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Syntaxin 3 is a member of a large protein family of syntaxin proteins that mediate fusion between vesicles and their target membranes. Mutations in the ubiquitously expressed syntaxin 3A splice form give rise to a serious gastrointestinal disorder in humans called microvillus inclusion disorder, while mutations that additionally involve syntaxin 3B, a splice form that is expressed primarily in retinal photoreceptors and bipolar cells, additionally give rise to an early onset severe retinal dystrophy. In this review, we discuss recent studies elucidating the roles of syntaxin 3B and the regulation of syntaxin 3B functionality in membrane fusion and neurotransmitter release in the vertebrate retina. [ABSTRACT FROM AUTHOR]

We generated a novel Cre mouse strain for cell-specific deletion of floxed genes in ribbon synapse-forming retinal neurons. Previous studies have shown that the RIBEYE promotor targets the expression of recombinant proteins such as fluorescently tagged RIBEYE to photoreceptors and retinal bipolar cells and generates fluorescent synaptic ribbons in situ in these neurons. Here, we used the same promotor to generate a novel transgenic mouse strain in which the RIBEYE promotor controls the expression of a Cre-ER(T2) recombinase (RIBEYE-Cre). To visualize Cre expression, the RIBEYE-Cre animals were crossed with ROSA26 tau-GFP (R26-τGFP) reporter mice. In the resulting RIBEYE-Cre/R26 τGFP animals, Cre-mediated removal of a transcriptional STOP cassette results in the expression of green fluorescent tau protein (tau-GFP) that binds to cellular microtubules. We detected robust tau-GFP expression in retinal bipolar cells. Surprisingly, we did not find fluorescent tau-GFP expression in mouse photoreceptors. The lack of tau-GFP reporter protein in these cells could be based on the previously reported absence of tau protein in mouse photoreceptors which could lead to the degradation of the recombinant tau protein. Consistent with this, we detected Cre and tau-GFP mRNA in mouse photoreceptor slices by RT-PCR. The transgenic RIBEYE-Cre mouse strain provides a new tool to study the deletion of floxed genes in ribbon synapse-forming neurons of the retina and will also allow for analyzing gene deletions that are lethal if globally deleted in neurons. [ABSTRACT FROM AUTHOR]

Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV. [ABSTRACT FROM AUTHOR]

Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons are presynaptic specializations of ribbon synapses and are anchored to the active zone. Synaptic ribbons bind many synaptic vesicles that are delivered to the active zone for continuous and faithful signalling. In the present study we demonstrate with independent antibodies at the light- and electron microscopic level that rabconnectin-3α (RC3α)—alternative name Dmx-like 2 (DMXL2)—is localized to the synaptic ribbons of rod photoreceptor synapses in the mouse retina. In the brain, RC3α-containing complexes are known to interact with important components of synaptic vesicles, including Rab3-activating/inactivating enzymes, priming proteins and the vesicular H+-ATPase that acidifies the synaptic vesicle lumen to promote full neurotransmitter loading. The association of RC3α/DMXL2 with rod synaptic ribbons of the mouse retina could enable these structures to deliver only fully signalling-competent synaptic vesicles to the active zone thus contributing to reliable synaptic communication. [ABSTRACT FROM AUTHOR]

Thyroid hormone deficiency can lead to abnormal auditory development of varying severity. Retardation of morphological development, including delays in degeneration of Kölliker's organ and subsequent delayed formation of the inner sulcus, along with delayed opening of the tunnel of Corti and malformation of the tectorial membrane, was consistently observed in an antithyroid drug-induced congenital hypothyroidism rodent model. Abnormal morphological development could partly explain impaired adult auditory function. However, whether the development of inner hair cell ribbon synapses is influenced by hypothyroidism remains unclear. In the present study, we characterize the normal degeneration pattern of Kölliker's organ along the basal-to-apical axis. Then, we verified the retardation of morphological development in congenital hypothyroid mice. Using this model, we found that twisted collagen is present in the major tectorial membrane and delayed separation from supporting cells affects the minor tectorial membrane. Finally, we found that the number of synaptic ribbons was not significantly altered but the ribbon synapse maturation process was significantly impaired in congenital hypothyroid mice. We conclude that thyroid hormone is involved in structural development of the tectorial membrane and the ribbon synapse maturation process. • Kölliker's organ began to degenerate between P4 and P5 at the basal turn and between P6 and P7 at the apical turn. • Despite delays, Kölliker's organ followed the typical basal-to-apical axis of degeneration in hypothyroid mice. • Impaired maturation of the tectorial membrane and ribbon synapse was observed in congenital hypothyroid mice. [ABSTRACT FROM AUTHOR]

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the β subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells. [ABSTRACT FROM AUTHOR]

Background: Obesity is an independent risk factor for hearing loss. Although attention has focused on major obesity comorbidities such as cardiovascular disease, stroke, and type 2 diabetes, the impact of obesity on sensorineural organs, including the auditory system, is unclear. Using a high-fat diet (HFD)-induced obese mouse model, we investigated the impact of diet-induced obesity on sexual dimorphism in metabolic alterations and hearing sensitivity. Methods: Male and female CBA/Ca mice were randomly assigned to three diet groups and fed, from weaning (at 28 days) to 14 weeks of age, a sucrose-matched control diet (10 kcal% fat content diet), or one of two HFDs (45 or 60 kcal% fat content diets). Auditory sensitivity was evaluated based on the auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and ABR wave 1 amplitude at 14 weeks of age, followed by biochemical analyses. Results: We found significant sexual dimorphism in HFD-induced metabolic alterations and obesity-related hearing loss. Male mice exhibited greater weight gain, hyperglycemia, increased ABR thresholds at low frequencies, elevated DPOAE, and lower ABR wave 1 amplitude compared to female mice. The hair cell (HC) ribbon synapse (CtBP2) puncta showed significant sex differences. The serum concentration of adiponectin, an otoprotective adipokine, was significantly higher in female than in male mice; cochlear adiponectin levels were elevated by HFD in female but not male mice. Adiponectin receptor 1 (AdipoR1) was widely expressed in the inner ear, and cochlear AdipoR1 protein levels were increased by HFD, in female but not male mice. Stress granules (G3BP1) were significantly induced by the HFD in both sexes; conversely, inflammatory (IL-1β) responses were observed only in the male liver and cochlea, consistent with phenotype HFD-induced obesity. Conclusions: Female mice are more resistant to the negative effects of an HFD on body weight, metabolism, and hearing. Females showed increased peripheral and intra-cochlear adiponectin and AdipoR1 levels, and HC ribbon synapses. These changes may mediate resistance to HFD-induced hearing loss seen in female mice. Highlights: Females are protected against HFD-induced metabolic alterations and hearing impairment. Sex differences were seen in the ABR wave 1 amplitude, which correlated with loss of hair-cell ribbon synapses (CtBP2). The serum adiponectin concentration was significantly higher in female compared to male mice, and the cochlear adiponectin levels were elevated by HFD in females, but not male mice. Cochlear and liver AdipoR1 expression evidenced significant sex differences in CBA/Ca mice. The core stress granule component, G3BP1, was induced by HFD in the cochleae of male and female mice. Significant sex differences were found in cochlear blood flow and IL-1β concentration. [ABSTRACT FROM AUTHOR]

As noise-induced hearing loss (NIHL) is a leading cause of occupational diseases, there is an urgent need for the development of preventive and therapeutic interventions. To avoid user-compliance-based problems occurring with conventional protection devices, the pharmacological prevention is currently in the focus of hearing research. Noise exposure leads to an increase in reactive oxygen species (ROS) in the cochlea. This way antioxidant agents are a promising option for pharmacological interventions. Previous animal studies reported preventive as well as therapeutic effects of Insulin-like growth factor 1 (IGF-1) in the context of NIHL. Unfortunately, in patients the time point of the noise trauma cannot always be predicted, and additive effects may occur. Therefore, continuous prevention seems to be beneficial. The present study aimed to investigate the preventive potential of continuous administration of low concentrations of IGF-1 to the inner ear in an animal model of NIHL. Guinea pigs were unilaterally implanted with an osmotic minipump. One week after surgery they received noise trauma, inducing a temporary threshold shift. Continuous IGF-1 delivery lasted for seven more days. It did not lead to significantly improved hearing thresholds compared to control animals. Quite the contrary, there is a hint for a higher noise susceptibility. Nevertheless, changes in the perilymph proteome indicate a reduced damage and better repair mechanisms through the IGF-1 treatment. Thus, future studies should investigate delivery methods enabling continuous prevention but reducing the risk of an overdosage. [ABSTRACT FROM AUTHOR]

Embryonic hyperglycemia negatively impacts retinal development, leading to abnormal visual behavior, altered timing of retinal progenitor differentiation, decreased numbers of retinal ganglion cells and Müller glia, and vascular leakage. Because synaptic disorganization is a prominent feature of many neurological diseases, the goal of the current work was to study the potential impact of hyperglycemia on retinal ribbon synapses during embryonic development. Our approach utilized reverse transcription quantitative PCR (RT-qPCR) and immunofluorescence labeling to compare the transcription of synaptic proteins and their localization in hyperglycemic zebrafish embryos, respectively. Our data revealed that the maturity of synaptic ribbons was compromised in hyperglycemic zebrafish larvae, where altered ribeye expression coincided with the delay in establishing retinal ribbon synapses and an increase in the immature synaptic ribbons. Our results suggested that embryonic hyperglycemia disrupts retinal synapses by altering the development of the synaptic ribbon, which can lead to visual defects. Future studies using zebrafish models of hyperglycemia will allow us to study the underlying mechanisms of retinal synapse development. [ABSTRACT FROM AUTHOR]

Retinal bipolar cells (BCs) compose the canonical vertical excitatory pathway that conveys photoreceptor output to inner retinal neurons. Although synaptic transmission from BC terminals is thought to rely almost exclusively on Ca21 influx through voltage-gated Ca21 (CaV) channels mediating L-type currents, the molecular identity of CaV channels in BCs is uncertain. Therefore, we combined molecular and functional analyses to determine the expression profiles of CaV a1, b, and a2d subunits in mouse rod bipolar (RB) cells, BCs from which the dynamics of synaptic transmission are relatively well-characterized. We found significant heterogeneity in CaV subunit expression within the RB population from mice of either sex, and significantly, we discovered that transmission from RB synapses was mediated by Ca21 influx through P/Q-type (CaV2.1) and N-type (CaV2.2) conductances as well as the previouslydescribed L-type (CaV1) and T-type (CaV3) conductances. Furthermore, we found both CaV1.3 and CaV1.4 proteins located near presynaptic ribbon-type active zones in RB axon terminals, indicating that the L-type conductance is mediated by multiple CaV1 subtypes. Similarly, CaV3 a1, b, and a2d subunits also appear to obey a "multisubtype" rule, i.e., we observed a combination of multiple subtypes, rather than a single subtype as previously thought, for each CaV subunit in individual cells. [ABSTRACT FROM AUTHOR]

• Effects of head and neck radiation therapy on auditory function were investigated. • Mice were irradiated to target the head and neck once at different doses. • Radiotherapy can cause SNHL via disruption of cochlear ribbon synapses. • A higher dose of radiotherapy can cause more serious hearing loss. To investigate the possible effects of head and neck radiotherapy on hearing function in mice. Adult C57BL/6J mice were irradiated to the head and neck once with cobalt-60 rays at doses of 10 Gy or 20 Gy. Hearing function was estimated by the detection of auditory brainstem response (ABR) thresholds and the suprathreshold function of cochlear was indicated by the peak amplitudes and latencies of wave I. The mice were tested on days 1, 7, 14, and 21 after radiation treatment, and untreated mice in littermates served as controls. The cochlear pre-synaptic ribbons were labeled using an anti-RIBEYE/CtBP2 antibody, and the synaptic vesicle membrane was traced using anti-vesicular glutamate transporter 3 (VGLUT-3) antibody. The number and size of the pre-synaptic ribbons were counted along the cochlear axis from the apex to the base. The expression of VGLUT-3 was measured by the intensity of immunofluorescence. Hematoxylin and eosin (H&E) staining was also performed to evaluate the structural changes in the cochlea. Compared with the controls, mice treated with 10 Gy and 20 Gy doses on days 1, 7, 14, and 21 were found to have significant disruptions in ABR thresholds and amplitudes (p < 0.05). Moreover, mice in the 20 Gy group, compared with the 10 Gy group, showed greater hearing loss and suprathreshold deficits (p < 0.05). Quantitative analysis revealed a decrease in the number and size of CtBP2-positive puncta in both the 10 Gy and 20 Gy groups compared with the controls (p < 0.05); in the 20 Gy group, the number and size of CtBP2-positive puncta were less than those in the 10 Gy group (p < 0.05). We observed a significant disruption in the expression of VGLUT-3 in the group treated with 20 Gy. However, compared with the control group, both immunofluorescence and H&E staining revealed no significant changes in the number of hair cells or the array for the 10 or 20 Gy treatments (p > 0.05). Radiation therapy targeting the head and neck can cause sensorineural hearing loss via disruption specific to the cochlear ribbon synapses. To our knowledge, this is the first study to demonstrate that cochlear ribbon synapses may be a subcellular target of radiation-induced hearing loss. [ABSTRACT FROM AUTHOR]

Glutamate release from rod and cone photoreceptor cells involves presynaptic ribbons composed largely of the protein RIBEYE. To examine roles of ribbons in rods and cones, we studied mice in which GCamP3 replaced the B-domain of RIBEYE. We discovered that ribbons were absent from rods and cones of both knock-in mice possessing GCamP3 and conditional RIBEYE knockout mice. The mice lacking ribbons showed reduced temporal resolution and contrast sensitivity assessed with optomotor reflexes. ERG recordings showed 50% reduction in scotopic and photopic b-waves. The readily releasable pool (RRP) of vesicles in rods and cones measured using glutamate transporter anion currents (IA(glu)) was also halved. We also studied the release from cones by stimulating them optogenetically with ChannelRhodopsin2 (ChR2) while recording postsynaptic currents in horizontal cells. Recovery of the release from paired pulse depression was twofold slower in the rods and cones lacking ribbons. The release from rods at −40 mV in darkness involves regularly spaced multivesicular fusion events. While the regular pattern of release remained in the rods lacking ribbons, the number of vesicles comprising each multivesicular event was halved. Our results support conclusions that synaptic ribbons in rods and cones expand the RRP, speed up vesicle replenishment, and augment some forms of multivesicular release. Slower replenishment and a smaller RRP in photoreceptors lacking ribbons may contribute to diminished temporal frequency responses and weaker contrast sensitivity. [ABSTRACT FROM AUTHOR]

This study was focused on impulse noise induces hidden hearing loss. This study was designed to determine the morphology changes of noise-induced hidden hearing loss (NIHHL). Fifteen guinea pigs were divided into three groups: noise-induced hidden hearing loss (NIHHL) group, noise-induced hearing loss (NIHL) group, and normal control group. For the NIHHL group, guinea pigs were exposed to 15 times of impulse noise with peak intensity of 163 dB SPL at one time. For the NIHL group, animals were exposed to two rounds of 100 times impulse noise, and the time interval is 24 h. Auditory brain response (ABR) was tested before, immediately, 24 h, one week, and one month after noise exposure to evaluate cochlear physiology changes. One month after noise exposure, all guinea pigs in three groups were sacrificed, and basement membranes were carefully dissected immediately after ABR tests. The cochlea samples were observed by transmission electron microscopy (TEM) to find out the morphology changes. The ABR results showed that 15 times of impulse noise exposure could cause NIHHL in guinea pigs and 200 times could cause completely hearing loss. Impulse noise exposure could cause a dramatic increase of mitochondria in the inner hair cell. The structures of ribbon synapse and heminode were also obviously impaired compared to the normal group. The nerve fiber and myelin sheath remained intact after impulse noise exposure. This research revealed that impulse noise could cause hidden hearing loss, and the changes in inner hair cells, ribbon synapse, and heminode all played a vital role in the pathogenesis of hidden hearing loss. [ABSTRACT FROM AUTHOR]

The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is crucial for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of bassoon to the OPL with each stage (P<0.0001). By applying these OPL staging criteria to human retinal organoids (HROs) derived from human embryonic and induced pluripotent stem cells, we observed comparable maturation from OPL-Stage 0 at day 100 in culture up to OPL-Stage 3 by day 160. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (P<0.0001). Overall, this study defines stages of human OPL development through mid-gestation and establishes HROs as a model system that recapitulates key aspects of human photoreceptor-bipolarcell synaptogenesis in vitro. [ABSTRACT FROM AUTHOR]

• Myosin VI haploinsufficiency leads to mild hearing loss in mice. • Heterozygous myosin VI knockout mice developed age-related stereocilia fusion. • The outer hair cells of aged mice decreased due to myosin VI haploinsufficiency. • The ribbon synapses of aged mice were decreased due to myosin VI haploinsufficiency. In human, myosin VI (MYO6) haploinsufficiency causes postlingual progressive hearing loss. Because the usefulness of mouse models remains unclear, we produced novel Myo6 null (−/−) mutant mice and analyzed the hearing phenotypes of Myo6 +/− (+/−) heterozygous mutants. We first recorded and compared the auditory brainstem responses and distortion product otoacoustic emissions in control Myo6 +/+ (+/+) wild-type and +/− mice. These hearing phenotypes of +/− mice were mild; however, we confirmed that +/− mice developed progressive hearing loss. In particular, the hearing loss of female +/− mice progressed faster than that of male +/− mice. The stereocilia bundles of +/− mice exhibited progressive taper loss in cochlear inner hair cells (IHCs) and outer hair cells (OHCs). The loss of OHCs in +/− heterozygotes occurred at an earlier age than in +/+ mice. In particular, the OHCs at the basal area of the cochlea were decreased in +/− mice. IHC ribbon synapses from the area at the base of the cochlea were significantly reduced in +/− mice. Thus, our study indicated that MYO6 haploinsufficiency affected the detection of sounds in mice, and we suggest that +/− mice with Myo6 null alleles are useful animal models for gene therapy and drug treatment in patients with progressive hearing loss due to MYO6 haploinsufficiency. [ABSTRACT FROM AUTHOR]

Cochlear synaptopathy is the noise-induced or age-related loss of ribbon synapses between inner hair cells (IHCs) and auditorynerve fibers (ANFs), first reported in CBA/CaJ mice. Recordings from single ANFs in anesthetized, noise-exposed guinea pigs suggested that neurons with low spontaneous rates (SRs) and high thresholds are more vulnerable than low-threshold, high-SR fibers. However, there is extensive postexposure regeneration of ANFs in guinea pigs but not in mice. Here, we exposed CBA/ CaJ mice to octave-band noise and recorded sound-evoked and spontaneous activity from single ANFs at least 2 wk later. Confocal analysis of cochleae immunostained for pre- and postsynaptic markers confirmed the expected loss of 40%-50% of ANF synapses in the basal half of the cochlea; however, our data were not consistent with a selective loss of low-SR fibers. Rather they suggested a loss of both SR groups in synaptopathic regions. Single-fiber thresholds and frequency tuning recovered to pre-exposure levels; however, response to tone bursts showed increased peak and steady-state firing rates, as well as decreased jitter in first-spike latencies. This apparent gain-of-function increased the robustness of tone-burst responses in the presence of continuous masking noise. This study suggests that the nature of noise-induced synaptic damage varies between different species and that, in mouse, the noise-induced hyperexcitability seen in central auditory circuits is also observed at the level of the auditory nerve. NEW & NOTEWORTHY Noise-induced damage to synapses between inner hair cells and auditory-nerve fibers (ANFs) can occur without permanent hair cell damage, resulting in pathophysiology that "hides" behind normal thresholds. Prior single-fiber neurophysiology in guinea pig suggested that noise selectively targets high-threshold ANFs. Here, we show that the lingering pathophysiology differs in mouse, with both ANF groups affected and a paradoxical gain-of-function in surviving low-threshold fibers, including increased onset rate, decreased onset jitter, and reduced maskability. [ABSTRACT FROM AUTHOR]

Glutamate is the most pivotal excitatory neurotransmitter in the central nervous system. Metabotropic glutamate receptors (mGluRs) dimerize and can couple to inhibitory intracellular signal cascades, thereby protecting glutamatergic neurons from excessive excitation and cell death. MGluR7 is correlated with age‐related hearing deficits and noise‐induced hearing loss; however its exact localization in the cochlea is unknown. Here, we analyzed the expression and localization of mGluR7a and mGluR7b in mouse cochlear wholemounts in detail, using confocal microscopy and 3D reconstructions. We observed a presynaptic localization of mGluR7a at inner hair cells (IHCs), close to the synaptic ribbon. To detect mGluR7b, newly generated antibodies were characterized and showed co‐localization with mGluR7a at IHC ribbon synapses. Compared to the number of synaptic ribbons, the numbers of mGluR7a and mGluR7b puncta were reduced at higher frequencies (48 to 64 kHz) and in older animals (6 and 12 months). Previously, we reported a presynaptic localization of mGluR4 and mGluR8b at this synapse type. This enables the possibility for the formation of homo‐ and/or heterodimeric receptors composed of mGluR4, mGluR7a, mGluR7b and mGluR8b at IHC ribbon synapses. These receptor complexes might represent new molecular targets suited for pharmacological concepts to protect the cochlea against noxious stimuli and excitotoxicity. [ABSTRACT FROM AUTHOR]

Aging, disease, and trauma can lead to loss of vestibular hair cells and permanent vestibular dysfunction. Previous work showed that, following acute destruction of; 95% of vestibular hair cells in adult mice,; 20% regenerate naturally (without exogenous factors) through supporting cell transdifferentiation. There is, however, no evidence for the recovery of vestibular function. To gain insight into the lack of functional recovery, we assessed functional differentiation in regenerated hair cells for up to 15months, focusing on key stages in stimulus transduction and transmission: hair bundles, voltage-gated conductances, and synaptic contacts. Regenerated hair cells had many features of mature type II vestibular hair cells, including polarized mechanosensitive hair bundles with zone-appropriate stereocilia heights, large voltage-gated potassium currents, basolateral processes, and afferent and efferent synapses. Regeneration failed, however, to recapture the full range of properties of normal populations, and many regenerated hair cells had some properties of immature hair cells, including small transduction currents, voltage-gated sodium currents, and small or absent HCN (hyperpolarizationactivated cyclic nucleotide-gated) currents. Furthermore, although mouse vestibular epithelia normally have slightly more type I hair cells than type II hair cells, regenerated hair cells acquired neither the low-voltage-activated potassium channels nor the afferent synaptic calyces that distinguish mature type I hair cells from type II hair cells and confer distinctive physiology. Thus, natural regeneration of vestibular hair cells in adult mice is limited in total cell number, cell type diversity, and extent of cellular differentiation, suggesting that manipulations are needed to promote full regeneration with the potential for recovery of vestibular function. [ABSTRACT FROM AUTHOR]

Light-evoked voltage responses of rod and cone photoreceptor cells in the vertebrate retina must be converted to a train of synaptic vesicle release events for transmission to downstream neurons. This review discusses the processes, proteins, and structures that shape this critical early step in vision, focusing on studies from salamander retina with comparisons to other experimental animals. Many mechanisms are conserved across species. In cones, glutamate release is confined to ribbon release sites although rods are also capable of release at non-ribbon sites. The role of non-ribbon release in rods remains unclear. Release from synaptic ribbons in rods and cones involves at least three vesicle pools: a readily releasable pool (RRP) matching the number of membrane-associated vesicles along the ribbon base, a ribbon reserve pool matching the number of additional vesicles on the ribbon, and an enormous cytoplasmic reserve. Vesicle release increases in parallel with Ca2+ channel activity. While the opening of only a few Ca2+ channels beneath each ribbon can trigger fusion of a single vesicle, sustained release rates in darkness are governed by the rate at which the RRP can be replenished. The number of vacant release sites, their functional status, and the rate of vesicle delivery in turn govern replenishment. Along with an overview of the mechanisms of exocytosis and endocytosis, we consider specific properties of ribbon-associated proteins and pose a number of remaining questions about this first synapse in the visual system. [ABSTRACT FROM AUTHOR]

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation. • Cochlear ribbon synapses undergo extensive morphological maturation after birth. • Early postnatal hypothyroidism impairs the overall maturation of ribbon synapses. • Early postnatal hyperthyroidism results in accelerated pruning of ribbon synapses. • Synaptic pruning by hyperthyroidism is reversible and does not affect hearing. [ABSTRACT FROM AUTHOR]

Ribbon‐class synapses in the ear achieve analog to digital transformation of a continuously graded membrane potential to all‐or‐none spikes. In mammals, several auditory nerve fibres (ANFs) carry information from each inner hair cell (IHC) to the brain in parallel. Heterogeneity of transmission among synapses contributes to the diversity of ANF sound‐response properties. In addition to the place code for sound frequency and the rate code for sound level, there is also a temporal code. In series with cochlear amplification and frequency tuning, neural representation of temporal cues over a broad range of sound levels enables auditory comprehension in noisy multi‐speaker settings. The IHC membrane time constant introduces a low‐pass filter that attenuates fluctuations of the receptor potential above 1–2 kHz. The ANF spike generator adds a high‐pass filter via its depolarization‐rate threshold that rejects slow changes in the postsynaptic potential and its phasic response property that ensures one spike per depolarization. Synaptic transmission involves several stochastic subcellular processes between IHC depolarization and ANF spike generation, introducing delay and jitter that limits the speed and precision of spike timing. ANFs spike at a preferred phase of periodic sounds in a process called phase‐locking that is limited to frequencies below a few kilohertz by both the IHC receptor potential and the jitter in synaptic transmission. During phase‐locking to periodic sounds of increasing intensity, faster and facilitated activation of synaptic transmission and spike generation may be offset by presynaptic depletion of synaptic vesicles, resulting in relatively small changes in response phase. Here we review encoding of spike‐timing at cochlear ribbon synapses. [ABSTRACT FROM AUTHOR]

Background: The SCN11A gene, encoded Nav1.9 TTX resistant sodium channels, is a main effector in peripheral inflammation related pain in nociceptive neurons. The role of SCN11A gene in the auditory system has not been well characterized. We therefore examined the expression of SCN11A in the murine cochlea, the morphological and physiological features of Nav1.9 knockout (KO) ICR mice.Results: Nav1.9 expression was found in the primary afferent endings beneath the inner hair cells (IHCs). The relative quantitative expression of Nav1.9 mRNA in modiolus of wild-type (WT) mice remains unchanged from P0 to P60. The number of presynaptic CtBP2 puncta in Nav1.9 KO mice was significantly lower than WT. In addition, the number of SGNs in Nav1.9 KO mice was also less than WT in the basal turn, but not in the apical and middle turns. There was no lesion in the somas and stereocilia of hair cells in Nav1.9 KO mice. Furthermore, Nav1.9 KO mice showed higher and progressive elevated ABR threshold at 16 kHz, and a significant increase in CAP thresholds.Conclusions: These data suggest a role of Nav1.9 in regulating the function of ribbon synapses and the auditory nerves. The impairment induced by Nav1.9 gene deletion mimics the characters of cochlear synaptopathy. [ABSTRACT FROM AUTHOR]

Aim: Off cone bipolar cells of the mammalian retina connect to cone photoreceptor synaptic terminals via non‐invaginating flat contacts at a considerable distance from the only established neurotransmitter release site so far, the synaptic ribbon. Diffusion from the ribbon synaptic active zone is considered the most likely mechanism for the neurotransmitter glutamate to reach postsynaptic receptors on the dendritic tips of Off cone bipolar cells. We used a mutant mouse with functionally impaired photoreceptor ribbon synapses to investigate the importance of intact ribbon synaptic active zones for signal transmission at Off cone bipolar cell contacts. Methods: Whole‐cell patch‐clamp recordings from Off cone bipolar cells in a horizontal slice preparation of wildtype (Bsnwt) and mutant (BsnΔEx4/5) mouse retina were applied to investigate signal transmission between cone photoreceptors and Off cone bipolar cells. The distribution of postsynaptic glutamate receptors in Off cone bipolar cell dendrites was studied using multiplex immunocytochemistry. Results: Tonic synaptic activity and evoked release were significantly reduced in mutant animals. Vesicle replenishment rates and the size of the readily releasable pool were likewise decreased. The precisely timed transient current response to light offset changed to a sustained response in the mutant, exemplified by random release events only loosely time‐locked to the stimulus. The kainate receptor distribution in postsynaptic Off cone bipolar cell dendritic contacts in BsnΔEx4/5 mice was largely disturbed. Conclusion: Our results suggest a major role of functional ribbon synaptic active zones for signal transmission and postsynaptic glutamate receptor organization at flat Off cone bipolar cell contacts. [ABSTRACT FROM AUTHOR]

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca2+ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca2+ imaging to examine Ca2+ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca2+ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca2+ ATPase (PMCA), the mitochondrial Ca2+ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca2+ ATPase (SERCA), but not the Na+/Ca2+ exchanger (NCX). Furthermore, we found that both the basal Ca2+ level and the Ca2+ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca2+ buffer, multiple Ca2+ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phase locking of auditory-nerve-fiber (ANF) responses to the temporal fine structure of acoustic stimuli, a hallmark of the auditory system's temporal precision, is important for many aspects of hearing. Previous work has shown that phase-locked period histograms are often well described by exponential transfer functions relating instantaneous stimulus pressure to instantaneous spike rate, with no observed clipping of the histograms. The operating points and slopes of these functions change with stimulus level. The mechanism underlying this apparent gain control is unclear but is distinct from mechanical compression, is independent of refractoriness and spike-rate adaptation, and is apparently instantaneous. Here we show that these findings can be accounted for by a model consisting of a static Boltzmann transducer function yielding a clipped output, followed by a lowpass filter and a static exponential transfer function. Using responses to tones of ANFs from cats of both sexes, we show that, for a given ANF, the period histograms obtained at all stimulus levels for a given stimulus frequency can be described using one set of levelindependent model parameters. The model also accounts for changes in the maximum and minimum instantaneous spike rates with changes in stimulus level. Notably, the estimated cutoff frequency is lower for low- than for high-spontaneous-rate ANFs, implying a synapse-specific contribution to lowpass filtering. These findings advance our understanding of ANF phase locking by highlighting the role of peripheral filtering mechanisms in shaping responses of individual ANFs. [ABSTRACT FROM AUTHOR]

• Autophagy is essential for the development of ribbon synapse. • Autophagy is involved in the refinement of ribbon synapse. • Autophagy contributes to the establishment of normal auditory function. Cochlear ribbon synapses formed between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are immature at birth and they require dramatic morphological and functional developments to achieve auditory maturation in postnatal mice. However, the mechanism underlying this remodeling process of cochlear ribbon synapse remains elusive. Here, we report that autophagy is necessary for the development and maturation of cochlear ribbon synapses in mice. In this study, significantly high levels of LC3B (a widespread marker of autophagy) were found in the cochlea from postnatal day 1 (P1) to P15, which then decreased at P28 to P30. Treatment of mice at P7 with rapamycin or 3-methyladenine (activator and inhibitor of autophagy, respectively) for 7 days led to the significant elevations of hearing threshold across frequencies from P15 to P30. Moreover, abnormal morphology of cochlear ribbon synapses and reduced IHC exocytosis function were detected from P15 to P30, which were likely associated to hearing impairment. Thus, our study demonstrated that autophagy was required for remodeling of cochlear ribbon synapses and provided a new insight into autophagy-related hearing disorder during auditory development. Furthermore, we implicated a novel therapeutic target for sensorineural hearing loss. [ABSTRACT FROM AUTHOR]

In vertebrate central nervous systems (CNSs), highly diverse neurons are selectively connected via synapses, which are essential for building an intricate neural network. The vertebrate retina is part of the CNS and is comprised of a distinct laminar organization, which serves as a good model system to study developmental synapse formation mechanisms. In the retina outer plexiform layer, rods and cones, two types of photoreceptor cells, elaborate selective synaptic contacts with ON- and/or OFF-bipolar cell terminals as well as with horizontal cell terminals. In the mouse retina, three photoreceptor subtypes and at least 15 bipolar subtypes exist. Previous and recent studies have significantly progressed our understanding of how selective synapse formation, between specific subtypes of photoreceptor and bipolar cells, is designed at the molecular level. In the ON pathway, photoreceptor-derived secreted and transmembrane proteins directly interact in trans with the GRM6 (mGluR6) complex, which is localized to ON-bipolar cell dendritic terminals, leading to selective synapse formation. Here, we review our current understanding of the key factors and mechanisms underlying selective synapse formation of photoreceptor cells with bipolar and horizontal cells in the retina. In addition, we describe how defects/mutations of the molecules involved in photoreceptor synapse formation are associated with human retinal diseases and visual disorders. [ABSTRACT FROM AUTHOR]

• We investigated whether repeated induction of temporary hearing threshold shifts can cause a permanent threshold shift. • Young adult C57BL/6J mice were exposed twice to moderate white noise (NE) in a design that minimized the effects of aging. • A first NE gave a reversible hearing loss as auditory brainstem response thresholds, amplitudes, and cochlear synapses. • A second NE with the same parameters caused persistent threshold shifts, amplitude reductions, and synaptic losses. • We found cumulative effects of repeated NE on hearing and synaptic plasticity not consistent with age-related hearing loss. Repeated induction of a temporary threshold shift (TTS) may result in a permanent threshold shift (PTS) and is thought to be associated with early onset of age-related hearing loss (ARHL). The possibility that a PTS might be induced by administration of repeated TTS-inducing noise exposures (NEs) over a short period during early adulthood has not been formally investigated. We aimed to investigate possible cumulative acoustic overstimulation effects that permanently shift the auditory threshold. Young adult C57BL/6J mice were exposed twice to moderate white noise in an experimental design that minimized the effects of aging. The first exposure resulted in a reversible noise-induced hearing loss (NIHL) measured as recoverable alterations in auditory brainstem response (ABR) thresholds, waveform amplitudes, and numbers of ribbon synapses. The second NE with the same parameters caused persistent threshold shifts, wave I amplitude reductions, wave IV/I ratio enhancements, and synaptic losses, even though recovery time sufficient for a TTS had been provided. The pattern of PTS resembled NIHL since the observed impairments tonotopically followed the power spectrum of the noise insult, rather than ARHL, which distributes at higher frequencies. No significant changes were observed in the control group as the mice aged. To conclude, our results demonstrate a cumulative effect of repetitive TTS-inducing NE on hearing function and synaptic plasticity that does not cause premature ARHL, thereby providing insight into the pathophysiological mechanisms underlying NIHL and ARHL. [ABSTRACT FROM AUTHOR]

Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel's activation threshold toward more hyperpolarized potentials. Taken together, the association with "slow" β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function. [ABSTRACT FROM AUTHOR]

• Rat models are established with prenatal high (stress) or low dB sound exposure. • Prenatal stress due to high sound exposure (95 dB) causes both hearing and ribbon synaptic impairments. • Prenatal low sound exposure (65 dB) sound did not induce these changes. 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. [ABSTRACT FROM AUTHOR]

More than 5% of the world population lives with a hearing impairment. The main factors responsible for hearing degeneration are ototoxic drugs, aging, continued exposure to excessive noise and infections. The pool of adult stem cells in the inner ear drops dramatically after birth, and therefore an endogenous cellular source for regeneration is absent. Hearing loss can emerge after the degeneration of different cochlear components, so there are multiple targets to be reached, such as hair cells (HCs), spiral ganglion neurons (SGNs), supporting cells (SCs) and ribbon synapses. Important discoveries in the hearing regeneration field have been reported regarding stem cell transplantation, migration and survival; genetic systems for cell fate monitoring; and stem cell differentiation to HCs, SGNs and SCs using adult stem cells, embryonic stem cells and induced pluripotent stem cells. Moreover, some molecular mediators that affect the establishment of functional synapses have been identified. In this review, we will focus on reporting the state of the art in the regenerative medicine field for hearing recovery. Stem cell research has enabled remarkable advances in regeneration, particularly in neuronal cells and synapses. Despite the progress achieved, there are certain issues that need a deeper development to improve the results already obtained, or to develop new approaches aiming for the clinical application. [ABSTRACT FROM AUTHOR]

Sensory hair cells of the inner ear are exposed to continuous mechanical stress, causing damage over time. The maintenance of hair cells is further challenged by damage from a variety of other ototoxic factors, including loud noise, aging, genetic defects, and ototoxic drugs. This damage can manifest in many forms, from dysfunction of the hair cell mechanotransduction complex to loss of specialized ribbon synapses, and may even result in hair cell death. Given that mammalian hair cells do not regenerate, the repair of hair cell damage is important for continued auditory function throughout life. Here, we discuss how several key hair cell structures can be damaged, and what is known about how they are repaired. Hair cells acquire damage from a variety of environmental and genetic factors. To fully maintain auditory function, damaged hair cells must be repaired. Broken tip links are repaired in both regenerating and nonregenerating hair cells. Intense noise exposure damages the stereocilia F-actin core, which may be repaired by localized F-actin remodeling. Ribbon synapse loss, which can reduce hearing ability in noisy environments, may or may not be reversible. Holes in the sensory epithelium caused by the extrusion of dying hair cells are sealed by projections from nearby supporting cells to preserve barrier integrity. [ABSTRACT FROM AUTHOR]

Abstract Strial dysfunction is commonly observed as a key consequence of aging in the cochlea. A large body of animal research, especially in the quiet-aged Mongolian gerbil, shows specific histopathological changes in the cochlear stria vascularis and the putatively corresponding effects on endocochlear potential and auditory nerve responses. However, recent work suggests that synaptopathy, or the loss of inner hair cell-auditory nerve fiber synapses, also presents as a consequence of aging. It is now believed that the loss of synapses is the earliest age-related degenerative event. The present review aims to integrate classic and novel research on age-related pathologies of the inner ear. First, we summarize current knowledge on age-related strial dysfunction and synaptopathy. We describe how these cochlear pathologies fit into the categories for presbyacusis, as first defined by Schuknecht in the '70s. Further, we discuss how strial dysfunction and synaptopathy affect sound coding by the auditory nerve and how they can be experimentally induced to study their specific contributions to age-related hearing deficits. As such, we aim to give an overview of the current literature on age-related cochlear pathologies and hope to inspire further research on the role of cochlear aging in age-related hearing deficits. Highlights • Strial dysfunction is a well-known degenerative manifestation of aging in the cochlea. • Loss of IHC-auditory nerve fiber synapses was recently recognized as an important age-related degenerative phenomenon. • Synapse loss is probably the earliest manifestation of Schuknecht's neural presbyacusis. • The relative contributions of synapse loss and strial dysfunction to changes in auditory-nerve coding are discussed. • The gerbil is an excellent model for addressing questions about functional consequences of the aging cochlea. [ABSTRACT FROM AUTHOR]

Encoding the wide range of audible sounds in the mammalian cochlea is collectively achieved by functionally diverse type I spiral ganglion neurons (SGNs) at each tonotopic position. The firing of each SGN is thought to be driven by an individual active zone (AZ) of a given inner hair cell (IHC). These AZs present distinct properties according to their position within the IHC, to some extent forming a gradient between the modiolar and the pillar IHC side. In this study, we investigated whether signaling involved in planar polarity at the apical surface can influence position-dependent AZ properties at the IHC base. Specifically, we tested the role of Gai proteins and their binding partner LGN/Gpsm2 implicated in cytoskeleton polarization and hair cell (HC) orientation along the epithelial plane. Using high and superresolution immunofluorescence microscopy as well as patch-clamp combined with confocal Ca2+ imaging we analyzed IHCs in which Gαi signaling was blocked by Cre-induced expression of the pertussis toxin catalytic subunit (PTXa). PTXa-expressing IHCs exhibited larger CaV1.3 Ca2+-channel clusters and consequently greater Ca2+ influx at the whole-cell and single-synapse levels, which also showed a hyperpolarized shift of activation. Moreover, PTXa expression collapsed the modiolar-pillar gradients of ribbon size and maximal synaptic Ca2+ influx. Finally, genetic deletion of Gai3 and LGN/Gpsm2 also disrupted the modiolar- pillar gradient of ribbon size. We propose a role for Gai proteins and LGN in regulating the position-dependent AZ properties in IHCs and suggest that this signaling pathway contributes to setting up the diverse firing properties of SGNs. [ABSTRACT FROM AUTHOR]

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones. [ABSTRACT FROM AUTHOR]

Hair cells transmit mechanical information by converting deflection of the hair bundle into synaptic release of glutamate. We have investigated this process in the lateral line of larval zebrafish (male and female) to understand how stimuli are encoded within a neuromast. Using multiphoton microscopy in vivo, we imaged synaptic release of glutamate using the reporter iGluSnFR as well as deflections of the cupula. We found that the neuromast is composed of a functionally diverse population of hair cells. Half the hair cells signaled cupula motion in both directions from rest, either by increasing glutamate release in response to a deflection in the positive direction or by reducing release in the negative direction. The relationship between cupula deflection and glutamate release demonstrated maximum sensitivity at displacements of just--40 nm in the positive direction. The remaining hair cells only signaled motion in one direction and were less sensitive, extending the operating range of the neuromast beyond 1 ju.ni. Adaptation of the synaptic output was also heterogeneous, with some hair cells generating sustained glutamate release in response to a steady deflection of the cupula and others generating transient outputs. Finally, a distinct signal encoded a return of the cupula to rest: a large and transient burst of glutamate release from hair cells unresponsive to the initial stimulus. A population of hair cells with these different sensitivities, operating ranges, and adaptive properties will allow the neuromast to encode weak stimuli while maintaining the dynamic range to signal the amplitude and duration of stronger deflections. [ABSTRACT FROM AUTHOR]

Computer models of the individual components of the peripheral auditory system – the outer, middle, and inner ears and the auditory nerve – have been developed in the past, with varying level of detail, breadth, and faithfulness of the underlying parameters. Building on previous work, we advance the modeling of the ear by presenting a complete, physiologically justified, bottom-up computer model based on up-to-date experimental data that integrates all of these parts together seamlessly. The detailed bottom-up design of the present model allows for the investigation of partial hearing mechanisms and their defects, including genetic, molecular, and microscopic factors. Also, thanks to the completeness of the model, one can study microscopic effects in the context of their implications on hearing as a whole, enabling the correlation with neural recordings and non-invasive psychoacoustic methods. Such a model is instrumental for advancing quantitative understanding of the mechanism of hearing, for investigating various forms of hearing impairment, as well as for devising next generation hearing aids and cochlear implants. • A globally coupled model of cochlear currents with tonotopically specialized hair cells is developed. • We employ a nonlinear 2D description of cochlear mechanics; the amplifier is driven by explicitly computed OHC potentials. • We present a detailed synapse morphology with Ca channel stochastic openings, currents, and diffusion. • The mechanism of hearing is described in detail allowing for modeling of effects of perturbations. • The model serves as a diagnostic tool correlating measurements with various sources of hearing loss. [ABSTRACT FROM AUTHOR]

Considerable evidence of reactive oxygen species (ROS) involvement in cochlear hair cell (HC) loss, leading to acquired sensorineural hearing loss (SNHL), were reported. Cochlear synaptopathy between HCs and spiral ganglion neurons has been gathering attention as a cochlear HC loss precursor not detectable by normal auditory evaluation. However, the molecular mechanisms linking ROS with HC loss, as well as the relationship between ROS and cochlear synaptopathy have not been elucidated. Here, we examined these linkages using NOX4 -TG mice, which constitutively produce ROS without stimulation. mRNA levels of Piccolo 1, a major component of the synaptic ribbon (a specialized structure surrounded by synaptic vesicles in HCs), were decreased in postnatal day 6 NOX4 -TG mice cochleae compared to those in WT mice; they were also decreased by noise exposure in 2-week-old WT cochleae. As noise exposure induces ROS production, this suggests that the synaptic ribbon is a target of ROS. The level of CtBP2, another synaptic ribbon component, was significantly lower in NOX4 -TG cochleae of 1-month-old and 4-month-old mice compared to that in WT mice, although no significant differences were noted at 1.5- and 2-months. The decrease in CtBP2 plateaued in 4-month-old NOX4 -TG, while it gradually decreased from 1 to 6 months in WT mice. Furthermore, CtBP2 level in 2-month-old NOX4 -TG mice decreased significantly after exposure to cisplatin and noise compared to that in WT mice. These findings suggest that ROS lead to developmental delays and early degeneration of synaptic ribbons, which could be potential targets for novel therapeutics for ROS-induced SNHL. [Display omitted] • Synaptic ribbons as well as Piccolo 1 are targets of ROS. • ROS lead to developmental delays and early degeneration of synaptic ribbons. • ROS are a cause of cochlear synaptopathy induced by aging. • Cisplatin and intense noise, aggravating factors of ARHL, cause synaptopathy. • Synaptic ribbons are a promising target for novel therapeutics for ROS-induced SNHL. [ABSTRACT FROM AUTHOR]

Spontaneous excitatory postsynaptic currents (sEPSCs) measured from the first synapse in the mammalian auditory pathway reach a large mean amplitude with a high level of variance (CV between 0.3 and 1). This has led some to propose that each inner hair cell (IHC) ribbon-type active zone (AZ), on average, releases ~6 synaptic vesicles (SVs) per sEPSC in a coordinated manner. If true, then the predicted change in membrane capacitance (Cm) for such multivesicular fusion events would equate to ~300 attofarads (aF). Here, we performed cell-attached Cm measurements to directly examine the size of fusion events at the basolateral membrane of IHCs where the AZs are located. The frequency of events depended on the membrane potential and the expression of Cav1.3, the principal Ca2+-channel type of IHCs. Fusion events averaged 40 aF, which equates to a normal-sized SV with an estimated diameter of 37 nm. The calculated SV volumes showed a high degree of variance (CV > 0.6). These results indicate that SVs fused individually with the plasma membrane during spontaneous and evoked release and SV volume may contribute more variability in EPSC amplitude than previously assumed. [ABSTRACT FROM AUTHOR]

Ribbon synapses located exclusively within retinal, cochlear and vestibular connections belong to the most interesting cellular structures but their molecular nature and functions had remained unclear. The study has provided a descriptive morphological analysis of rat eye ribbon synapses using high‐resolution transmission electron microscopy (TEM). An original collection of untypical, rarely present in the literature sagittal or tangential sections through the single RIBEYE domain of the particular ribbon have been delivered. [ABSTRACT FROM AUTHOR]

The timely and reliable processing of auditory and vestibular information within the inner ear requires highly sophisticated sensory transduction pathways. On a cellular level, these demands are met by hair cells, which respond to sound waves – or alterations in body positioning – by releasing glutamate‐filled synaptic vesicles (SVs) from their presynaptic active zones with unprecedented speed and exquisite temporal fidelity, thereby initiating the auditory and vestibular pathways. In order to achieve this, hair cells have developed anatomical and molecular specializations, such as the characteristic and name‐giving 'synaptic ribbons' – presynaptically anchored dense bodies that tether SVs prior to release – as well as other unique or unconventional synaptic proteins. The tightly orchestrated interplay between these molecular components enables not only ultrafast exocytosis, but similarly rapid and efficient compensatory endocytosis. So far, the knowledge of how endocytosis operates at hair cell ribbon synapses is limited. In this Review, we summarize recent advances in our understanding of the SV cycle and molecular anatomy of hair cell ribbon synapses, with a focus on cochlear inner hair cells. [ABSTRACT FROM AUTHOR]

Background: Perineuronal nets (PNNs) are specialized aggregations of extracellular matrix (ECM) molecules surrounding specific neurons in the central nervous system (CNS). PNNs are supposed to control synaptic transmission and are frequently associated with neurons firing at high rates, including principal neurons of auditory brainstem nuclei. The origin of high-frequency activity of auditory brainstem neurons is the indefatigable sound-driven transmitter release of inner hair cells (IHCs) in the cochlea. Results: Here, we show that synaptic poles of IHCs are ensheathed by basket-like ECM complexes formed by the same molecules that constitute PNNs of neurons in the CNS, including brevican, aggreccan, neurocan, hyaluronan, and proteoglycan link proteins 1 and 4 and tenascin-R. Genetic deletion of brevican, one of the main components, resulted in a massive degradation of ECM baskets at IHCs, a significant impairment in spatial coupling of pre- and postsynaptic elements and mild impairment of hearing. Conclusions: These ECM baskets potentially contribute to control of synaptic transmission at IHCs and might be functionally related to PNNs of neurons in the CNS. [ABSTRACT FROM AUTHOR]

Cochlear synaptopathy produced by exposure to noise levels that cause only transient auditory threshold elevations is a condition that affects many people and is believed to contribute to poor speech discrimination in noisy environments. These functional deficits in hearing, without changes in sensitivity, have been called hidden hearing loss (HHL). It has been proposed that activity of the medial olivocochlear (MOC) system can ameliorate acoustic trauma effects. Here we explore the role of theMOCsystem inHHLby comparing the performance of two different mouse models: an α9 nicotinic receptor subunit knock-out (KO; Chrna9 KO), which lacks cholinergic transmission between efferent neurons and hair cells; and a gain-of-function knock-in (KI; Chrna9L9'T KI) carrying an α9 point mutation that leads to enhanced cholinergic activity. Animals of either sex were exposed to sound pressure levels that in wild-type produced transient cochlear threshold shifts and a decrease in neural response amplitudes, together with the loss of ribbon synapses, which is indicative of cochlear synaptopathy. Moreover, a reduction in the number of efferent contacts to outer hair cells was observed. In Chrna9 KO ears, noise exposure produced permanent auditory threshold elevations together with cochlear synaptopathy. In contrast, the Chrna9L9'T KI was completely resistant to the same acoustic exposure protocol. These results show a positive correlation between the degree of HHL prevention and the level of cholinergic activity. Notably, enhancement of the MOC feedback promoted new afferent synapse formation, suggesting that it can trigger cellular and molecular mechanisms to protect and/or repair the inner ear sensory epithelium. [ABSTRACT FROM AUTHOR]

α2δ-4 is an auxiliary subunit of voltage-gated Cav1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the CACNA2D4 gene encoding α2δ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of α2δ-4, we used genome editing to generate an α2δ-4 knock-out (α2δ-4 KO) mouse. In male and female α2δ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in α2δ-4 KO retina. In reconstructions of α2δ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in α2δ-4 KO mice correlates with a progressive loss of Cav1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in α2δ-4 KO mice and better under photopic than scotopic conditions. We conclude that α2δ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with CACNA2D4 mutations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The initial neural encoding of acoustic information occurs by means of spikes in primary auditory afferents. Each mammalian primary auditory afferent (type-I auditory-nerve fiber; ANF) is associated with only one ribbon synapse in one receptor cell (inner hair cell; IHC). The properties of ANF spike trains therefore provide an indirect view of the operation of individual IHC synapses. We showed previously that a point process model of presynaptic vesicle pool depletion and deterministic exponential replenishment, combined with short postsynaptic neural refractoriness, accounts for the interspike interval (ISI) distributions, serial ISI correlations, and spike-count statistics of a population of cat-ANF spontaneous spike trains. Here, we demonstrate that this previous synapse model produces unrealistic properties when spike rates are high and show that this problem can be resolved if the replenishment of each release site is stochastic and independent. We assume that the depletion probability varies between synapses to produce differences in spontaneous rate and that the other model parameters are constant across synapses. We find that this model fits best with only four release sites per IHC synapse, a mean replenishment time of 17 ms, and absolute and mean relative refractory periods of 0.6 ms each. This model accounts for ANF spontaneous spike timing better than two influential, comprehensive models of the auditory periphery. It also reproduces ISI distributions from spontaneous and steady-state driven activity from other studies and other mammalian species. Adding fractal noise to the rate of depletion of each release site can yield long-range correlations as typically observed in long spike trains. We also examine two model variants having more complex vesicle cycles, but neither variant yields a markedly improved fit or a different estimate of the number of release sites. In addition, we examine a model variant having both short and long relative refractory components and find that it cannot account for all aspects of the data. These model results will be beneficial for understanding ribbon synapses and ANF responses to acoustic stimulation. [ABSTRACT FROM AUTHOR]

Key points: The synaptic ribbon is an organelle that coordinates rapid and sustained vesicle release to enable hearing and balance. Ribeye a and b proteins are major constituents of the synaptic ribbon in hair cells. In this study, we use optically clear transgenic zebrafish to examine the potential dynamics of ribeye proteins in vivo. We demonstrate that ribeye proteins are inherently dynamic but are stabilized at the ribbons of hair cells in the ear and the lateral line system. Abstract: Ribeye protein is a major constituent of the synaptic ribbon, an organelle that coordinates rapid and sustained vesicle release to enable hearing and balance. The ribbon is considered to be a stable structure. However, under certain physiological conditions such as acoustic overexposure that results in temporary noise‐induced hearing loss or perturbations of ion channels, ribbons may change shape or vanish altogether, suggesting greater plasticity than previously appreciated. The dynamic properties of ribeye proteins are unknown. Here we use transgenesis and imaging to explore the behaviours of ribeye proteins within the ribbon and also their intrinsic properties outside the context of the ribbon synapse in a control cell type, the skin cell. By fluorescence recovery after photobleaching (FRAP) on transgenic zebrafish larvae, we test whether ribeye proteins are dynamic in vivo in real time. In the skin, a cell type devoid of synaptic contacts, Ribeye a‐mCherry exchanges with ribbon‐like structures on a time scale of minutes (t1/2 = 3.2 min). In contrast, Ribeye a of the ear and lateral line and Ribeye b of the lateral line each exchange at ribbons of hair cells an order of magnitude slower (t1/2 of 125.6 min, 107.0 min and 95.3 min, respectively) than Ribeye a of the skin. These basal exchange rates suggest that long‐term ribbon presence may require ribeye renewal. Our studies demonstrate that ribeye proteins are inherently dynamic but are stabilized at the ribbons of sensory cells in vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]