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BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. METHODS: Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. RESULTS: We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.
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Cardiomegalia , Disferlina , Camundongos Knockout , Miócitos Cardíacos , Retículo Sarcoplasmático , Animais , Disferlina/metabolismo , Disferlina/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Humanos , Camundongos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patologia , Camundongos Endogâmicos C57BL , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proliferação de Células , Células Cultivadas , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Quinase de Cadeia Leve de MiosinaRESUMO
BACKGROUND/OBJECTIVE: Observational real-world study to analyze the clinical effects of alemtuzumab (ALEM) and subsequent disease-modifying therapy (DMT) usage in multiple sclerosis (MS). METHODS: Data retrieved from the Austrian MS treatment registry (AMSTR) included baseline (BL) characteristics (at ALEM start), annualized relapse rate (ARR), 6-month confirmed progression independent of relapse activity (PIRA; ≥ 0.5-point Expanded Disability Status Scale (EDSS) score increase), 6-month confirmed disability improvement (CDI; ≥ 0.5-point EDSS decrease), and safety outcomes until initiation of a subsequent DMT. The EDSS was re-baselined at 30 days from ALEM start (BL EDSS). RESULTS: Eighty-seven ALEM-treated patients (median age: 32 years, 72% female, 14% treatment-naïve) were followed for a median of 55 (interquartile range 31-68) months. We found significant reductions in the ARR from 1.16 before ALEM to 0.15 throughout Years 1-9 (p < 0.001). Subsequent DMTs were initiated in 19 patients (22%, 74% anti-CD20 monoclonal antibodies). At Year 5 (n = 53), more patients achieved CDI (58%, 95% confidence interval (CI) 45%-71%) than had experienced PIRA (14%, CI 7.5%-24%), and 58% remained relapse-free. Shorter MS duration (p < 0.001, hazard ratio (HR) 0.86 (CI 0.80-0.93)) and no previous high-efficacy treatment (p < 0.001, HR 5.16 (CI 2.66-10.0)) were the best predictors of CDI, while PIRA was associated with a higher number of previous DMTs (p = 0.04, HR 3.06, CI 1.05-8.89). We found no new safety signals. INTERPRETATION: ALEM had long-lasting beneficial effects on the ARR and disability improvement, especially when initiated early in the course of the disease. Only a subset of patients received subsequent DMTs.
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Alemtuzumab , Humanos , Feminino , Alemtuzumab/farmacologia , Alemtuzumab/administração & dosagem , Masculino , Áustria , Adulto , Esclerose Múltipla/tratamento farmacológico , Fatores Imunológicos/farmacologia , Fatores Imunológicos/administração & dosagem , Sistema de Registros , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Resultado do Tratamento , Pessoa de Meia-Idade , Progressão da DoençaRESUMO
BACKGROUND AND PURPOSE: This study was undertaken to raise awareness of a role of B cells in immune checkpoint inhibitor (ICI)-associated neurological immune-related adverse events (nirAE). METHODS: A systematic literature review was made, with case observations of a melanoma and a non-small cell lung cancer (NSCLC) patient who developed ICI-associated nirAE with cerebrospinal fluid (CSF) findings indicating B cell involvement. RESULTS: Two patients receiving ipilimumab/nivolumab for melanoma and chemotherapy/pembrolizumab for NSCLC developed nirAE in the form of myocarditis/myositis/myasthenia gravis overlap syndrome (triple M) and cerebellitis plus longitudinal transverse myelitis (c-LETM), respectively. Intrathecal inflammation with chemokine C-X-C motif ligand (CXCL13) elevation was present in both patients; the triple M case had acetylcholine receptor antibodies, antititin reactivity, altered CD4/CD8 T cell ratio in blood, and depressed programmed death-1 (PD-1) expression on CSF T cells; the c-LETM case showed intrathecal antibody production and plasma cells. Both patients insufficiently responded to first-line treatment. The NSCLC case improved upon administration of B cell-depleting therapy with rituximab, whereas the melanoma patient died before escalation therapy was initiated. Literature research revealed one additional ICI-associated LETM case with intrathecal CXCL13 elevation, three cases with ICI-associated aquaporin-4 antibody neuromyelitis spectrum disorder, and evidence of B cell-mediated toxicity based on antibody-mediated immune pathologies in ICI-associated immune-related adverse events. CONCLUSIONS: The case observations highlight the plethora of uncertainties in diagnosis and treatment of ICI-associated nirAE, exemplify the heterogeneity of immune mechanisms involved, and suggest a role of B cells, which may be underdiagnosed. Intrathecal CXCL13 may serve as a biomarker of B cell involvement in nirAE, supported by intrathecal immunoglobulin synthesis, presence of plasma cells, and/or recruitment of cognate immune cells.
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Linfócitos B , Quimiocina CXCL13 , Inibidores de Checkpoint Imunológico , Idoso , Feminino , Humanos , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/administração & dosagem , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Quimiocina CXCL13/líquido cefalorraquidiano , Inibidores de Checkpoint Imunológico/efeitos adversos , Ipilimumab/efeitos adversos , Ipilimumab/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Melanoma/tratamento farmacológico , Mielite Transversa/induzido quimicamente , Mielite Transversa/imunologia , Nivolumabe/efeitos adversos , Nivolumabe/administração & dosagem , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
The cochlear implant (CI) is considered the most successful neuroprosthesis as it enables speech comprehension in the majority of the million otherwise deaf patients. In hearing by electrical stimulation of the auditory nerve, the broad spread of current from each electrode acts as a bottleneck that limits the transfer of sound frequency information. Hence, there remains a major unmet medical need for improving the quality of hearing with CIs. Recently, optogenetic stimulation of the cochlea has been suggested as an alternative approach for hearing restoration. Cochlear optogenetics promises to transfer more sound frequency information, hence improving hearing, as light can conveniently be confined in space to activate the auditory nerve within smaller tonotopic ranges. In this review, we discuss the latest experimental and technological developments of optogenetic hearing restoration and outline remaining challenges en route to clinical translation.
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Implantes Cocleares , Optogenética , Optogenética/métodos , Humanos , Animais , Perda Auditiva/fisiopatologia , Audição/fisiologia , Cóclea/fisiologiaRESUMO
With the growth of optogenetic research, the demand for optical probes tailored to specific applications is ever rising. Specifically, for applications like the coiled cochlea of the inner ear, where planar, stiff, and nonconformable probes can hardly be used, transitioning from commonly used stiff glass fibers to flexible probes is required, especially for long-term use. Following this demand, polydimethylsiloxane (PDMS) with its lower Young's modulus compared to glass fibers can serve as material of choice. Hence, the long-term usability of PDMS as a waveguide material with respect to variations in transmission and refractive index over time is investigated. Different manufacturing methods for PDMS-based flexible waveguides are established and compared with the aim to minimize optical losses and thus maximize optical output power. Finally, the waveguides with lowest optical losses (-4.8 dB cm-1 ± 1.3 dB cm-1 at 472 nm) are successfully inserted into the optogenetically modified cochlea of a Mongolian gerbil (Meriones unguiculatus), where optical stimuli delivered by the waveguides evoked robust neuronal responses in the auditory pathway.
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Dimetilpolisiloxanos , Gerbillinae , Optogenética , Dimetilpolisiloxanos/química , Animais , Optogenética/métodos , Cóclea/fisiologiaRESUMO
Introduction: Recently developed fluorescent neurotransmitter indicators have enabled direct measurements of neurotransmitter in the synaptic cleft. Precise optical measurements of neurotransmitter release may be used to make inferences about presynaptic function independent of electrophysiological measurements. Methods: Here, we express iGluSnFR, a genetically encoded glutamate reporter in mouse spiral ganglion neurons to compare electrophysiological and optical readouts of presynaptic function and short-term synaptic plasticity at the endbulb of Held synapse. Results: We show iGluSnFR robustly and approximately linearly reports glutamate release from the endbulb of Held during synaptic transmission and allows assessment of short-term plasticity during high-frequency train stimuli. Furthermore, we show that iGluSnFR expression slightly alters the time course of spontaneous postsynaptic currents, but is unlikely to impact measurements of evoked synchronous release of many synaptic vesicles. Discussion: We conclude that monitoring glutamate with optical sensors at fast and large central synapses like the endbulb of Held is feasible and allows robust quantification of some, but not all aspects of glutamate release.
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OBJECTIVE: Repeated intravenous administration of anti-CD20 depleting monoclonal antibodies 6 months apart is among the highly effective treatment options in multiple sclerosis (MS). Here, we aimed to investigate peripheral immune cell subset depletion kinetics following either rituximab (RTX) or ocrelizumab (OCR) infusions in people with MS (pwMS). METHODS: We studied pwMS treated de-novo with either RTX (n = 7) or OCR (n = 8). The examinations were scheduled before the initiation of anti-CD20 therapy and every 12 weeks for up to 15 months. Immunophenotyping of immune cell subsets in peripheral blood was performed by multiparametric fluorescence cytometry. RESULTS: A significant, persistent decrease of CD19+ B cells was observed already with the first anti-CD20 infusion (p < 0.0001). A significant proportional reduction of memory B cells within the B-cell pool was achieved only after two treatment cycles (p = 0.005). We observed a proportional increase of immature (p = 0.04) and naive B cells (p = 0.004), again only after the second treatment cycle. As for the peripheral T-cell pool, we observed a continuous proportional increase of memory T helper (TH) cells/central memory TH cells (p = 0.02/p = 0.008), while the number of regulatory T cells (Treg) decreased (p = 0.007). The percentage of B-cell dependent TH17.1 central memory cells dropped after the second treatment cycle (p = 0.02). No significant differences in the depletion kinetics between RTX and OCR were found. INTERPRETATION: Peripheral immune cell profiling revealed more differentiated insights into the prompt and delayed immunological effects of repeated intravenous anti-CD20 treatment. The observation of proportional changes of some pathogenetically relevant immune cell subsets only after two infusion cycles deserves further attention.
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Esclerose Múltipla , Humanos , Rituximab/farmacologia , Rituximab/uso terapêutico , Anticorpos Monoclonais/farmacologia , Linfócitos B , Antígenos CD19/metabolismo , Antígenos CD19/farmacologiaRESUMO
BACKGROUND/OBJECTIVE: The use of natalizumab (NAT) in multiple sclerosis (MS) may be complicated by progressive multifocal leukoencephalopathy (PML), a rare and life-threatening opportunistic brain infection. We aimed to analyze the course of MS after PML recovery together with the long-term outcome of NAT-associated PML (NAT-PML) in Austria. METHODS: Retrospective study based on identification of cases in the nationwide Austrian MS treatment registry (AMSTR) and MS centers with review of patient records. The expanded disability status scale (EDSS) was used to measure neurological disability and outcome. RESULTS: As of December 2022, we identified 15 NAT-PML cases in Austria; only 20% occurred after 2016, when increased vigilance commenced. Two patients did not survive acute PML, and an additional patient died five years later, yielding a mortality rate of 20%. Seizures occurred exclusively in patients with pronounced EDSS increase. Gadolinium (Gd)-enhancement on brain magnetic resonance imaging (MRI) on PML suspicion was associated with minor changes of post-PML neurological disability. Long-term follow-up of up to 132 months (median 76 months) was available in 11/15. The overall median EDSS increased from 3.5 at pre-PML to 6.5 at the last assessment. Regarding inflammatory MS-related disease activity during the observation period, one single individual experienced an MS relapse and another patient had two Gd-enhancing brain lesions. Three patients converted to progressive MS within three years from PML and the EDSS further increased in 6/11. CONCLUSIONS: The number of NAT-PML cases is decreasing over time. While many patients accumulated severe persistent neurological deficits compared to pre-PML, inflammatory MS-related disease activity after PML recovery was rare.
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Leucoencefalopatia Multifocal Progressiva , Esclerose Múltipla , Humanos , Leucoencefalopatia Multifocal Progressiva/epidemiologia , Leucoencefalopatia Multifocal Progressiva/etiologia , Natalizumab/efeitos adversos , Estudos Retrospectivos , Áustria/epidemiologia , Fatores Imunológicos/efeitos adversosRESUMO
In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.
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Células Ciliadas Auditivas Internas , Proteômica , Camundongos , Animais , Células Ciliadas Auditivas Internas/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Afferent synapses of cochlear inner hair cells (IHCs) employ a unique molecular machinery. Otoferlin is a key player in this machinery, and its genetic defects cause human auditory synaptopathy. We employed site-directed mutagenesis in mice to investigate the role of Ca2+ binding to the C2F domain of otoferlin. Substituting two aspartate residues of the C2F top loops, which are thought to coordinate Ca2+-ions, by alanines (OtofD1841/1842A) abolished Ca2+-influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca2+ influx of IHCs and their resting membrane capacitance, reflecting IHC size, as well as the number of IHC synapses were maintained. The mutant otoferlin showed a strong apex-to-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C2F domain in otoferlin targeting and of Ca2+ binding by the C2F domain for IHC exocytosis and hearing.
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The journal retracts the article, An Innovative Tool for Evidence-Based, Personalized Treatment Trials in Mucopolysaccharidosis [...].
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In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.
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Células Ciliadas Auditivas Internas , Células Ciliadas Vestibulares , Animais , Camundongos , Células Ciliadas Auditivas Internas/metabolismo , Ácido Glutâmico/metabolismo , Audição/fisiologia , Células Ciliadas Vestibulares/metabolismo , Sinapses/metabolismo , Cóclea/metabolismo , Cálcio/metabolismoRESUMO
Cochlear implants (CIs) restore activity in the deafened auditory system via electrical stimulation of the auditory nerve. As the spread of electric current in biological tissues is rather broad, the spectral information provided by electrical CIs is limited. Optogenetic stimulation of the auditory nerve has been suggested for artificial sound coding with improved spectral selectivity, as light can be conveniently confined in space. Yet, the foundations for optogenetic sound coding strategies remain to be established. Here, we parametrized stimulus-response-relationships of the auditory pathway in gerbils for optogenetic stimulation. Upon activation of the auditory pathway by waveguide-based optogenetic stimulation of the spiral ganglion, we recorded neuronal activity of the auditory midbrain, in which neural representations of spectral, temporal, and intensity information can be found. Screening a wide range of optical stimuli and taking the properties of optical CI emitters into account, we aimed to optimize stimulus paradigms for potent and energy-efficient activation of the auditory pathway. We report that efficient optogenetic coding builds on neural integration of millisecond stimuli built from microsecond light pulses, which optimally accommodate power-efficient laser diode operation. Moreover, we performed an activity-level-dependent comparison of optogenetic and acoustic stimulation in order to estimate the dynamic range and the maximal stimulation intensity amenable to single channel optogenetic sound encoding, and indicate that it complies well with speech comprehension in a typical conversation (65 dB). Our results provide a first framework for the development of coding strategies for future optogenetic hearing restoration.
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Implante Coclear , Implantes Cocleares , Vias Auditivas/fisiologia , Optogenética/métodos , Mesencéfalo , Estimulação Acústica , Estimulação ElétricaRESUMO
Our sense of hearing enables the processing of stimuli that differ in sound pressure by more than six orders of magnitude. How to process a wide range of stimulus intensities with temporal precision is an enigmatic phenomenon of the auditory system. Downstream of dynamic range compression by active cochlear micromechanics, the inner hair cells (IHCs) cover the full intensity range of sound input. Yet, the firing rate in each of their postsynaptic spiral ganglion neurons (SGNs) encodes only a fraction of it. As a population, spiral ganglion neurons with their respective individual coding fractions cover the entire audible range. How such "dynamic range fractionation" arises is a topic of current research and the focus of this review. Here, we discuss mechanisms for generating the diverse functional properties of SGNs and formulate testable hypotheses. We postulate that an interplay of synaptic heterogeneity, molecularly distinct subtypes of SGNs, and efferent modulation serves the neural decomposition of sound information and thus contributes to a population code for sound intensity.
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Cóclea , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Internas/fisiologia , Som , Sinapses/fisiologia , Gânglio Espiral da CócleaRESUMO
Hearing loss is the most common human sensory deficit. Severe-to-complete sensorineural hearing loss is often treated by electrical cochlear implants (eCIs) bypassing dysfunctional or lost hair cells by direct stimulation of the auditory nerve. The wide current spread from each intracochlear electrode array contact activates large sets of tonotopically organized neurons limiting spectral selectivity of sound coding. Despite many efforts, an increase in the number of independent eCI stimulation channels seems impossible to achieve. Light, which can be better confined in space than electric current may help optical cochlear implants (oCIs) to overcome eCI shortcomings. In this review, we present the current state of the optogenetic sound encoding. We highlight optical sound coding strategy development capitalizing on the optical stimulation that requires fine-grained, fast, and power-efficient real-time sound processing controlling dozens of microscale optical emitters as an emerging research area.
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Neural sound encoding in the mammalian cochlea faces the challenge of representing audible sound pressures that vary over six orders of magnitude. The cochlea meets this demand through the use of active micromechanics as well as the diversity and adaptation of afferent neurons and their synapses. Mechanisms underlying neural diversity likely include heterogeneous presynaptic input from inner hair cells (IHCs) to spiral ganglion neurons (SGNs) as well as differences in the molecular profile of SGNs and in their efferent control. Here, we tested whether glutamate release from IHCs, previously found to be critical for maintaining different molecular SGN profiles, is required for establishing heterogeneity of active zones (AZs) in IHCs. We analyzed structural and functional heterogeneity of IHC AZs in mouse mutants with disrupted glutamate release from IHCs due to lack of a vesicular glutamate transporter (Vglut3) or impaired exocytosis due to defective otoferlin. We found the variance of the voltage-dependence of presynaptic Ca2+ influx to be reduced in exocytosis-deficient IHCs of otoferlin mutants. Yet, the spatial gradients of maximal amplitude and voltage-dependence of Ca2+ influx along the pillar-modiolar IHC axis were maintained in both mutants. Further immunohistochemical analysis showed an intact spatial gradient of ribbon size in Vglut3-/- mice. These results indicate that IHC exocytosis and glutamate release are not strictly required for establishing the heterogeneity of IHC AZs.