of SGNs and SGSs, promoting cells proliferation, and inhibiting cells apoptosis by activating the Nrf2/antioxidant response elements (ARE) signal pathway. However, knockdown of Nrf2 rescued the protective effect of shikonin on SGNs and SGSs damage. In addition, we injected shikonin pretreatment into mouse that ouabain-induced hearing loss and found that shikonin pretreatment has a defensive effect on auditory nerve damage. In summary, the results of this study indicate that shikonin could attenuate the level of oxidative stress in SGNs and SGSs through the Nrf2-ARE signaling pathway activated, induce the proliferation and differentiation of SGNs, and thereby improve the neurological hearing damage in mice. Therefore, shikonin may be a candidate therapeutic drug for endogenous antioxidants that can be used to treat neurological deafness.

Aminoglycosides (AGs) are commonly used antibiotics that cause deafness through the irreversible loss of cochlear sensory hair cells (HCs). How AGs enter the cochlea and then target HCs remains unresolved. Here, we performed time-lapse multicellular imaging of cochlea in live adult hearing mice via a chemo-mechanical cochleostomy. The in vivo tracking revealed that systemically administered Texas Red–labeled gentamicin (GTTR) enters the cochlea via the stria vascularis and then HCs selectively. GTTR uptake into HCs was completely abolished in transmembrane channel-like protein 1 (TMC1) knockout mice, indicating mechanotransducer channel-dependent AG uptake. Blockage of megalin, the candidate AG transporter in the stria vascularis, by binding competitor cilastatin prevented GTTR accumulation in HCs. Furthermore, cilastatin treatment markedly reduced AG-induced HC degeneration and hearing loss in vivo. Together, our in vivo real-time tracking of megalin-dependent AG transport across the blood–labyrinth barrier identifies new therapeutic targets for preventing AG-induced ototoxicity.

Abnormal levels of acoustic activity can result in hearing problems such as tinnitus and language processing disorders, but the underlying cellular and synaptic changes triggered by abnormal activity are not well understood. To address this issue, we studied the time course of activity-dependent changes that occur at auditory nerve synapses in mice of both sexes after noise exposure and conductive hearing loss. We found that EPSC amplitude and synaptic depression decreased within 2 d of noise exposure through a decrease in the probability of vesicle release ( P r ). This was followed by a gradual increase in EPSC amplitude through a larger pool of releasable vesicles ( N ). Occlusion of the ear canal led to a rapid decrease in EPSC amplitude through a decrease in N , which was followed by an increase in EPSC amplitude and synaptic depression through an increase in P r . After returning to normal sound levels, synaptic depression recovered to control levels within 1–2 d. However, repeated exposure to noise for as little as 8 h/d caused synaptic changes after 7 d, suggesting recovery did not fully offset changes. Thus, there appear to be three activity-dependent mechanisms in auditory nerve synapses—bidirectional changes in P r in 1–2 d, slower bidirectional changes in N through synaptic growth or retraction, and rapid downregulation of N with low activity. The dynamic changes indicate that multiple mechanisms are present to fine-tune synaptic fidelity across different acoustic conditions in a simple relay. SIGNIFICANCE STATEMENT Hearing impairments can arise from exposure to noise or conductive hearing loss. This appears to result from changes in the brain, but the mechanisms are not well understood. We study this issue by studying the synapses made by auditory nerve fibers called endbulbs of Held. These synapses undergo bidirectional changes in size and release probability of neurotransmitter in response to increased or decreased activity. Here, we made a close examination of how quickly these synaptic characteristics change, which indicates there are at least three cellular mechanisms underlying changes. Furthermore, repeated exposure to brief periods of noise can produce cumulative effects. These changes could significantly affect hearing, especially because they occur at the start of the central auditory pathway.

Functional hair cell regeneration in the adult mammalian inner ear remains challenging. This study aimed to study the function of new hair cells induced by a DNA demethylating agent 5-azacytidine. Adult mice were deafened chemically, followed by injection of 5-azacytidine or vehicle into the inner ear. Functionality of regenerated hair cells was evaluated by expression of hair cell proteins, auditory brainstem response (ABR), and distortion-product otoacoustic emission (DPOAE) tests for 6 weeks. In the vehicle-treated group, no cells expressed the hair cell-specific protein myosin VIIa in the cochlea, whereas numerous myosin VIIa-expressing cells were found in the 5-azacytidine-treated cochlea, suggesting the regeneration of auditory hair cells. Moreover, regenerated hair cells were co-labeled with functional proteins espin and prestin. Expression of ribbon synapse proteins suggested synapse formation between new hair cells and neurons. In hearing tests, progressive improvements in ABR [5–30 dB sound pressure level (SPL)] and DPOAE (5–20 dB) thresholds were observed in 5-azacytidine-treated mice. In vehicle-treated mice, there were <5 dB threshold changes in hearing tests. This study demonstrated the ability of 5-azacytidine to promote the functional regeneration of auditory hair cells in a mature mouse model via DNA demethylation, which may provide insights into hearing regeneration using an epigenetic approach.

Background To localize sound sources accurately in a reverberant environment, human binaural hearing strongly favors analyzing the initial wave front of sounds. Behavioral studies of this “precedence effect” have so far largely been confined to human subjects, limiting the scope of complementary physiological approaches. Similarly, physiological studies have mostly looked at neural responses in the inferior colliculus, the main relay point between the inner ear and the auditory cortex, or used modeling of cochlear auditory transduction in an attempt to identify likely underlying mechanisms. Studies capable of providing a direct comparison of neural coding and behavioral measures of sound localization under the precedence effect are lacking. Results We adapted a “temporal weighting function” paradigm previously developed to quantify the precedence effect in human for use in laboratory rats. The animals learned to lateralize click trains in which each click in the train had a different interaural time difference. Computing the “perceptual weight” of each click in the train revealed a strong onset bias, very similar to that reported for humans. Follow-on electrocorticographic recording experiments revealed that onset weighting of interaural time differences is a robust feature of the cortical population response, but interestingly, it often fails to manifest at individual cortical recording sites. Conclusion While previous studies suggested that the precedence effect may be caused by early processing mechanisms in the cochlea or inhibitory circuitry in the brainstem and midbrain, our results indicate that the precedence effect is not fully developed at the level of individual recording sites in the auditory cortex, but robust and consistent precedence effects are observable only in the auditory cortex at the level of cortical population responses. This indicates that the precedence effect emerges at later cortical processing stages and is a significantly “higher order” feature than has hitherto been assumed.

Cell cycle associated protein 1 (Caprin1) is an RNA-binding protein that can regulate the cellular post-transcriptional response to stress. It is a component of both stress granules and neuronal RNA granules and is implicated in neurodegenerative disease, synaptic plasticity and long-term memory formation. Our previous work suggested that Caprin1 also plays a role in the response of the cochlea to stress. Here, targeted inner ear-deletion of Caprin1 in mice leads to an early onset, progressive hearing loss. Auditory brainstem responses from Caprin1 -deficient mice show reduced thresholds, with a significant reduction in wave-I amplitudes compared to wildtype. Whilst hair cell structure and numbers were normal, the inner hair cell-spiral ganglion neuron (IHC-SGN) synapse revealed abnormally large post-synaptic GluA2 receptor puncta, a defect consistent with the observed wave-I reduction. Unlike wildtype mice, mild-noise-induced hearing threshold shifts in Caprin1 -deficient mice did not recover. Oxidative stress triggered TIA-1/HuR-positive stress granule formation in ex-vivo cochlear explants from Caprin1- deficient mice, showing that stress granules could still be induced. Taken together, these findings suggest that Caprin1 plays a key role in maintenance of auditory function, where it regulates the normal status of the IHC-SGN synapse.

Survival relies on an organism’s intrinsic ability to instinctively react to stimuli such as food, water, and threats, ensuring the fundamental ability to feed, drink, and avoid danger even in the absence of prior experience. These natural, unconditioned stimuli can also facilitate associative learning, where pairing them consistently with neutral cues will elicit responses to those cue. Threat conditioning, a well-explored form of associative learning, commonly employs painful electric shocks, mimicking injury, as unconditioned stimuli. It remains elusive whether actual injury or pain is necessary for effective learning, or whether the threat of harm is sufficient. Approaching predators create looming shadows and sounds, triggering strong innate defensive responses like escape and freezing. This study investigates whether visual looming stimuli can induce learned freezing or learned escape responses to a conditioned stimulus in rats. Surprisingly, pairing a neutral tone with a looming stimulus only weakly evokes learned defensive responses, in contrast to the strong responses observed when the looming stimulus is replaced by a shock. This dissociation sheds light on the boundaries for learned defensive responses thereby impacting our comprehension of learning processes and defensive strategies.

The excessive generation of reactive oxygen species (ROS) and mitochondrial damage have been widely reported in noise-induced hearing loss (NIHL). However, the specific mechanism of noise-induced mitochondrial damage remains largely unclear. In this study, we showed that acoustic trauma caused oxidative damage to mitochondrial DNA (mtDNA), leading to the reduction of mtDNA content, mitochondrial gene expression and ATP level in rat cochleae. The expression level and mtDNA-binding function of mitochondrial transcription factor A (TFAM) were impaired following acoustic trauma without affecting the upstream PGC-1α and NRF-1. The mitochondria-target antioxidant mito-TEMPO (MT) was demonstrated to enter the inner ear after the systemic administration. MT treatment significantly alleviated noise-induced auditory threshold shifts 3d and 14d after noise exposure. Furthermore, MT significantly reduced outer hair cell (OHC) loss, cochlear ribbon synapse loss, and auditory nerve fiber (ANF) degeneration after the noise exposure. In addition, we found that MT treatment effectively attenuated noise-induced cochlear oxidative stress and mtDNA damage, as indicated by DHE, 4-HNE, and 8-OHdG. MT treatment also improved mitochondrial biogenesis, ATP generation, and TFAM-mtDNA interaction in the cochlea. These findings suggest that MT has protective effects against NIHL via maintaining TFAM-mtDNA interaction and mitochondrial biogenesis based on its ROS scavenging capacity.

The ability to determine a sound’s location is critical in everyday life. However, sound source localization is severely compromised for patients with hearing loss who receive bilateral cochlear implants (BiCIs). Several patient factors relate to poorer performance in listeners with BiCIs, associated with auditory deprivation, experience, and age. Critically, characteristic errors are made by patients with BiCIs (e.g., medial responses at lateral target locations), and the relationship between patient factors and the type of errors made by patients has seldom been investigated across individuals. In the present study, several different types of analysis were used to understand localization errors and their relationship with patient-dependent factors (selected based on their robustness of prediction). Binaural hearing experience is required for developing accurate localization skills, auditory deprivation is associated with degradation of the auditory periphery, and aging leads to poorer temporal resolution. Therefore, it was hypothesized that earlier onsets of deafness would be associated with poorer localization acuity and longer periods without BiCI stimulation or older age would lead to greater amounts of variability in localization responses. A novel machine learning approach was introduced to characterize the types of errors made by listeners with BiCIs, making them simple to interpret and generalizable to everyday experience. Sound localization performance was measured in 48 listeners with BiCIs using pink noise trains presented in free-field. Our results suggest that older age at testing and earlier onset of deafness are associated with greater average error, particularly for sound sources near the center of the head, consistent with previous research. The machine learning analysis revealed that variability of localization responses tended to be greater for individuals with earlier compared to later onsets of deafness. These results suggest that early bilateral hearing is essential for best sound source localization outcomes in listeners with BiCIs.

Before hearing onset (postnatal day 12 in mice), inner hair cells (IHCs) spontaneously fire action potentials, thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISCs) of Kölliker’s organ through the activity of the Ca 2+ -activated Cl - -channel TMEM16A (ANO1). Here, we show that conditional deletion of Ano1 ( Tmem16a ) in mice disrupts Ca 2+ waves within Kölliker’s organ, reduces the burst-firing activity and the frequency selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the functional refinement of MNTB projections to the lateral superior olive. These results reveal the importance of the activity of Kölliker’s organ for the refinement of central auditory connectivity. In addition, our study suggests the involvement of TMEM16A in the propagation of Ca 2+ waves, which may also apply to other tissues expressing TMEM16A.

Several studies identified hearing loss as a risk factor for aging-related processes, including neurodegenerative diseases, as dementia and age-related hearing loss (ARHL). Although the association between hearing impairment in midlife and ARHL has been widely documented by epidemiological and experimental studies, the molecular mechanisms underlying this association are not fully understood. In this study, we used an established animal model of ARHL (C57BL/6 mice) to evaluate if early noise-induced hearing loss (NIHL) could affect the onset or progression of age-related cochlear dysfunction. We found that hearing loss can exacerbate ARHL, damaging sensory-neural cochlear epithelium and causing synaptopathy. Moreover, we studied common pathological markers shared between hearing loss and ARHL, demonstrating that noise exposure can worsen/accelerate redox status imbalance [increase of reactive oxygen species (ROS) production, lipid peroxidation, and dysregulation of endogenous antioxidant response] and vascular dysfunction [increased expression of hypoxia-inducible factor-1alpha (HIF-1α) and vascular endothelial growth factor C (VEGFC)] in the cochlea. Unveiling the molecular mechanisms underlying the link between hearing loss and aging processes could be valuable to identify effective therapeutic strategies to limit the effect of environmental risk factors on age-related diseases.

In the mammalian brain, auditory brainstem nuclei are arranged topographically according to acoustic frequency responsiveness. During postnatal development, the axon initial segment (AIS) of principal neurons undergoes structural refinement depending on location along the tonotopic axis within the medial nucleus of the trapezoid body (MNTB). However, the molecular mechanisms underlying the structural refinement of the AIS along the tonotopic axis in the auditory brainstem have not been explored. We tested the hypothesis that brain-derived neurotrophic factor (BDNF) is a molecular mediator of the structural development of the MNTB in an activity-dependent manner. Using BDNF heterozygous mutant ( BDNF +/– ) mice, we examined the impact of global BDNF reduction on structural and functional development of MNTB neurons by assessing AIS structure and associated intrinsic neuronal properties. BDNF reduction inhibits the structural and functional differentiation of principal neurons along the tonotopic axis in the MNTB. Augmented sound input during the critical period of development has been shown to enhance the structural refinement of the AIS of MNTB neurons. However, in BDNF +/– mice, MNTB neurons did not show this activity-dependent structural modification of the AIS following repeated sound stimulation. In addition, BDNF +/– mice lacked a defined isofrequency band of neuronal activity following exposure to 16 kHz sound, suggesting degradation of tonotopy. Taken together, structural development and functional refinement of auditory brainstem neurons require physiological levels of BDNF to establish proper tonotopic gradients.