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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]

Deafness is the prevailing sensory impairment among humans, impacting every aspect of one's existence. Half of congenital deafness cases are attributed to genetic factors. Studies have shown that Luzp2 is expressed in hair cells (HCs) and supporting cells of the inner ear, but its specific role in hearing remains unclear. To determine the importance of Luzp2 in auditory function, we generated mice deficient in Luzp2. Our results revealed that Luzp2 has predominant expression within the HCs and pillar cells. However, the loss of Luzp2 did not result in any changes in auditory threshold. HCs or synapse number and HC stereocilia morphology in Luzp2 knockout mice did not show any notable distinctions. This was the first study of the role of Luzp2 in hearing in mice, and our results provide important guidance for the screening of deafness genes. [ABSTRACT FROM AUTHOR]

Optomotor responses are a popular way to assess sub-cortical visual responses in mice. We studied photoreceptor inputs into optomotor circuits using genetically-modified mice lacking the exocytotic calcium sensors synaptotagmin 1 (Syt1) and 7 (Syt7) in rods or cones. We also tested mice that in which cone transducin, GNAT2, had been eliminated. We studied spatial frequency sensitivity under mesopic conditions by varying the spatial frequency of a grating rotating at 12 deg/s and contrast sensitivity by varying luminance contrast of 0.2c/deg gratings. We found that eliminating Syt1 from rods reduced responses to a low spatial frequency grating (0.05c/deg) consistent with low resolution in this pathway. Conversely, eliminating the ability of cones to respond to light (by eliminating GNAT2) or transmit light responses (by selectively eliminating Syt1) showed weaker responses to a high spatial frequency grating (3c/deg). Eliminating Syt7 from the entire optomotor pathway in a global knockout had no significant effect on optomotor responses. We isolated the secondary rod pathway involving transmission of rod responses to cones via gap junctions by simultaneously eliminating Syt1 from rods and GNAT2 from cones. We found that the secondary rod pathway is sufficient to drive robust optomotor responses under mesopic conditions. Finally, eliminating Syt1 from both rods and cones almost completely abolished optomotor responses, but we detected weak responses to large, bright rotating gratings that are likely driven by input from intrinsically photosensitive retinal ganglion cells.

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]

Afferent synapses of cochlear inner hair cells (IHCs) employ a unique molecular machinery. Otoferlin is a key player in this machinery, and its genetic defects cause human auditory synaptopathy. We employed site-directed mutagenesis in mice to investigate the role of Ca2+ binding to the C2F domain of otoferlin. Substituting two aspartate residues of the C2F top loops, which are thought to coordinate Ca2+- ions, by alanines (OtofD1841/1842A) abolished Ca2+-influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca2+ influx of IHCs and their resting membrane capacitance, reflecting IHC size, as well as the number of IHC synapses were maintained. The mutant otoferlin showed a strong apexto-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C2F domain in otoferlin targeting and of Ca2+ binding by the C2F domain for IHC exocytosis and hearing. [ABSTRACT FROM AUTHOR]

Introduction: Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs. Methods: We examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements. Results: The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents. Discussion: TPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function. [ABSTRACT FROM AUTHOR]

Vision is our primary sense, and maintaining it throughout our lifespan is crucial for our well-being. However, the retina, which initiates vision, suffers from an age-related, irreversible functional decline. What causes this functional decline, and how it might be treated, is still unclear. Synapses are the functional hub for signal transmission between neurons, and studies have shown that aging is widely associated with synaptic dysfunction. In this study, we examined the first synapse of the visual system - the rod and cone photoreceptor ribbon synapse - in the mouse retina using light and electron microscopy at 2-3 months, ~1 year, and >2 years of age. We asked, whether age-related changes in key synaptic components might be a driver of synaptic dysfunction and ultimately age-related functional decline during normal aging. We found sprouting of horizontal and bipolar cells, formation of ectopic photoreceptor ribbon synapses, and a decrease in the number of rod photoreceptors and photoreceptor ribbon synapses in the aged retina. However, the majority of the photoreceptors did not show obvious changes in the structural components and protein composition of their ribbon synapses. Noteworthy is the increase in mitochondrial size in rod photoreceptor terminals in the aged retina. [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]

Afferent synapses of cochlear inner hair cells (IHCs) employ a unique molecular machinery. Otoferlin is a key player in this machinery, and its genetic defects cause human auditory synaptopathy. We employed site-directed mutagenesis in mice to investigate the role of Ca2+ binding to the C2F domain of otoferlin. Substituting two aspartate residues of the C2F top loops, which are thought to coordinate Ca2+-ions, by alanines (OtofD1841/1842A) abolished Ca2+-influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca2+ influx of IHCs and their resting membrane capacitance, reflecting IHC size, as well as the number of IHC synapses were maintained. The mutant otoferlin showed a strong apex-to-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C2F domain in otoferlin targeting and of Ca2+ binding by the C2F domain for IHC exocytosis and hearing.

Vision is our primary sense, and maintaining it throughout our lifespan is crucial for our well-being. However, the retina, which initiates vision, suffers from an age-related, irreversible functional decline. What causes this functional decline, and how it might be treated, is still unclear. Synapses are the functional hub for signal transmission between neurons, and studies have shown that aging is widely associated with synaptic dysfunction. In this study, we examined the first synapse of the visual system – the rod and cone photoreceptor ribbon synapse – in the mouse retina using light and electron microscopy at 2–3 months, ~1 year, and >2 years of age. We asked, whether age-related changes in key synaptic components might be a driver of synaptic dysfunction and ultimately age-related functional decline during normal aging. We found sprouting of horizontal and bipolar cells, formation of ectopic photoreceptor ribbon synapses, and a decrease in the number of rod photoreceptors and photoreceptor ribbon synapses in the aged retina. However, the majority of the photoreceptors did not show obvious changes in the structural components and protein composition of their ribbon synapses. Noteworthy is the increase in mitochondrial size in rod photoreceptor terminals in the aged retina.

IntroductionRod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs.MethodsWe examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements.ResultsThe scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents.DiscussionTPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function.

The ability of the visual system to relay meaningful information over a wide range of lighting conditions is critical to functional vision, and relies on mechanisms of adaptation within the retina that adjust sensitivity and gain as ambient light changes. Photoreceptor synapses represent the first stage of image processing in the visual system, thus activity-driven changes at this site are a potentially powerful, yet under-studied means of adaptation. To gain insight into these mechanisms, the abundance and distribution of key synaptic proteins involved in photoreceptor to ON-bipolar cell transmission were compared between light-adapted mice and mice subjected to prolonged dark exposure (72 hours), by immunofluorescence confocal microscopy and immunoblotting. We also tested the effects on protein abundance and distribution of 0.5-4 hours of light exposure following prolonged darkness. Proteins examined included the synaptic ribbon protein, ribeye, and components of the ON-bipolar cell signal transduction pathway (mGluR6, TRPM1, RGS11, GPR179, Goα). The results indicate a reduction in immunoreactivity for ribeye, TRPM1, mGluR6, and RGS11 following prolonged dark exposure compared to the light-adapted state, but a rapid restoration of the light-adapted pattern upon light exposure. Electron microscopy revealed similar ultrastructure of light-adapted and dark-adapted photoreceptor terminals, with the exception of electron dense vesicles in dark-adapted but not light-adapted ON-bipolar cell dendrites. To assess synaptic transmission from photoreceptors to ON-bipolar cells, we recorded electroretinograms after different dark exposure times (2, 16, 24, 48, 72 hours) and measured the b-wave to a-wave ratios. Consistent with the reduction in synaptic proteins, the b/a ratios were smaller following prolonged dark exposure (48-72 hours) compared to 16 hours dark exposure (13-21%, depending on flash intensity). Overall, the results provide evidence of light/dark-dependent plasticity in photoreceptor synapses at the biochemical, morphological, and physiological levels.

Neural sound encoding in the mammalian cochlea faces the challenge of representing audible sound pressures that vary over six orders of magnitude. The cochlea meets this demand through the use of active micromechanics as well as the diversity and adaptation of afferent neurons and their synapses. Mechanisms underlying neural diversity likely include heterogeneous presynaptic input from inner hair cells (IHCs) to spiral ganglion neurons (SGNs) as well as differences in the molecular profile of SGNs and in their efferent control. Here, we tested whether glutamate release from IHCs, previously found to be critical for maintaining different molecular SGN profiles, is required for establishing heterogeneity of active zones (AZs) in IHCs.We analyzed structural and functional heterogeneity of IHC AZs in mouse mutants with disrupted glutamate release from IHCs due to lack of a vesicular glutamate transporter (Vglut3) or impaired exocytosis due to defective otoferlin. We found the variance of the voltage-dependence of presynaptic Ca2+ influx to be reduced in exocytosis-deficient IHCs of otoferlin mutants. Yet, the spatial gradients of maximal amplitude and voltage-dependence of Ca2+ influx along the pillar-modiolar IHC axis were maintained in both mutants. Further immunohistochemical analysis showed an intact spatial gradient of ribbon size in Vglut3-/- mice. These results indicate that IHC exocytosis and glutamate release are not strictly required for establishing the heterogeneity of IHC AZs. [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]

Neural sound encoding in the mammalian cochlea faces the challenge of representing audible sound pressures that vary over six orders of magnitude. The cochlea meets this demand through the use of active micromechanics as well as the diversity and adaptation of afferent neurons and their synapses. Mechanisms underlying neural diversity likely include heterogeneous presynaptic input from inner hair cells (IHCs) to spiral ganglion neurons (SGNs) as well as differences in the molecular profile of SGNs and in their efferent control. Here, we tested whether glutamate release from IHCs, previously found to be critical for maintaining different molecular SGN profiles, is required for establishing heterogeneity of active zones (AZs) in IHCs. We analyzed structural and functional heterogeneity of IHC AZs in mouse mutants with disrupted glutamate release from IHCs due to lack of a vesicular glutamate transporter (Vglut3) or impaired exocytosis due to defective otoferlin. We found the variance of the voltage-dependence of presynaptic Ca2+ influx to be reduced in exocytosis-deficient IHCs of otoferlin mutants. Yet, the spatial gradients of maximal amplitude and voltage-dependence of Ca2+ influx along the pillar-modiolar IHC axis were maintained in both mutants. Further immunohistochemical analysis showed an intact spatial gradient of ribbon size in Vglut3–/– mice. These results indicate that IHC exocytosis and glutamate release are not strictly required for establishing the heterogeneity of IHC AZs.

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, the underlying mechanisms are not well understood and the possible accompanying morphological changes in the VHC synapses have not yet been systematically examined. We investigated the effects of maturation and aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to 1 year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters, mostly consisting of newly synthesized ribbon material. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

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]

Background: Proper connectivity between type I spiral ganglion neurons (SGNs) and inner hair cells (IHCs) in the cochlea is necessary for conveying sound information to the brain in mammals. Previous studies have shown that type I SGNs are heterogeneous in form, function and synaptic location on IHCs, but factors controlling their patterns of connectivity are not well understood. Results: During development, cochlear supporting cells and SGNs express Semaphorin‐3A (SEMA3A), a known axon guidance factor. Mice homozygous for a point mutation that attenuates normal SEMA3A repulsive activity (Sema3aK108N) show cochleae with grossly normal patterns of IHC innervation. However, genetic sparse labeling and three‐dimensional reconstructions of individual SGNs show that cochleae from Sema3aK108N mice lacked the normal synaptic distribution of type I SGNs. Additionally, Sema3aK108N cochleae show a disrupted distribution of GLUA2 postsynaptic patches around the IHCs. The addition of SEMA3A‐Fc to postnatal cochleae led to increases in SGN branching, similar to the effects of inhibiting glutamate receptors. Ca2+ imaging studies show that SEMA3A‐Fc decreases SGN activity. Conclusions: Contrary to the canonical view of SEMA3A as a guidance ligand, our results suggest SEMA3A may regulate SGN excitability in the cochlea, which may influence the morphology and synaptic arrangement of type I SGNs. Key Findings: The cochlear components responsible for peripheral auditory transduction include hair cells and afferent spiral ganglion neurons (SGNs).Genetic sparse labeling at different development time points shows the progression of the synaptic location of type I SGNs of different fiber diameters.SEMA3A is expressed by SGNs and epithelial cells adjacent to the hair cells, whereas NRP1 (a SEMA3A receptor) is expressed by SGNs.We have found that Semaphorin‐3A (SEMA3A), a secreted factor known in axon chemo‐repulsion, regulates normal SGN‐hair cell innervation patterns, possibly by modulating SGN activity.SEMA3A is also necessary for the normal distribution of postsynaptic glutamate receptor patches found between type I SGNs and inner hair cells.The results in this report provide new knowledge regarding the maturation of type I SGNs along with an unconventional role for SEMA3A. [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.SIGNIFICANCE STATEMENT In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for α2δ-4, a subunit of voltage-gated Ca2+ channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca2+ channels are progressively lost and that rod and cone synapses are disrupted in mice that lack α2δ-4. Our results suggest that alterations in presynaptic Ca2+ signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the CACNA2D4 gene encoding α2δ-4.

Mammalian cone photoreceptors enable through their sophisticated synapse the high-fidelity transfer of visual information to second-order neurons in the retina. The synapse contains a proteinaceous organelle, called the synaptic ribbon, which tethers synaptic vesicles (SVs) at the active zone (AZ) close to voltage-gated Ca2C channels. However, the exact contribution of the synaptic ribbon to neurotransmission is not fully understood, yet. In mice, precursors to synaptic ribbons appear within photoreceptor terminals shortly after birth as free-floating spherical structures, which progressively elongate and then attach to the AZ during the following days. Here, we took advantage of the process of synaptic ribbon maturation to study their contribution to SV release. We performed whole-cell patch-clamp recordings from cone photoreceptors at three postnatal (P) development stages (P8-- 9, P12--13, >P30) and measured evoked SV release, SV replenishment rate, recovery from synaptic depression, domain organization of voltage-sensitive Ca2C channels, and Ca2C-sensitivity of exocytosis. Additionally, we performed electron microscopy to determine the density of SVs at ribbon-free and ribbon-occupied AZs. Our results suggest that ribbon attachment does not organize the voltage-sensitive Ca2C channels into nanodomains or control SV release probability. However, ribbon attachment increases SV density at the AZ, increases the pool size of readily releasable SVs available for evoked SV release, facilitates SV replenishment without changing the SV pool refilling time, and increases the Ca2C- sensitivity of glutamate release. [ABSTRACT FROM AUTHOR]

Mammalian cone photoreceptors enable through their sophisticated synapse the high-fidelity transfer of visual information to second-order neurons in the retina. The synapse contains a proteinaceous organelle, called the synaptic ribbon, which tethers synaptic vesicles (SVs) at the active zone (AZ) close to voltage-gated Ca2+ channels. However, the exact contribution of the synaptic ribbon to neurotransmission is not fully understood, yet. In mice, precursors to synaptic ribbons appear within photoreceptor terminals shortly after birth as free-floating spherical structures, which progressively elongate and then attach to the AZ during the following days. Here, we took advantage of the process of synaptic ribbon maturation to study their contribution to SV release. We performed whole-cell patch-clamp recordings from cone photoreceptors at three postnatal (P) development stages (P8–9, P12–13, >P30) and measured evoked SV release, SV replenishment rate, recovery from synaptic depression, domain organization of voltage-sensitive Ca2+ channels, and Ca2+-sensitivity of exocytosis. Additionally, we performed electron microscopy to determine the density of SVs at ribbon-free and ribbon-occupied AZs. Our results suggest that ribbon attachment does not organize the voltage-sensitive Ca2+ channels into nanodomains or control SV release probability. However, ribbon attachment increases SV density at the AZ, increases the pool size of readily releasable SVs available for evoked SV release, facilitates SV replenishment without changing the SV pool refilling time, and increases the Ca2+- sensitivity of glutamate release.

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.

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]

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

The maintenance of balance and gaze relies on the faithful and rapid signaling of head movements to the brain. In mammals, vestibular organs contain two types of sensory hair cells, type-I and type-II, which convert the head motion-induced movement of their hair bundles into a graded receptor potential that drives action potential activity in their afferent fibers. While signal transmission in both hair cell types involves Ca2+-dependent quantal release of glutamate at ribbon synapses, type-I cells appear to also exhibit a non-quantal mechanism that is believed to increase transmission speed. However, the reliance of mature type-I hair cells on non-quantal transmission remains unknown. Here we investigated synaptic transmission in mammalian utricular hair cells using patch-clamp recording of Ca2+ currents and changes in membrane capacitance (ΔCm). We found that mature type-II hair cells showed robust exocytosis with a high-order dependence on Ca2+ entry. By contrast, exocytosis was approximately 10 times smaller in type-I hair cells. Synaptic vesicle exocytosis was largely absent in mature vestibular hair cells of CaV1.3 (CaV1.3-/-) and otoferlin (Otof-/- knockout mice. Even though Ca2+-dependent exocytosis was small in type-I hair cells of wild-type mice, or absent in CaV1.3-/- and Otof-/-) mice, these cells were able to drive action potential activity in the postsynaptic calyces. This supports a functional role for non-quantal synaptic transmission in type-I cells. The large vesicle pools in type-II cells would facilitate sustained transmission of tonic or low-frequency signals. In type-I cells, the restricted vesicle pool size, together with a rapid non-quantal mechanism, could allow them to sustain high-frequency phasic signal transmission at their specialized large calyceal synapses. [ABSTRACT FROM AUTHOR]

Objectives: This study aims to investigate the effects of multiple sevoflurane exposures in neonatal mice on hearing function in the later life and explores the underlying mechanisms and protective strategies. Materials and Methods: Neonatal Kunming mice were exposed to sevoflurane for 3 days. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) tests, immunofluorescence, patch-clamp recording, and quantitative real-time PCR were performed to observe hearing function, hair cells, ribbon synapses, nerve fibers, spiral ganglion neurons, and oxidative stress. Results: Compared to control group, multiple sevoflurane exposures during the neonatal time significantly elevated ABR thresholds at 8 kHz (35.42 ± 1.57 vs. 41.76 ± 1.97 dB, P = 0.0256), 16 kHz (23.33 ± 1.28 vs. 33.53 ± 2.523 dB, P = 0.0012), 24 kHz (30.00 ± 2.04 vs. 46.76 ± 3.93 dB, P = 0.0024), and 32 kHz (41.25 ± 2.31 vs. 54.41 ± 2.94 dB, P = 0.0028) on P30, caused ribbon synapse loss on P15 (13.10 ± 0.43 vs. 10.78 ± 0.52, P = 0.0039) and P30 (11.24 ± 0.56 vs. 8.50 ± 0.84, P = 0.0141), and degenerated spiral ganglion neuron (SGN) nerve fibers on P30 (110.40 ± 16.23 vs. 55.04 ± 8.13, P = 0.0073). In addition, the Vhalf of calcium current become more negative (In addition, the Vhalf of calcium current become more negative (-21.99 ± 0.70 vs. -27.17 ± 0.60 mV, P < 0.0001), exocytosis was reduced (105.40 ± 19.97 vs. 59.79 ± 10.60 fF, P < 0.0001), and Lpo was upregulated (P = 0.0219) in sevoflurane group than those in control group. N-acetylcysteine (NAC) reversed hearing impairment induced by sevoflurane. Conclusion: The findings suggest that multiple sevoflurane exposures during neonatal time may cause hearing impairment in adult mice. The study also demonstrated that elevated oxidative stress led to ribbon synapses impairment and SGN nerve fibers degeneration, and the interventions of antioxidants alleviated the sevoflurane-induced hearing impairment. [ABSTRACT FROM AUTHOR]

Neural circuit wiring relies on selective synapse formation whereby a presynaptic release apparatus is matched with its cognate postsynaptic machinery. At metabotropic synapses, the molecular mechanisms underlying this process are poorly understood. In the mammalian retina, rod photoreceptors form selective contacts with rod ON-bipolar cells by aligning the presynaptic voltage-gated Ca2+ channel directing glutamate release (CaV1.4) with postsynaptic mGluR6 receptors. We show this coordination requires an extracellular protein, α2δ4, which complexes with CaV1.4 and the rod synaptogenic mediator, ELFN1, for trans-synaptic alignment with mGluR6. Eliminating α2δ4 in mice abolishes rod synaptogenesis and synaptic transmission to rod ON-bipolar cells, and disrupts postsynaptic mGluR6 clustering. We further find that in rods, α2δ4 is crucial for organizing synaptic ribbons and setting CaV1.4 voltage sensitivity. In cones, α2δ4 is essential for CaV1.4 function, but is not required for ribbon organization, synaptogenesis, or synaptic transmission. These findings offer insights into retinal pathologies associated with α2δ4 dysfunction.

The maintenance of balance and gaze relies on the faithful and rapid signaling of head movements to the brain. In mammals, vestibular organs contain two types of sensory hair cells, type-I and type-II, which convert the head motion-induced movement of their hair bundles into a graded receptor potential that drives action potential activity in their afferent fibers. While signal transmission in both hair cell types involves Ca2+-dependent quantal release of glutamate at ribbon synapses, type-I cells appear to also exhibit a non-quantal mechanism that is believed to increase transmission speed. However, the reliance of mature type-I hair cells on non-quantal transmission remains unknown. Here we investigated synaptic transmission in mammalian utricular hair cells using patch-clamp recording of Ca2+ currents and changes in membrane capacitance (ΔCm). We found that mature type-II hair cells showed robust exocytosis with a high-order dependence on Ca2+ entry. By contrast, exocytosis was approximately 10 times smaller in type-I hair cells. Synaptic vesicle exocytosis was largely absent in mature vestibular hair cells of CaV1.3 (CaV1.3−/−) and otoferlin (Otof−/−) knockout mice. Even though Ca2+-dependent exocytosis was small in type-I hair cells of wild-type mice, or absent in CaV1.3−/− and Otof−/−mice, these cells were able to drive action potential activity in the postsynaptic calyces. This supports a functional role for non-quantal synaptic transmission in type-I cells. The large vesicle pools in type-II cells would facilitate sustained transmission of tonic or low-frequency signals. In type-I cells, the restricted vesicle pool size, together with a rapid non-quantal mechanism, could allow them to sustain high-frequency phasic signal transmission at their specialized large calyceal synapses.

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.

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]

Objective: To establish an animal model of noise-induced hidden hearing loss (NIHHL), evaluate the dynamic changes in cochlear ribbon synapses and cochlear hair cell morphology, and observe the involvement of the SIRT1/PGC-1α signaling pathway in NIHHL. Methods: Male guinea pigs were randomly divided into three groups: control group, noise exposure group, and resveratrol treatment group. Each group was divided into five subgroups: the control group and 1 day, 1 week, 2 weeks, and 1 month post noise exposure groups. The experimental groups received noise stimulation at 105 dB SPL for 2 h. Hearing levels were examined by auditory brainstem response (ABR). Ribbon synapses were evaluated by inner ear basilar membrane preparation and immunofluorescence. The cochlear morphology was observed using scanning electron microscopy. Western blotting analysis and immunofluorescence was performed to assess the change of SIRT1/PGC-1α signaling. Levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), ATP and SIRT1 activity were measured using commercial testing kits. Results: In the noise exposure group, hearing threshold exhibited a temporary threshold shift (TTS), and amplitude of ABR wave I decreased irreversibly. Ribbon synapse density decreased after noise exposure, and the stereocilia were chaotic and then returned to normal. The expression and activity of SIRT1 and PGC-1α protein was lower than that in the control group. SOD, CAT and ATP were also influenced by noise exposure and were lower than those in the control group, but MDA showed no statistical differences compared with the control group. After resveratrol treatment, SIRT1 expression and activity showed a significant increase after noise exposure, compared with the noise exposure group. In parallel, the PGC-1α and antioxidant proteins were also significantly altered after noise exposure, compared with the noise exposure group. The damage to the ribbon synapses and the stereocilia were attenuated by resveratrol as well. More importantly, the auditory function, especially ABR wave I amplitudes, was also promoted in the resveratrol treatment group. Conclusion: The SIRT1/PGC-1α signaling pathway and oxidative stress are involved in the pathogenesis of NIHHL and could be potential therapeutical targets in the future. [ABSTRACT FROM AUTHOR]

Abstract 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.

Objective: To establish an animal model of noise-induced hidden hearing loss (NIHHL), evaluate the dynamic changes in cochlear ribbon synapses and cochlear hair cell morphology, and observe the involvement of the SIRT1/PGC-1α signaling pathway in NIHHL.Methods: Male guinea pigs were randomly divided into three groups: control group, noise exposure group, and resveratrol treatment group. Each group was divided into five subgroups: the control group and 1 day, 1 week, 2 weeks, and 1 month post noise exposure groups. The experimental groups received noise stimulation at 105 dB SPL for 2 h. Hearing levels were examined by auditory brainstem response (ABR). Ribbon synapses were evaluated by inner ear basilar membrane preparation and immunofluorescence. The cochlear morphology was observed using scanning electron microscopy. Western blotting analysis and immunofluorescence was performed to assess the change of SIRT1/PGC-1α signaling. Levels of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), ATP and SIRT1 activity were measured using commercial testing kits.Results: In the noise exposure group, hearing threshold exhibited a temporary threshold shift (TTS), and amplitude of ABR wave I decreased irreversibly. Ribbon synapse density decreased after noise exposure, and the stereocilia were chaotic and then returned to normal. The expression and activity of SIRT1 and PGC-1α protein was lower than that in the control group. SOD, CAT and ATP were also influenced by noise exposure and were lower than those in the control group, but MDA showed no statistical differences compared with the control group. After resveratrol treatment, SIRT1 expression and activity showed a significant increase after noise exposure, compared with the noise exposure group. In parallel, the PGC-1α and antioxidant proteins were also significantly altered after noise exposure, compared with the noise exposure group. The damage to the ribbon synapses and the stereocilia were attenuated by resveratrol as well. More importantly, the auditory function, especially ABR wave I amplitudes, was also promoted in the resveratrol treatment group.Conclusion: The SIRT1/PGC-1α signaling pathway and oxidative stress are involved in the pathogenesis of NIHHL and could be potential therapeutical targets in the future.

Recent studies have revealed great functional and structural heterogeneity in the ribbon-type synapses at the basolateral pole of the isopotential inner hair cell (IHC). This feature is believed to be critical for audition over a wide dynamic range, but whether the spatial gradient of ribbon morphology is fine-tuned in each IHC and how the mitochondrial network is organized to meet local energy demands of synaptic transmission remain unclear. By means of three-dimensional electron microscopy and artificial intelligence-based algorithms, we demonstrated the cell-wide structural quantification of ribbons and mitochondria in mature mid-cochlear IHCs of mice. We found that adjacent IHCs in staggered pairs differ substantially in cell body shape and ribbon morphology gradient as well as mitochondrial organization. Moreover, our analysis argues for a location-specific arrangement of correlated ribbon and mitochondrial function at the basolateral IHC pole. [ABSTRACT FROM AUTHOR]

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

Synaptic ribbons are presynaptic specializations that define eponymous ribbon synapses. Synaptic ribbons are largely composed of RIBEYE, a protein containing an N-terminal A-domain and a carboxyterminal B-domain that is identical with CtBP2, a NAD(H)-binding transcriptional co-repressor. Previously we showed that synaptic ribbons are completely absent in RIBEYE knockout mice in which the RIBEYE A-domain-encoding exon had been deleted, but CtBP2 is still made, demonstrating that the A-domain is required for synaptic ribbon assembly. In the present study, we asked whether the RIBEYE B-domain also has an essential role in the assembly of synaptic ribbons. For this purpose, we made use of RIBEYE knockin mice in which the RIBEYE B-domain was replaced by a fluorescent protein domain, whereas the RIBEYE A-domain was retained unchanged. We found that replacing the RIBEYE B-domain with a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons. [ABSTRACT FROM AUTHOR]

Xia Yuan,1,* Hongjun Liu,1,* Yufeng Li,1 Wen Li,2 Huiqian Yu,3 Xia Shen1 1Department of Anesthesiology, Shanghai Eye, Ear, Nose, and Throat Hospital, Fudan University, Shanghai 200031, People’s Republic of China; 2Research Center, Shanghai Eye, Ear, Nose, and Throat Hospital, Fudan University, Shanghai 200031, People’s Republic of China; 3Department of Otorhinolaryngology, Shanghai Eye, Ear, Nose, and Throat Hospital, Fudan University, Shanghai 200031, People’s Republic of China*These authors contributed equally to this workCorrespondence: Huiqian YuDepartment of Otorhinolaryngology, Shanghai Eye, Ear, Nose, and Throat Hospital, Fudan University, Shanghai 200031, People’s Republic of ChinaTel +86-21-64377134Fax +86-21-64373416Email yhq925@163.comXia ShenDepartment of Anesthesiology, Shanghai Eye, Ear, Nose, and Throat Hospital, Fudan University, Shanghai 200031, People’s Republic of ChinaTel +86-21-64377134Fax +86-21-64373416Email zlsx@yahoo.comIntroduction: In utero, exposure to sevoflurane (a commonly used inhalation anesthetic) can lead to hearing impairment in offspring mice, but the underlying impairment mechanism is not known.Materials and Methods: Day-15 pregnant mice were treated with 2.5% sevoflurane for 2 h to investigate sevoflurane ototoxicity. Cochleae from offspring mice were harvested for hair-cell and ribbon-synapse assessments. Hearing in offspring mice was assessed at postnatal day 30 using an auditory brainstem-response (ABR) test. Cochlear-explant cultures from offspring mice were exposed to 2.5% sevoflurane for 6 h. Immediately after treatment, explants were assessed for hair-cell morphology, mitochondrial oxidative stress, and autophagy.Results: In utero, sevoflurane exposure impaired hearing in the offspring is demonstrated by a decrease in ABR wave I amplitudes, a marker for ribbon-synapse functionality. Sevoflurane exposure caused no obvious damage to hair cells, but cochlear ribbon synapses were reduced in postnatal day 15 offspring, and partially recovered by postnatal day 30. Sevoflurane treatment also increased mitochondrial reactive-oxygen species stress and decreased autophagy in the cochlear explants.Conclusion: These results suggest that oxidative stress and reduced autophagy may underly ribbon-synapse involvement in sevoflurane-induced hearing loss.Keywords: sevoflurane, hearing impairment, ribbon synapse, hair cells, cochlea

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

Synaptic ribbons are presynaptic specializations that define eponymous ribbon synapses. Synaptic ribbons are largely composed of RIBEYE, a protein containing an N-terminal A-domain and a carboxyterminal B-domain that is identical with CtBP2, a NAD(H)-binding transcriptional co-repressor. Previously we showed that synaptic ribbons are completely absent in RIBEYE knockout mice in which the RIBEYE A-domain-encoding exon had been deleted, but CtBP2 is still made, demonstrating that the A-domain is required for synaptic ribbon assembly. In the present study, we asked whether the RIBEYE B-domain also has an essential role in the assembly of synaptic ribbons. For this purpose, we made use of RIBEYE knockin mice in which the RIBEYE B-domain was replaced by a fluorescent protein domain, whereas the RIBEYE A-domain was retained unchanged. We found that replacing the RIBEYE B-domain with a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons.

Synaptotagmin-9 (Syt9) is a Ca 2+ sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed Syt9 fl/fl mice with Rho-iCre, HRGP-Cre, and CMV-Cre mice to generate mice in which Syt9 was eliminated from rods (rod Syt9CKO ), cones (cone Syt9CKO ), or whole animals (CMV Syt9 ). CMV Syt9 mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Cone-driven photopic ERG b-waves were not significantly different in CMV Syt9 knockout mice and selective elimination of Syt9 from cones had no effect on ERGs. However, selective elimination from rods decreased scotopic and photopic b-waves as well as oscillatory potentials. These changes occurred only with bright flashes where cone responses contribute. Synaptic release was measured in individual rods by recording anion currents activated by glutamate binding to presynaptic glutamate transporters. Loss of Syt9 from rods had no effect on spontaneous or depolarization-evoked release. Our data show that Syt9 acts at multiple sites in the retina and suggest that it may play a role in regulating transmission of cone signals by rods.

In congenital stationary night blindness type 2 (CSNB2)-a disorder involving the Ca v 1.4 (L-type) Ca 2+ channel-visual impairment is mild considering that Ca v 1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Ca v 1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Ca v 1.4. Surprisingly, Ca v 3 (T-type) Ca 2+ currents were detected in cones of G369i KI mice and Ca v 1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Ca v 1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Ca v 1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Ca v 1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Ca v 3 channels maintain cone synaptic output provided that the nonconducting role of Ca v 1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Optomotor responses are a popular way to assess sub-cortical visual responses in mice. We studied photoreceptor inputs into optomotor circuits using genetically-modified mice lacking the exocytotic calcium sensors synaptotagmin 1 (Syt1) and 7 (Syt7) in rods or cones. We also tested mice that in which cone transducin, GNAT2, had been eliminated. We studied spatial frequency sensitivity under mesopic conditions by varying the spatial frequency of a grating rotating at 12 deg/s and contrast sensitivity by varying luminance contrast of 0.2c/deg gratings. We found that eliminating Syt1 from rods reduced responses to a low spatial frequency grating (0.05c/deg) consistent with low resolution in this pathway. Conversely, eliminating the ability of cones to respond to light (by eliminating GNAT2) or transmit light responses (by selectively eliminating Syt1) showed weaker responses to a high spatial frequency grating (3c/deg). Eliminating Syt7 from the entire optomotor pathway in a global knockout had no significant effect on optomotor responses. We isolated the secondary rod pathway involving transmission of rod responses to cones via gap junctions by simultaneously eliminating Syt1 from rods and GNAT2 from cones. We found that the secondary rod pathway is sufficient to drive robust optomotor responses under mesopic conditions. Finally, eliminating Syt1 from both rods and cones almost completely abolished optomotor responses, but we detected weak responses to large, bright rotating gratings that are likely driven by input from intrinsically photosensitive retinal ganglion cells., Competing Interests: The authors have no conflicts of interest to declare regarding the study described in this article and preparation of the article., (© 2024 The Authors.)

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]