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.

Sensorineural hearing loss (SNHL) is caused by an irreversible impairment of cochlear hair cells and subsequent progressive degeneration of spiral ganglion neurons (SGNs). Eighty-five requiring 3 (Efr3) is a plasma membrane protein conserved from yeast to human, and knockout of Efr3a was reported to facilitate the survival of hippocampal newborn neurons in adult mice. Previously, we found Efr3a expression in the auditory neural pathway is upregulated soon after the destruction of hair cells. Here we conducted a time-course analysis of drug-caused damage to hearing ability, hair cells and SGNs in Efr3a knocking down mice (Efr3a-/+, Efr3a KD) and their wild type littermates. Functional examination showed that both groups of mice suffered from serious hearing loss with a higher level of severity in wild type (WT) mice. Morphologic observation following drugs administration showed that both WT and Efr3a KD mice went through progressive loss of hair cells and SGNs, in association with degenerative changes in the perikarya, intracellular organelles, cell body conformation in SGNs, and the changes of SGNs in WT mice were more severe than in Efr3a KD mice. These beneficial effects of Efr3a KD could be ascribed to an increase in the expression of some neurotrophic factors and their receptors in Efr3a KD mice. Our results indicate that Efr3a insufficiency suppresses drug-caused SNHL neurodegeneration in association with an increase in the expression of some neurotrophic factors and their receptors, which may be targeted in the treatment of neurodegeneration.

Optogenetic stimulation of type I spiral ganglion neurons (SGNs) promises an alternative to the electrical stimulation by current cochlear implants (CIs) for improved hearing restoration by future optical CIs (oCIs). Most of the efforts in using optogenetic stimulation in the cochlea so far used early postnatal injection of viral vectors carrying blue-light activated channelrhodopsins (ChRs) into the cochlea of mice. However, preparing clinical translation of the oCI requires (i) reliable and safe transduction of mature SGNs of further species and (ii) use of long-wavelength light to avoid phototoxicity. Here, we employed a fast variant of the red-light activated channelrhodopsin Chrimson (f-Chrimson) and different AAV variants to implement optogenetic SGN stimulation in Mongolian gerbils. We compared early postnatal (p8) and adult (>8 weeks) AAV administration, employing different protocols for injection of AAV-PHP.B and AAV2/6 into the adult cochlea. Success of the optogenetic manipulation was analyzed by optically evoked auditory brainstem response (oABR) and immunohistochemistry of mid-modiolar cryosections of the cochlea. In order to most efficiently evaluate the immunohistochemical results a semi-automatic procedure to identify transduced cells in confocal images was developed. Our results indicate that the rate of SGN transduction is significantly lower for AAV administration into the adult cochlea compared to early postnatal injection. SGN transduction upon AAV administration into the adult cochlea was largely independent of the chosen viral vector and injection approach. The higher the rate of SGN transduction, the lower were oABR thresholds and the larger were oABR amplitudes. Our results highlight the need to optimize viral vectors and virus administration for efficient optogenetic manipulation of SGNs in the adult cochlea for successful clinical translation of SGN-targeting gene therapy and of the oCI.

In the mature cochlea, each inner hair cell (IHC) is innervated by multiple spiral ganglion neurons of type I (SGNI). SGNIs are morphologically and electro-physiologically diverse. Also, they differ in their susceptibility to noise insult. However, the molecular underpinnings of their identity and physiological differences remain poorly understood. In this study, we developed a novel triple transgenic mouse, which enabled the isolation of pure populations of SGNIs and the analysis of a 96-gene panel via single-cell qPCR. We found three distinct populations of Type I SGNs, which were marked by their exclusive expression of Lmx1a, Slc4a4, or Mfap4/Fzd2, respectively, at postnatal days P3, P8, and P12. Our data suggest that afferent SGN subtypes are established genetically before the onset of hearing and that the expression of key physiological markers, such as ion channels, is heterogeneous and may be underlying the heterogeneous firing proprieties of SGNIs.

The fibroblast growth factor 2 (FGF2) is a member of the FGF family which is involved in key biological processes including development, cellular proliferation, wound healing, and angiogenesis. Although the utility of the FGF family as therapeutic agents has attracted attention, and FGF2 has been studied in several clinical contexts, there remains an incomplete understanding of the molecular and clinical function of FGF2 in the auditory system. In this review, we highlight the role of FGF2 in inner ear development and hearing protection and present relevant clinical studies for tympanic membrane (TM) repair. We conclude by discussing the future implications of FGF2 as a potential therapeutic agent.

Sensorineural hearing loss (SNHL) caused by noise exposure and attendant loss of glutamatergic synapses between cochlear spiral ganglion neurons (SGNs) and hair cells is the most common sensory deficit worldwide. We show here that systemic administration of a bisphosphonate to mice 24 h after synaptopathic noise exposure regenerated synapses between inner hair cells and SGNs and restored cochlear function. We further demonstrate that this effect is mediated by inhibition of the mevalonate pathway. These results are highly significant because they suggest that bisphosphonates could reverse cochlear synaptopathy for the treatment of SNHL.

Aminoglycosides (AG) antibiotics are a common treatment for recurrent infections in cystic fibrosis (CF) patients. AGs are highly ototoxic, resulting in a range of auditory dysfunctions. It was recently shown that the acoustic startle reflex (ASR) can assess behavioral evidence of hyperacusis and tinnitus in an amikacin cochleotoxicity mouse model. The goal of this study was to establish if tobramycin treatment led to similar changes in ASR behavior and to establish whether ebselen can prevent the development of these maladaptive neuroplastic symptoms. CBA/Ca mice were divided into three groups: Group 1 served as a control and did not receive tobramycin or ebselen, Group 2 received tobramycin (200 mg/kg/s.c.) and the vehicle (DMSO/saline/i.p.) daily for 14 continuous days, and Group 3 received the same dose/schedule of tobramycin as Group 2 and ebselen at (20 mg/kg/i.p.). Auditory brainstem response (ABR) and ASR hearing assessments were collected at baseline and 2, 6, 10, 14, and 18 weeks from the start of treatment. ASR tests included input/output (I/O) functions which assess general hearing and hyperacusis, and Gap-induced prepulse inhibition of the acoustic startle (GPIAS) to assess tinnitus. At 18 weeks, histologic analysis showed predominantly normal appearing hair cells and spiral ganglion neuron (SGN) synapses. Following 14 days of tobramycin injections, 16 kHz thresholds increased from baseline and fluctuated over the 18-week recovery period. I/O functions revealed exaggerated startle response magnitudes in 50% of mice over the same period. Gap detection deficits, representing behavioral evidence of tinnitus, were observed in a smaller subset (36%) of animals. Interestingly, increases in ABR wave III/wave I amplitude ratios were observed. These tobramycin data corroborate previous findings that AGs can result in hearing dysfunctions. We show that a 14-day course of tobramycin treatment can cause similar levels of hearing loss and tinnitus, when compared to a 14-day course of amikacin, but less hyperacusis. Evidence suggests that tinnitus and hyperacusis might be common side effects of AG antibiotics.

The afferent synapses between inner hair cells (IHC) and spiral ganglion neurons are specialized to faithfully encode sound with sub-millisecond precision over prolonged periods of time. Here, we studied the role of Rab3 interacting molecule-binding proteins (RIM-BP) 1 and 2 - multidomain proteins of the active zone known to directly interact with RIMs, Bassoon and Ca V 1.3 - in IHC presynaptic function and hearing. Recordings of auditory brainstem responses and otoacoustic emissions revealed that genetic disruption of RIM-BPs 1 and 2 in mice (RIM-BP1/2-/- ) causes a synaptopathic hearing impairment exceeding that found in mice lacking RIM-BP2 (RIM-BP2-/- ). Patch-clamp recordings from RIM-BP1/2-/- IHCs indicated a subtle impairment of exocytosis from the readily releasable pool of synaptic vesicles that had not been observed in RIM-BP2-/- IHCs. In contrast, the reduction of Ca2+-influx and sustained exocytosis was similar to that in RIMBP2-/- IHCs. We conclude that both RIM-BPs are required for normal sound encoding at the IHC synapse, whereby RIM-BP2 seems to take the leading role.

In rodents, massive initial synapses are formed in the auditory peripheral nervous system at the early postnatal stage, and one of the major phenomena is that the number of afferent synapses in the cochlea is significantly reduced in the duration of development. This raises the hypothesis that the number of cochlear ribbon synapses are dramatically changed with hearing development and maturation. In this study, several tracers identifying activities of autophagy were applied to estimate the level of autophagy activity in the process of ribbon synapse development in mice; further, changes in the synaptic number and spiral ganglion nerve (SGN) fibers were quantitatively measured. We found robust expression of LC3B and lysosomal-associated membrane protein 1 as well as LysoTracker in or near inner hair cells and cochlear ribbon synapses in the early stage of postnatal development. Moreover, we found a significant loss in the intensity of SGN fibers at ribbon synaptic development and hearing onset. Thus, this study demonstrates that ribbon synaptic refinement and SGN fibers pruning are closely associated with the morphological and functional maturation of ribbon synapses and that synaptic refinement and SGN fiber pruning are regulated by the robust activities of autophagy in the earlier stages of auditory development.

Ribbon synapses are important structures in transmitting auditory signals from the inner hair cells (IHCs) to their corresponding spiral ganglion neurons (SGNs). Over the last few decades, deafness has been primarily attributed to the deterioration of cochlear hair cells rather than ribbon synapses. Hearing dysfunction that cannot be detected by the hearing threshold is defined as hidden hearing loss (HHL). The relationship between ribbon synapses and FGF22 deletion remains unknown. In this study, we used a 6-week-old FGF22 knockout mice model (Fgf22 -/-) and mainly focused on alteration in ribbon synapses by applying the auditory brainstem response (ABR) test, the immunofluorescence staining, the patch-clamp recording, and quantitative real-time PCR. In Fgf22 -/- mice, we found the decreased amplitude of ABR wave I, the reduced vesicles of ribbon synapses, and the decreased efficiency of exocytosis, which was suggested by a decrease in the capacitance change. Quantitative real-time PCR revealed that Fgf22 - / - led to dysfunction in ribbon synapses by downregulating SNAP-25 and Gipc3 and upregulating MEF2D expression, which was important for the maintenance of ribbon synapses' function. Our research concluded that FGF22 deletion caused HHL by affecting the function of IHC ribbon synapses and may offer a novel therapeutic target to meet an ever-growing demand for deafness treatment.

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.

Hearing relies on the transmission of auditory information from sensory hair cells (HCs) to the brain through the auditory nerve. This relay of information requires HCs to be innervated by spiral ganglion neurons (SGNs) in an exclusive manner and SGNs to be ensheathed by myelinating and non-myelinating glial cells. In the developing auditory nerve, mistargeted SGN axons are retracted or pruned and excessive cells are cleared in a process referred to as nerve refinement. Whether auditory glial cells are eliminated during auditory nerve refinement is unknown. Using early postnatal mice of either sex, we show that glial cell numbers decrease after the first postnatal week, corresponding temporally with nerve refinement in the developing auditory nerve. Additionally, expression of immune-related genes was upregulated and macrophage numbers increase in a manner coinciding with the reduction of glial cell numbers. Transient depletion of macrophages during early auditory nerve development, using transgenic CD11bDTR/EGFP mice, resulted in the appearance of excessive glial cells. Macrophage depletion caused abnormalities in myelin formation and transient edema of the stria vascularis. Macrophage-depleted mice also showed auditory function impairment that partially recovered in adulthood. These findings demonstrate that macrophages contribute to the regulation of glial cell number during postnatal development of the cochlea and that glial cells play a critical role in hearing onset and auditory nerve maturation.

Plants of the genus Cannabis have been used by humans for millennia for a variety of purposes. Perhaps most notable is the use of certain Cannabis strains for their psychoactive effects. More recently, several biologically active molecules within the plants of these Cannabis strains, called phytocannabinoids or simply cannabinoids, have been identified. Furthermore, within human cells, endogenous cannabinoids, or endocannabinoids, as well as the receptors and secondary messengers that give rise to their neuromodulatory effects, have also been characterized. This endocannabinoid system (ECS) is composed of two primary ligands-anandamide and 2-arachidonyl glycerol; two primary receptors-cannabinoid receptors 1 and 2; and several enzymes involved in biosynthesis and degradation of endocannabinoid ligands including diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL). Here we briefly summarize cannabinoid signaling and review what has been discerned to date with regard to cannabinoid signaling in the auditory system and its roles in normal physiological function as well as pathological conditions. While much has been uncovered regarding cannabinoid signaling in the central nervous system, less attention has been paid to the auditory system specifically. Still, evidence is emerging to suggest that cannabinoid signaling is critical for the development, maturation, function, and survival of cochlear hair cells (HCs) and spiral ganglion neurons (SGNs). Furthermore, cannabinoid signaling can have profound effects on synaptic connectivity in CNS structures related to auditory processing. While clinical cases demonstrate that endogenous and exogenous cannabinoids impact auditory function, this review highlights several areas, such as SGN development, where more research is warranted.

Sound encoding relies on Ca2+-mediated exocytosis at the ribbon synapse between cochlear inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs). Otoferlin, a multi-C2 domain protein, is proposed to regulate Ca2+-triggered exocytosis at this synapse, but the precise mechanisms of otoferlin function remain to be elucidated. Here, performing whole-cell voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from SGNs in otoferlin mutant mice, we investigated the impact of Otof disruption at individual synapses with single release event resolution. Otof deletion decreased the spontaneous release rate and abolished the stimulus-secretion coupling. This was evident from failure of potassium-induced IHC depolarization to stimulate release and supports the proposed role of otoferlin in Ca2+ sensing for fusion. A missense mutation in the Otof gene (pachanga), in which otoferlin level at the IHC plasma membrane was lowered without changing its Ca2+ binding, also reduced the spontaneous release rate but spared the stimulus-secretion coupling. The slowed stimulated release rate supports the hypothesis that a sufficient abundance of otoferlin at the plasma membrane is crucial for the vesicle supply. Large-sized monophasic EPSCs remained present upon Otof deletion despite the drastic reduction of the rate of exocytosis. However, EPSC amplitude, on average, was modestly decreased. Moreover, a reduced contribution of multiphasic EPSC was observed in both Otof mutants. We argue that the presence of large monophasic EPSCs despite the exocytic defect upon Otof deletion supports the uniquantal hypothesis of transmitter release at the IHC ribbon synapse. Based upon the reduced contribution of multiphasic EPSC, we propose a role of otoferlin in regulating the mode of exocytosis in IHCs.

Environmental exposure to heavy metal lead, a public health hazard in many post-industrial cities, causes hearing impairment upon long-term exposure. Lead-induced cochlear and vestibular dysfunction is well-documented in animal models. Although short-term exposure to lead at concentrations relevant to environmental settings does not cause significant shifts in hearing thresholds in adults, moderate- to low-level lead exposures induce neuronal damage and synaptic dysfunction. We reported that lead exposure induces oxidative stress in the mouse cochlea. However, lead-induced nitrative stress and potential damage to cochlear ribbon synapses are yet to be fully understood. Therefore, this study has evaluated cochlear synaptopathy and nitrative stress in young-adult mice exposed to 2 mM lead acetate for 28 days. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated that this exposure significantly increased the blood lead levels. Assessment of hair cell loss by immunohistochemistry analysis and outer hair cell (OHC) activity by recording distortion product otoacoustic emissions (DPOAEs) indicated that the structure and function of the hair cells were not affected by lead exposure. However, this exposure significantly decreased the expression of C-terminal-binding protein-2 (CtBP2) and GluA2, pre- and post-synaptic protein markers in the inner hair cell synapses, particularly in the basal turn of the organ of Corti, suggesting lead-induced disruption of ribbon synapses. In addition, lead exposure significantly increased the nitrotyrosine levels in spiral ganglion cells, suggesting lead-induced nitrative stress in the cochlea. Collectively, these findings suggest that lead exposure even at levels that do not affect the OHCs induces cochlear nitrative stress and causes cochlear synaptopathy.

Voltage-gated L-type Ca(2+) channels (L-VGCCs) like CaV1.2 are assumed to play a crucial role for controlling release of trophic peptides including brain-derived neurotrophic factor (BDNF). In the inner ear of the adult mouse, besides the well-described L-VGCC CaV1.3, CaV1.2 is also expressed. Due to lethality of constitutive CaV1.2 knock-out mice, the function of this ion channel as well as its putative relationship to BDNF in the auditory system is entirely elusive. We recently described that BDNF plays a differential role for inner hair cell (IHC) vesicles release in normal and traumatized condition. To elucidate a presumptive role of CaV1.2 during this process, two tissue-specific conditional mouse lines were generated. To distinguish the impact of CaV1.2 on the cochlea from that on feedback loops from higher auditory centers CaV1.2 was deleted, in one mouse line, under the Pax2 promoter (CaV1.2(Pax2)) leading to a deletion in the spiral ganglion neurons, dorsal cochlear nucleus, and inferior colliculus. In the second mouse line, the Egr2 promoter was used for deleting CaV1.2 (CaV1.2(Egr2)) in auditory brainstem nuclei. In both mouse lines, normal hearing threshold and equal number of IHC release sites were observed. We found a slight reduction of auditory brainstem response wave I amplitudes in the CaV1.2(Pax2) mice, but not in the CaV1.2(Egr2) mice. After noise exposure, CaV1.2(Pax2) mice had less-pronounced hearing loss that correlated with maintenance of ribbons in IHCs and less reduced activity in auditory nerve fibers, as well as in higher brain centers at supra-threshold sound stimulation. As reduced cochlear BDNF mRNA levels were found in CaV1.2(Pax2) mice, we suggest that a CaV1.2-dependent step may participate in triggering part of the beneficial and deteriorating effects of cochlear BDNF in intact systems and during noise exposure through a pathway that is independent of CaV1.2 function in efferent circuits.