Presynaptic Ribbons Research Articles

Disruption of Atg7-dependent autophagy causes electromotility disturbances, outer hair cell loss, and deafness in mice

Atg7 is an indispensable factor that plays a role in canonical nonselective autophagy. Here we show that genetic ablation of Atg7 in outer hair cells (OHCs) in mice caused stereocilium damage, somatic electromotility disturbances, and presynaptic ribbon degeneration over time, which led to the gradual wholesale loss of OHCs and subsequent early-onset profound hearing loss. Impaired autophagy disrupted OHC mitochondrial function and triggered the accumulation of dysfunctional mitochondria that would otherwise be eliminated in a timely manner. Atg7-independent autophagy/mitophagy processes could not compensate for Atg7 deficiency and failed to rescue the terminally differentiated, non-proliferating OHCs. Our results show that OHCs orchestrate intricate nonselective and selective autophagic/mitophagy pathways working in concert to maintain cellular homeostasis. Overall, our results demonstrate that Atg7-dependent autophagy plays a pivotal cytoprotective role in preserving OHCs and maintaining hearing function.

Exocytosis in mouse vestibular Type II hair cells shows a high-order Ca2+ dependence that is independent of synaptotagmin-4.

Mature hair cells transduce information over a wide range of stimulus intensities and frequencies for prolonged periods of time. The efficiency of such a demanding task is reflected in the characteristics of exocytosis at their specialized presynaptic ribbons. Ribbons are electron‐dense structures able to tether a large number of releasable vesicles allowing them to maintain high rates of vesicle release. Calcium entry through rapidly activating, non‐inactivating CaV1.3 (L‐type) Ca2+ channels in response to cell depolarization causes a local increase in Ca2+ at the ribbon synapses, which is detected by the exocytotic Ca2+ sensors. The Ca2+ dependence of vesicle exocytosis at mammalian vestibular hair cell (VHC) ribbon synapses is believed to be linear, similar to that observed in mature cochlear inner hair cells (IHCs). The linear relation has been shown to correlate with the presence of the Ca2+ sensor synaptotagmin‐4 (Syt‐4). Therefore, we studied the exocytotic Ca2+ dependence, and the release kinetics of different vesicle pool populations, in Type II VHCs of control and Syt‐4 knockout mice using patch‐clamp capacitance measurements, under physiological recording conditions. We found that exocytosis in mature control and knockout Type II VHCs displayed a high‐order dependence on Ca2+ entry, rather than the linear relation previously observed. Consistent with this finding, the Ca2+ dependence and release kinetics of the ready releasable pool (RRP) of vesicles were not affected by an absence of Syt‐4. However, we did find that Syt‐4 could play a role in regulating the release of the secondary releasable pool (SRP) in these cells. Our findings show that the coupling between Ca2+ influx and neurotransmitter release at mature Type II VHC ribbon synapses is faithfully described by a nonlinear relation that is likely to be more appropriate for the accurate encoding of low‐frequency vestibular information, consistent with that observed at low‐frequency mammalian auditory receptors.

Auditory synaptopathy in mice lacking the glutamate transporter GLAST and its impact on brain activity.

Neurotransmission of acoustic signals from the hair cells to the auditory nerve relies on a tightly controlled communication between pre-synaptic ribbons and post-synaptic glutamatergic terminals. After noise overexposure, de-afferentation occurs as a consequence of excessive glutamate release. What maintains synaptic integrity in the cochlea is poorly understood. The objective of this study is to evaluate the role of GLAST in maintaining synaptic integrity in the cochlea in absence or presence of noise, and its impact on sound-evoked brain activity using manganese-enhanced MRI (MeMRI). The glutamate aspartate transporter GLAST is present in supporting cells near the afferent synapse and its genetic deletion leads to greater synaptic swelling after noise overexposure. At baseline, GLAST knockout (GLAST KO) mice displayed two-fold lower wave 1 amplitude of the auditory brainstem response (ABR) when compared to their wild-type littermates in spite of similar ABR and distortion product otoacoustic emissions (DPOAE) thresholds. While the abundance of ribbons was not affected by the loss of GLAST function, the number of paired synapses was halved in GLAST KO mice, suggestive of a pre-existing auditory synaptopathy. Immediately after the noise exposure ABR thresholds rose by 41-62dB to a similar degree in GLAST WT and KO mice and DPOAE remained unaffected. In the acute phase following noise exposure, GLAST KO mice showed near complete de-afferentation unlike WT mice which maintained four to seven paired synapses per IHC. Brain activity using MeMRI found noise exposure to cause greater activity in the inferior colliculus in GLAST KO but not in WT mice. No changes in brain activity was found in GLAST KO mice at baseline in spite of affected afferent synapses, suggesting that auditory synaptopathy may not be sufficient to alter brain activity in the absence of noise exposure.

Preventing presbycusis in mice with enhanced medial olivocochlear feedback

"Growing old" is the most common cause of hearing loss. Age-related hearing loss (ARHL) (presbycusis) first affects the ability to understand speech in background noise, even when auditory thresholds in quiet are normal. It has been suggested that cochlear denervation ("synaptopathy") is an early contributor to age-related auditory decline. In the present work, we characterized age-related cochlear synaptic degeneration and hair cell loss in mice with enhanced α9α10 cholinergic nicotinic receptors gating kinetics ("gain of function" nAChRs). These mediate inhibitory olivocochlear feedback through the activation of associated calcium-gated potassium channels. Cochlear function was assessed via distortion product otoacoustic emissions and auditory brainstem responses. Cochlear structure was characterized in immunolabeled organ of Corti whole mounts using confocal microscopy to quantify hair cells, auditory neurons, presynaptic ribbons, and postsynaptic glutamate receptors. Aged wild-type mice had elevated acoustic thresholds and synaptic loss. Afferent synapses were lost from inner hair cells throughout the aged cochlea, together with some loss of outer hair cells. In contrast, cochlear structure and function were preserved in aged mice with gain-of-function nAChRs that provide enhanced olivocochlear inhibition, suggesting that efferent feedback is important for long-term maintenance of inner ear function. Our work provides evidence that olivocochlear-mediated resistance to presbycusis-ARHL occurs via the α9α10 nAChR complexes on outer hair cells. Thus, enhancement of the medial olivocochlear system could be a viable strategy to prevent age-related hearing loss.

Volume gradients in inner hair cell-auditory nerve fiber pre- and postsynaptic proteins differ across mouse strains

In different animal models, auditory nerve fibers display variation in spontaneous activity and response threshold. Functional and structural differences among inner hair cell ribbon synapses are believed to contribute to this variation. The relative volumes of synaptic proteins at individual synapses might be one such difference. This idea is based on the observation of opposing volume gradients of the presynaptic ribbons and associated postsynaptic glutamate receptor patches in mice along the pillar modiolar axis of the inner hair cell, the same axis along which fibers were shown to vary in their physiological properties. However, it is unclear whether these opposing gradients are expressed consistently across animal models. In addition, such volume gradients observed for separate populations of presynaptic ribbons and postsynaptic glutamate receptor patches suggest different relative volumes of these synaptic structures at individual synapses; however, these differences have not been examined in mice. Furthermore, it is unclear whether such gradients are limited to these synaptic proteins. Therefore, we analyzed organs of Corti isolated from CBA/CaJ, C57BL/6, and FVB/NJ mice using immunofluorescence, confocal microscopy, and quantitative image analysis. We find consistent expression of presynaptic volume gradients across strains of mice and inconsistent expression of postsynaptic volume gradients. We find differences in the relative volume of synaptic proteins, but these are different between CBA/CaJ mice, and C57BL/6 and FVB/NJ mice. We find similar results in C57BL/6 and FVB/NJ mice when using other postsynaptic density proteins (Shank1, Homer, and PSD95). These results have implications for the mechanisms by which volumes of synaptic proteins contribute to variations in the physiology of individual auditory nerve fibers and their vulnerability to excitotoxicity.

LRRTM4: A Novel Regulator of Presynaptic Inhibition and Ribbon Synapse Arrangements of Retinal Bipolar Cells

LRRTM4 is a transsynaptic adhesion protein regulating glutamatergic synapse assembly on dendrites of central neurons. In the mouse retina, we find that LRRTM4 is enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs). Knockout of LRRTM4 reduces RBC axonal GABAA and GABAC receptor clustering and disrupts presynaptic inhibition onto RBC terminals. LRRTM4 removal also perturbs the stereotyped output synapse arrangement at RBC terminals. Synaptic ribbons are normally apposed to two distinct postsynaptic "dyad" partners, but in the absence of LRRTM4, "monad" and "triad" arrangements are also formed. RBCs from retinas deficient in GABA release also demonstrate dyad mis-arrangements but maintain LRRTM4 expression, suggesting that defects in dyad organization in the LRRTM4 knockout could originate from reduced GABA receptor function. LRRTM4 is thus a key synapse organizing molecule at RBC terminals, where it regulates function of GABAergic synapses and assembly of RBC synaptic dyads.

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea

Recent studies have revealed great diversity in the structure, function and efferent innervation of afferent synaptic connections between the cochlear inner hair cells (IHCs) and spiral ganglion neurons (SGNs), which likely enables audition to process a wide range of sound pressures. By performing an extensive electron microscopic reconstruction of the neural circuitry in the mature mouse organ of Corti, we demonstrate that afferent SGN-dendrites differ in strength and composition of efferent innervation in a manner dependent on their afferent synaptic connectivity with IHCs. SGNs that sample glutamate release from several presynaptic ribbons receive more efferent innervation from lateral olivocochlear projections than those driven by a single ribbon. Next to the prevailing unbranched SGN-dendrites, we found branched SGN-dendrites that can contact several ribbons of 1-2 IHCs. Unexpectedly, medial olivocochlear neurons provide efferent innervation of SGN-dendrites, preferring those contacting single-ribbon, pillar-side synapses. We propose a fine-tuning of afferent and efferent SGN-innervation.

Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses.

Glutamate is the major excitatory neurotransmitter in the CNS binding to a variety of glutamate receptors. Metabotropic glutamate receptors (mGluR1 to mGluR8) can act excitatory or inhibitory, depending on associated signal cascades. Expression and localization of inhibitory acting mGluRs at inner hair cells (IHCs) in the cochlea are largely unknown. Here, we analyzed expression of mGluR2, mGluR3, mGluR4, mGluR6, mGluR7, and mGluR8 and investigated their localization with respect to the presynaptic ribbon of IHC synapses. We detected transcripts for mGluR2, mGluR3, and mGluR4 as well as for mGluR7a, mGluR7b, mGluR8a, and mGluR8b splice variants. Using receptor-specific antibodies in cochlear wholemounts, we found expression of mGluR2, mGluR4, and mGluR8b close to presynaptic ribbons. Super resolution and confocal microscopy in combination with 3-dimensional reconstructions indicated a postsynaptic localization of mGluR2 that overlaps with postsynaptic density protein 95 on dendrites of afferent type I spiral ganglion neurons. In contrast, mGluR4 and mGluR8b were expressed at the presynapse close to IHC ribbons. In summary, we localized in detail 3 mGluR types at IHC ribbon synapses, providing a fundament for new therapeutical strategies that could protect the cochlea against noxious stimuli and excitotoxicity.-Klotz, L., Wendler, O., Frischknecht, R., Shigemoto, R., Schulze, H., Enz, R. Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses.

Rod Bipolar Cells Require Horizontal Cells for Invagination Into the Terminals of Rod Photoreceptors.

In the central nervous system, neuronal processing relies on the precisely orchestrated formation of synapses during development. The first synapse of the visual system is a triad synapse, comprising photoreceptors, horizontal cells and bipolar cells. During the second postnatal week, the axon terminal processes of horizontal cells invaginate rod spherules, followed by rod bipolar cell dendrites. Both elements finally oppose the synaptic ribbon (the release site of glutamate). However, it has not been fully elucidated whether horizontal cells are essential for rod bipolar cell dendrites to find their way into the rod terminal. In the present study, we investigated this question by specifically ablating horizontal cells from the early postnatal mouse retina. We monitored the formation of the rod-to-rod bipolar cell synapse during retinal maturation until postnatal day 21. Based on quantitative electron microscopy, we found that without horizontal cells, the dendrites of rod bipolar cells never entered rod terminals. Furthermore, rods displayed significantly fewer and shorter presynaptic ribbons, suggesting that glutamate release is decreased, which coincided with significantly reduced expression of postsynaptic proteins (mGluR6, GPR179) in rod bipolar cells. Collectively, our findings uncover that horizontal cells are indeed necessary guideposts for rod bipolar cells. Whether horizontal cells release diffusible guidance cues or provide structural guidance by expressing specific cell adhesion molecules remains to be seen.

Maternal high-decibel acoustic exposure elevates prenatal stress, impairing postnatal hearing thresholds associated with decreasing ribbon synapses in young rats

Maternal stress may affect the fetal auditory system than direct sound exposure. The objective of this study was to evaluate the role of prenatal stress due to high-decibel (dB) sound exposure on postnatal hearing and cochlear structure. Pregnant rats were exposed to 95 or 65 dB noise or music for 2 h once a day from gestational day 15 until delivery. The serum corticosterone was measured in the pregnant dams and pups. On postnatal day 22, pups underwent auditory brainstem response (ABR) testing. Then, the cochleae were immediately harvested for biochemical and molecular investigations. Prenatal stress impaired reproductive parameters, increased serum corticosterone and ABR thresholds with the decrease in wave I peak amplitude and the number of pre-synaptic ribbon. Thus, prenatal stress induces postnatal hearing loss in young rats, which are related to the reduction of ribbon synapses.

Actin Dynamics, Regulated by RhoA-LIMK-Cofilin Signaling, Mediates Rod Photoreceptor Axonal Retraction After Retinal Injury.

PurposeRetraction of the axon terminals of rod photoreceptors after retinal detachment breaks the first synapse in the visual pathway, resulting in visual impairment. Previous work showed that the mechanism of axonal retraction involves RhoA signaling and its downstream effector LIM Kinase (LIMK) activation. We examined the response of the downstream component cofilin, a direct binding protein of actin filaments, as well as the regulation by RhoA-LIMK-Cofilin signaling of actin assembly/disassembly, in the presynaptic ribbon terminal of injured rod cells.MethodsInjury was produced by retinal detachment or rod cell isolation. Detached porcine retina was probed for levels and localization of phosphorylated cofilin with Western blots and confocal microscopy, whereas rod cell cultures of dissociated salamander retina were examined for filamentous actin assembly/disassembly with a barbed end assay and phalloidin staining.ResultsA detachment increased phosphorylation of cofilin in retinal explants; phosphorylation occurred in rod terminals in sections of detached retina. Isolation of rod cells resulted in axon retraction accompanied by an increase in actin barbed ends and a decrease in net filament labeling. All changes were significantly reduced by either Rho kinase (ROCK) or LIMK inhibition, using Y27632 or BMS-5, respectively. Cytochalasin D also reduced retraction and stabilized filaments in isolated rod cells.ConclusionsThese results indicate that actin depolymerization via activation of RhoA downstream kinases and cofilin contributes to axon retraction. Preventing depolymerization, in addition to actomyosin contraction, may stabilize ribbon synapses after trauma.

Vesicular Glutamatergic Transmission in Noise-Induced Loss and Repair of Cochlear Ribbon Synapses.

Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3 (Vglut3KO ), at 9-11 weeks, approximately half the number of synapses found in Vglut3WT were maintained as postsynaptic AMPA receptors juxtaposed with presynaptic ribbons and voltage-gated calcium channels (CaV1.3). Synapses were larger in Vglut3KO than Vglut3WT In Vglut3WT and Vglut3+/- mice, 8-16 kHz octave-band noise exposure at 100 dB sound pressure level caused a threshold shift (∼40 dB) and a loss of synapses (>50%) at 24 h after exposure. Hearing threshold and synapse number partially recovered by 2 weeks after exposure as ribbons became larger, whereas recovery was significantly better in Vglut3WT Noise exposure at 94 dB sound pressure level caused auditory threshold shifts that fully recovered in 2 weeks, whereas suprathreshold hearing recovered faster in Vglut3WT than Vglut3+/- These results, from mice of both sexes, suggest that spontaneous repair of synapses after noise depends on the level of Vglut3 protein or the level of glutamate release during the recovery period. Noise-induced loss of presynaptic ribbons or postsynaptic AMPA receptors was not observed in Vglut3KO , demonstrating its dependence on vesicular glutamate release. In Vglut3WT and Vglut3+/-, noise exposure caused unpairing of presynaptic ribbons and presynaptic CaV1.3, but not in Vglut3KO where CaV1.3 remained clustered with ribbons at presynaptic active zones. These results suggest that, without glutamate release, noise-induced presynaptic Ca2+ influx was insufficient to disassemble the active zone. However, synapse volume increased by 2 weeks after exposure in Vglut3KO , suggesting glutamate-independent mechanisms.SIGNIFICANCE STATEMENT Hearing depends on glutamatergic transmission mediated by Vglut3, but the role of glutamate in synapse loss and repair is unclear. Here, using mice of both sexes, we show that one copy of the Vglut3 gene is sufficient for noise-induced threshold shift and loss of ribbon synapses, but both copies are required for normal recovery of hearing function and ribbon synapse number. Impairment of the recovery process in mice having only one functional copy suggests that glutamate release may promote synapse regeneration. At least one copy of the Vglut3 gene is necessary for noise-induced synapse loss. Although the excitotoxic mechanism remains unknown, these findings are consistent with the presumption that glutamate is the key mediator of noise-induced synaptopathy.

Noise-Induced Cochlear Synaptopathy and Ribbon Synapse Regeneration: Repair Process and Therapeutic Target.

The synapse between the inner hair cells (IHCs) and the spiral ganglion neurons (SGNs) in mammalian cochleae is characterized as having presynaptic ribbons and therefore is called ribbon synapse. The special molecular organization is reviewed in this chapter in association with the functional feature of this synapse in signal processing. This is followed by the review on noise-induced damage to this synapse with a focus on recent reports in animal models in which the effect of brief noise exposures is observed without causing significant permanent threshold shift (PTS). In this regard, the potential mechanism of the synaptic damage by noise and the impact of this damage on hearing are summarized to clarify the concept of noise-induced hidden hearing loss, which is defined as the functional deficits in hearing without threshold elevation. A controversial issue is addressed in this review as whether the disrupted synapses can be regenerated. Moreover, the review summarizes the work of therapeutic research to protect the synapses or to promote the regeneration of the synapse after initial disruption. Lastly, several unresolved issues are raised for investigation in the future.

Voltage-Gated Calcium Influx Modifies Cholinergic Inhibition of Inner Hair Cells in the Immature Rat Cochlea.

Until postnatal day (P) 12, inner hair cells of the rat cochlea are invested with both afferent and efferent synaptic connections. With the onset of hearing at P12, the efferent synapses disappear, and afferent (ribbon) synapses operate with greater efficiency. This change coincides with increased expression of voltage-gated potassium channels, the loss of calcium-dependent electrogenesis, and the onset of graded receptor potentials driven by sound. The transient efferent synapses include near-membrane postsynaptic cisterns thought to regulate calcium influx through the hair cell's α9-containing and α10-containing nicotinic acetylcholine receptors. This influx activates small-conductance Ca2+-activated K+ (SK) channels. Serial-section electron microscopy of inner hair cells from two 9-d-old (male) rat pups revealed many postsynaptic efferent cisterns and presynaptic afferent ribbons whose average minimal separation in five cells ranged from 1.1 to 1.7 μm. Efferent synaptic function was studied in rat pups (age, 7-9 d) of either sex. The duration of these SK channel-mediated IPSCs was increased by enhanced calcium influx through L-type voltage-gated channels, combined with ryanodine-sensitive release from internal stores-presumably the near-membrane postsynaptic cistern. These data support the possibility that inner hair cell calcium electrogenesis modulates the efficacy of efferent inhibition during the maturation of inner hair cell synapses.SIGNIFICANCE STATEMENT Strict calcium buffering is essential for cellular function. This problem is especially acute for compact hair cells where increasing cytoplasmic calcium promotes the opposing functions of closely adjoining afferent and efferent synapses. The near-membrane postsynaptic cistern at efferent synapses segregates synaptic calcium signals by acting as a dynamic calcium store. The hair cell serves as an informative model for synapses with postsynaptic cisterns (C synapses) found in central neurons.

Concurrent gradients of ribbon volume and AMPA-receptor patch volume in cochlear afferent synapses on gerbil inner hair cells

The Mongolian gerbil is a classic animal model for age-related hearing loss. As a prerequisite for studying age-related changes, we characterized cochlear afferent synaptic morphology in young adult gerbils, using immunolabeling and quantitative analysis of confocal microscopic images. Cochlear wholemounts were triple-labeled with a hair-cell marker, a marker of presynaptic ribbons, and a marker of postsynaptic AMPA-type glutamate receptors. Seven cochlear positions covering an equivalent frequency range from 0.5 – 32 kHz were evaluated. The spatial positions of synapses were determined in a coordinate system with reference to their individual inner hair cell. Synapse numbers confirmed previous reports for gerbils (on average, 20–22 afferents per inner hair cell). The volumes of presynaptic ribbons and postsynaptic glutamate receptor patches were positively correlated: larger ribbons associated with larger receptor patches and smaller ribbons with smaller patches. Furthermore, the volumes of both presynaptic ribbons and postsynaptic receptor patches co-varied along the modiolar-pillar and the longitudinal axes of their hair cell. The gradients in ribbon volume are consistent with previous findings in cat, guinea pig, mouse and rat and further support a role in differentiating the physiological properties of type I afferents. However, the positive correlation between the volumes of pre- and postsynaptic elements in the gerbil is different to the opposing gradients found in the mouse, suggesting species-specific differences in the postsynaptic AMPA receptors that are unrelated to the fundamental classes of type I afferents.

The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse.

The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon's contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles.

Role of Neuropilin-1/Semaphorin-3A signaling in the functional and morphological integrity of the cochlea.

Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.

Effects of Aging and Noise Exposure on Auditory Brainstem Responses and Number of Presynaptic Ribbons in Inner Hair Cells of C57BL/6J Mice

C57BL/6J mice develop age-related hearing loss (HL) early in life. The influence of aging and noise exposure on the number of presynaptic structures in inner hair cells of C57BL/6J mice has not been studied. We monitored auditory brainstem responses, ABRs, in C57BL/6J mice over time and assessed changes in the number of inner hair cell presynaptic ribbons. Multifaceted verification of the effects of aging and noise exposure on hearing in the C57BL/6J strain was performed. The HL was additively increased by noise exposure in 5-week-old mice. The earliest change observed was a decrease in the amplitude of the ABR first wave. Hair cells and spiral ganglion neurons were also lost. Immunohistochemistry and high-resolution confocal microscopy revealed decreased numbers of CtBP2-positive structures in the inner hair cells localized in other than those in the low-frequency region of the cochlea. On the other hand, the influence of acute noise exposure on the inner hair cell ribbons was observed only within the highest-frequency area.

Maximal number of pre-synaptic ribbons are formed in cochlear region corresponding to middle frequency in mice

Objective: To investigate whether there are more quantitative pre-synaptic ribbons formed in the cochlear region corresponding to middle-frequency in cochlea of mice.Methods: Counts of pre-synaptic ribbons were performed using immunostaining and laser confocal microscopy. Hearing thresholds and function of ribbon synapses were estimated by auditory brain response (ABR) and compound action potential (CAP). Cochlear mapping has been achieved to match the frequencies and corresponding regions along the cochlear spiral.Results: The number of pre-synaptic ribbons in per inner hair cell (IHC) has been found to increase gradually from the base turn, the maximal quantity appeared at the region of 50–70% from the apex. Next, ABR thresholds showed that there was the lowest ABR threshold in the frequency around 8–16 kHz, corresponding to the region of 50–70% from the apex according to the cochlear mapping. Further, CAP amplitudes were estimated, and the maximal value identified at the same frequency (8–16 kHz).Conclusions: Maximal number of pre-synaptic ribbons is formed in the cochlear region of middle frequency in mice, coupling with the lowest ABR threshold and highest CAP amplitudes. Our study shows that the middle frequency (8–16 kHz) could be the most sensitive region to sound stimuli in mice.

Afferent synaptogenesis between ectopic hair-cell-like cells and neurites of spiral ganglion induced by Atoh1 in mammals in vitro

Newly formed ectopic hair-cell-like cells (EHCLCs) induced by overexpression of atonal homolog 1 (Atoh1) in vitro were found to possess features of endogenous hair cells (HCs) in previous reports and in the present study. However, limited information is available regarding whether EHCLCs and native spiral ganglion neurons (SGNs) form afferent synapses, which are important for the restoration of hearing. In the current study, we focused on the afferent synaptogenesis between EHCLCs and SGN-derived dendrites. Cochlear explants of auditory epithelia with native SGNs retained were cultured in vitro, and human adenovirus serotype 5 (Ad5) vectors encoding Atoh1 were used to overexpress Atoh1 and induce EHCLCs. We observed that the neurites of the original SGNs extended toward the lesser epithelial ridge (LER) and innervated the EHCLCs. Immunohistochemical analyses revealed the expression of presynaptic ribbon C-terminal-binding protein 2 (CtBP2) and postsynaptic density protein (PSD)-95 in the nerve endings of SGN-derived neurons adjacent to EHCLCs. PSD-95 was located directly opposite CtBP2-positive puncta in the terminals of branches of SGNs, demonstrating that the neurites of SGNs formed afferent-like synaptic connections with EHCLCs. However, the expression of glutamate receptor type 2 (GluR2) could not be detected in the terminals of branches of SGNs surrounding EHCLCs. In addition, we found that the presynaptic ribbon (CtBP2) formation in EHCLCs preceded neural innervation. Furthermore, CtBP2-positive puncta increased and then decreased in EHCLCs, similar to the changes observed in endogenous HCs in terms of their number and distribution. Our finding of the generation of cochlear afferent synapses between EHCLCs and original SGNs will lay the foundation for regenerative approaches to restoring hearing after hair cell loss.