Presynaptic Ribbons Research Articles

Noise-induced cochlear synaptopathy in C57BL/6 N mice as a function of trauma strength: ribbons are more vulnerable than postsynapses.

Noise-induced cochlear synaptopathy is characterized by irreversible loss of synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) despite normal hearing thresholds. We analyzed hearing performance and cochlear structure in C57BL/6 N mice exposed to 100, 106, or 112 dB SPL broadband noise (8-16 kHz) for 2 h. Auditory brainstem responses (ABRs) were assessed before, directly after, and up to 28 days post-trauma. Finally, the number, size, and pairing of IHC presynaptic (CtBP2-positive) ribbons and postsynaptic AMPA receptor scaffold (Homer1-positive) clusters were analyzed along the cochlea. Four weeks after the 100 dB SPL trauma, a permanent threshold shift (PTS) was observed at 45 kHz, which after the higher traumata extended toward middle to low frequencies. Loss in ABR wave I amplitudes scaled with trauma strength indicating loss of functional IHC synaptic connections. Latencies of wave I mostly increased with trauma strength. No trauma-related OHC loss was found. The number of synaptic pairs was reduced in the midbasal and basal cochlear region in all trauma conditions, with ribbon loss amounting up to 46% of control. Ribbons surviving the trauma were paired, whereas 4-6 unpaired postsynapses/IHC were found in the medial, midbasal, and basal regions irrespective of trauma strength, contrasting findings in CBA/CaJ mice. Our data confirm the susceptibility of ribbon synapses and ABR wave I amplitudes to a noise trauma of 100 dB SPL or larger. Notably, peripheral dendrites bearing IHC postsynapses were less vulnerable than presynaptic ribbons in C57BL/6 N mice.

Piplartine attenuates aminoglycoside-induced TRPV1 activity and protects from hearing loss in mice.

Hearing loss is a major health concern in our society, affecting more than 400 million people worldwide. Among the causes, aminoglycoside therapy can result in permanent hearing loss in 40% to 60% of patients receiving treatment, and despite these high numbers, no drug for preventing or treating this type of hearing loss has yet been approved by the US Food and Drug Administration. We have previously conducted high-throughput screenings of bioactive compounds, using zebrafish as our discovery platform, and identified piplartine as a potential therapeutic molecule. In the present study, we expanded this work and characterized piplartine's physicochemical and therapeutic properties. We showed that piplartine had a wide therapeutic window and neither induced nephrotoxicity in vivo in zebrafish nor interfered with aminoglycoside antibacterial activity. In addition, a fluorescence-based assay demonstrated that piplartine did not inhibit cytochrome C activity in microsomes. Coadministration of piplartine protected from kanamycin-induced hair cell loss in zebrafish and protected hearing function, outer hair cells, and presynaptic ribbons in a mouse model of kanamycin ototoxicity. Last, we investigated piplartine's mechanism of action by phospho-omics, immunoblotting, immunohistochemistry, and molecular dynamics experiments. We found an up-regulation of AKT1 signaling in the cochleas of mice cotreated with piplartine. Piplartine treatment normalized kanamycin-induced up-regulation of TRPV1 expression and modulated the gating properties of this receptor. Because aminoglycoside entrance to the inner ear is, in part, mediated by TRPV1, these results suggested that by regulating TRPV1 expression, piplartine blocked aminoglycoside's entrance, thereby preventing the long-term deleterious effects of aminoglycoside accumulation in the inner ear compartment.

Synaptic ribbon dynamics after noise exposure in the hearing cochlea

Moderate noise exposure induces cochlear synaptopathy, the loss of afferent ribbon synapses between cochlear hair cells and spiral ganglion neurons, which is associated with functional hearing decline. Prior studies have demonstrated noise-induced changes in the distribution and number of synaptic components, but the dynamic changes that occur after noise exposure have not been directly visualized. Here, we describe a live imaging model using RIBEYE-tagRFP to enable direct observation of pre-synaptic ribbons in mature hearing mouse cochleae after synaptopathic noise exposure. Ribbon number does not change, but noise induces an increase in ribbon volume as well as movement suggesting unanchoring from synaptic tethers. A subgroup of basal ribbons displays concerted motion towards the cochlear nucleus with subsequent migration back to the cell membrane after noise cessation. Understanding the immediate dynamics of synaptic damage after noise exposure may facilitate identification of specific target pathways to treat cochlear synaptopathy.

Cochlear Ribbon Synapses in Aged Gerbils.

In mammalian hearing, type-I afferent auditory nerve fibers comprise the basis of the afferent auditory pathway. They are connected to inner hair cells of the cochlea via specialized ribbon synapses. Auditory nerve fibers of different physiological types differ subtly in their synaptic location and morphology. Low-spontaneous-rate auditory nerve fibers typically connect on the modiolar side of the inner hair cell, while high-spontaneous-rate fibers are typically found on the pillar side. In aging and noise-damaged ears, this fine-tuned balance between auditory nerve fiber populations can be disrupted and the functional consequences are currently unclear. Here, using immunofluorescent labeling of presynaptic ribbons and postsynaptic glutamate receptor patches, we investigated changes in synaptic morphology at three different tonotopic locations along the cochlea of aging gerbils compared to those of young adults. Quiet-aged gerbils showed about 20% loss of afferent ribbon synapses. While the loss was random at apical, low-frequency cochlear locations, at the basal, high-frequency location it almost exclusively affected the modiolar-located synapses. The subtle differences in volumes of pre- and postsynaptic elements located on the inner hair cell's modiolar versus pillar side were unaffected by age. This is consistent with known physiology and suggests a predominant, age-related loss in the low-spontaneous-rate auditory nerve population in the cochlear base, but not the apex.

Ultrastructure of noise-induced cochlear synaptopathy

Acoustic overexposure can eliminate synapses between inner hair cells (IHCs) and auditory nerve fibers (ANFs), even if hair-cell function recovers. This synaptopathy has been extensively studied by confocal microscopy, however, understanding the nature and sequence of damage requires ultrastructural analysis. Here, we used focused ion-beam scanning electron microscopy to mill, image, segment and reconstruct ANF terminals in mice, 1 day and 1 week after synaptopathic exposure (8–16 kHz, 98 dB SPL). At both survivals, ANF terminals were normal in number, but 62% and 53%, respectively, lacked normal synaptic specializations. Most non-synapsing fibers (57% and 48% at 1 day and 1 week) remained in contact with an IHC and contained healthy-looking organelles. ANFs showed a transient increase in mitochondrial content (51%) and efferent innervation (34%) at 1 day. Fibers maintaining synaptic connections showed hypertrophy of pre-synaptic ribbons at both 1 day and 1 week. Non-synaptic fibers were lower in mitochondrial content and typically on the modiolar side of the IHC, where ANFs with high-thresholds and low spontaneous rates are normally found. Even 1 week post-exposure, many ANF terminals remained in IHC contact despite loss of synaptic specializations, thus, regeneration efforts at early post-exposure times should concentrate on synaptogenesis rather than neurite extension.

Three-Dimensional Ultrastructure of the Normal Rod Photoreceptor Synapse and Degenerative Changes Induced by Retinal Detachment.

The rod photoreceptor synapse is the first synapse of dim-light vision and one of the most complex in the mammalian CNS. The components of its unique structure, a presynaptic ribbon and a single synaptic invagination enclosing several postsynaptic processes, have been identified, but disagreements about their organization remain. Here, we have used EM tomography to generate high-resolution images of 3-D volumes of the rod synapse from the female domestic cat. We have resolved the synaptic ribbon as a single structure, with a single arciform density, indicating the presence of one long site of transmitter release. The organization of the postsynaptic processes, which has been difficult to resolve with past methods, appears as a tetrad arrangement of two horizontal cell and two rod bipolar cell processes. Retinal detachment severely disrupts this organization. After 7 d, EM tomography reveals withdrawal of rod bipolar dendrites from most spherules; fragmentation of synaptic ribbons, which lose their tight association with the presynaptic membrane; and loss of the highly branched telodendria of the horizontal cell axon terminals. After detachment, the hilus, the opening through which postsynaptic processes enter the invagination, enlarges, exposing the normally sequestered environment within the invagination to the extracellular space of the outer plexiform layer. Our use of EM tomography provides the most accurate description to date of the complex rod synapse and details changes it undergoes during outer segment degeneration. These changes would be expected to disrupt the flow of information in the rod pathway.SIGNIFICANCE STATEMENT Ribbon-type synapses transmit the first electrical signals of vision and hearing. Despite their crucial role in sensory physiology, the three-dimensional ultrastructure of these synapses, especially the complex organization of the rod photoreceptor synapse, is not well understood. We used EM tomography to obtain 3-D imaging at nanoscale resolution to help resolve the organization of rod synapses in normal and detached retinas. This approach has enabled us to show that in the normal retina a single ribbon and arciform density oppose a tetrad of postsynaptic processes. In addition, it enabled us to provide a 3-D perspective of the ultrastructural changes that occur in response to retinal detachment.

Protective Effects of Infliximab Against Kanamycin-Induced Ototoxicity in Rats.

To examine the protective effects of infliximab (INF) against kanamycin (KM)-induced hearing loss. Tumor necrosis factor α blockers can reduce cellular inflammatory reactions and decrease cell death. Thirty-six rats with normal hearing were randomly divided into six groups. The first group was injected with 400 mg/kg KM intramuscularly (IM), the second group with 7 mg/kg INF intraperitoneally (IP) and 400 mg/kg KM IM, the third group with 7 mg/kg INF IP and 200 mg/kg KM IM, and the fourth group with 1 mg/kg 6-methylprednisolone (MP) IP and 400 mg/kg KM IM. Group 5 was injected with 1 mg/kg MP IP and 200 mg/kg KM IM, and group 6 with saline IP once. Auditory brain-stem response (ABR) for hearing thresholds was performed on days 7 and 14. From the frozen sections of the cochlea, the area of the stria vascularis, the number of neurons in the spiral ganglion, the fluorescence intensity of hair cells (FIHC), postsynaptic density (PSD), and presynaptic ribbons (PSRs) were calculated. The KM-induced increase in hearing thresholds was detected on the 14th day. Hearing was only preserved in the group treated with INF after low-dose KM exposure but not in the groups that received high-dose KM. The FIHC, excitatory PSD, and PSR were preserved only in the INF-treated group after half-dose KM exposure. In MP groups, FIHC, excitatory PSD, and PSR were significantly lower than in the control group. Our results support that tumor necrosis factor-based inflammation may play a role in the ototoxicity mechanism.

GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses and for presynaptic ribbon modiolar-pillar morphology.

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3-knockout mice (i.e., subunit GluA3KO) we determined cochlear function, synapse ultrastructure, and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3KO and wild-type (GluA3WT) mice reared in ambient sound pressure level (SPL) of 55-75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes, and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3WT, but not GluA3KO, demonstrating GluA3 is required for modiolar-pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3KO and GluA2-lacking synapses were observed only in GluA3KO. GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3KO, despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3KO than GluA3WT. However, GluA3KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3WT (threefold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca2+-permeable AMPARs.

Putative COVID-19 therapies imatinib, lopinavir, ritonavir, and ivermectin cause hair cell damage: A targeted screen in the zebrafish lateral line.

The biomedical community is rapidly developing COVID-19 drugs to bring much-need therapies to market, with over 900 drugs and drug combinations currently in clinical trials. While this pace of drug development is necessary, the risk of producing therapies with significant side-effects is also increased. One likely side-effect of some COVID-19 drugs is hearing loss, yet hearing is not assessed during preclinical development or clinical trials. We used the zebrafish lateral line, an established model for drug-induced sensory hair cell damage, to assess the ototoxic potential of seven drugs in clinical trials for treatment of COVID-19. We found that ivermectin, lopinavir, imatinib, and ritonavir were significantly toxic to lateral line hair cells. By contrast, the approved COVID-19 therapies dexamethasone and remdesivir did not cause damage. We also did not observe damage from the antibiotic azithromycin. Neither lopinavir nor ritonavir altered the number of pre-synaptic ribbons per surviving hair cell, while there was an increase in ribbons following imatinib or ivermectin exposure. Damage from lopinavir, imatinib, and ivermectin was specific to hair cells, with no overall cytotoxicity noted following TUNEL labeling. Ritonavir may be generally cytotoxic, as determined by an increase in the number of TUNEL-positive non-hair cells following ritonavir exposure. Pharmacological inhibition of the mechanotransduction (MET) channel attenuated damage caused by lopinavir and ritonavir but did not alter imatinib or ivermectin toxicity. These results suggest that lopinavir and ritonavir may enter hair cells through the MET channel, similar to known ototoxins such as aminoglycoside antibiotics. Finally, we asked if ivermectin was ototoxic to rats in vivo. While ivermectin is not recommended by the FDA for treating COVID-19, many people have chosen to take ivermectin without a doctor’s guidance, often with serious side-effects. Rats received daily subcutaneous injections for 10 days with a clinically relevant ivermectin dose (0.2 mg/kg). In contrast to our zebrafish assays, ivermectin did not cause ototoxicity in rats. Our research suggests that some drugs in clinical trials for COVID-19 may be ototoxic. This work can help identify drugs with the fewest side-effects and determine which therapies warrant audiometric monitoring.

Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment.

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.

Head and neck radiotherapy causes significant disruptions of cochlear ribbon synapses and consequent sensorineural hearing loss

PurposeTo investigate the possible effects of head and neck radiotherapy on hearing function in mice. MethodAdult 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. ResultsCompared 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). ConclusionRadiation 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.

Synaptic Release Potentiation at Aging Auditory Ribbon Synapses.

Age-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin-23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50% loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs' cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast-repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis.

Hearing Impairment in a Mouse Model of Diabetes Is Associated with Mitochondrial Dysfunction, Synaptopathy, and Activation of the Intrinsic Apoptosis Pathway.

Although previous studies continuously report an increased risk of hearing loss in diabetes patients, the impact of the disease on the inner ear remains unexplored. Herein, we examine the pathophysiology of diabetes-associated hearing impairment and cochlear synaptopathy in a mouse model of diabetes. Male B6.BKS(D)-Leprdb/J (db/db, diabetes) and heterozygote (db/+, control) mice were assigned into each experimental group (control vs. diabetes) based on the genotype and tested for hearing sensitivity every week from 6 weeks of age. Each cochlea was collected for histological and biological assays at 14 weeks of age. The diabetic mice exerted impaired hearing and a reduction in cochlear blood flow and C-terminal-binding protein 2 (CtBP2, a presynaptic ribbon marker) expression. Ultrastructural images revealed severely damaged mitochondria from diabetic cochlea accompanied by a reduction in Cytochrome c oxidase subunit 4 (COX4) and CR6-interacting factor 1 (CRIF1). The diabetic mice presented significantly decreased levels of platelet endothelial cell adhesion molecule (PECAM-1), B-cell lymphoma 2 (BCL-2), and procaspase-9, but not procaspase-8. Importantly, significant changes were not found in necroptotic programmed cell death markers (receptor-interacting serine/threonine-protein kinase 1, RIPK1; RIPK3; and mixed lineage kinase domain-like pseudokinase, MLKL) between the groups. Taken together, diabetic hearing loss is accompanied by synaptopathy, microangiopathy, damage to the mitochondrial structure/function, and activation of the intrinsic apoptosis pathway. Our results imply that mitochondrial dysfunction is deeply involved in diabetic hearing loss, and further suggests the potential benefits of therapeutic strategies targeting mitochondria.

Cochlear Synaptic Degeneration and Regeneration After Noise: Effects of Age and Neuronal Subgroup

In CBA/CaJ mice, confocal analysis has shown that acoustic overexposure can immediately destroy synapses between auditory-nerve fibers (ANFs) and their peripheral targets, the inner hair cells (IHCs), and that years later, a corresponding number of ANF cell bodies degenerate. In guinea pig, post-exposure disappearance of pre-synaptic ribbons can be equally dramatic, however, post-exposure recovery to near-baseline counts has been reported. Since confocal counts are confounded by thresholding issues, the fall and rise of synaptic ribbon counts could represent “regeneration,” i.e., terminal retraction, re-extension and synaptogenesis, or “recovery,” i.e., down- and subsequent up-regulation of synaptic markers. To clarify, we counted pre-synaptic ribbons, assessed their juxtaposition with post-synaptic receptors, measured the extension of ANF terminals, and quantified the spatial organization and size gradients of these synaptic elements around the hair cell. Present results in guinea pigs exposed as adults (14 months), along with prior results in juveniles (1 month), suggest there is post-exposure neural regeneration in the guinea pig, but not the CBA/CaJ mouse, and that this regenerative capacity extends into adulthood. The results also show, for the first time, that the acute synaptic loss is concentrated on the modiolar side of IHCs, consistent with a selective loss of the high-threshold ANFs with low spontaneous rates. The morphological similarities between the post-exposure neurite extension and synaptogenesis, seen spontaneously in the guinea pig, and in CBA/CaJ only with forced overexpression of neurotrophins, suggest that the key difference may be in the degree of sustained or injury-induced expression of these signaling molecules in the cochlea.

Developmental maturation of presynaptic ribbon numbers in chicken basilar-papilla hair cells and its perturbation by long-term overexpression of Wnt9a.

The avian basilar papilla is a valuable model system for exploring the developmental determination and differentiation of sensory hair cells and their innervation. In the mature basilar papilla, hair cells form a well-known continuum between two extreme types-tall and short hair cells-that differ strikingly in their innervation. Previous work identified Wnt9a as a crucial factor in this differentiation. Here, we quantified the number and volume of immunolabelled presynaptic ribbons in tall and short hair cells of chickens, from developmental stages shortly after ribbons first appear to the mature posthatching condition. Two longitudinal locations were sampled, responding to best frequencies of approximately 1kHz and approximately 5.5kHz when mature. We found significant reductions of ribbon number during normal development in the tall-hair-cell domains, but stable, low numbers in the short-hair-cell domains. Exposing developing hair cells to continuous, excessive Wnt9a levels (through virus-mediated overexpression) led to transiently abnormal high numbers of ribbons and a delayed reduction of ribbon numbers at all sampled locations. Thus, (normally) short-hair-cell domains also showed tall-hair-cell like behaviour, confirming previous findings (Munnamalai etal., 2017). However, at 3weeks posthatching, ribbon numbers had decreased to the location-specific typical values of control hair cells at all sampled locations. Furthermore, as shown previously, mature hair cells at the basal, high-frequency location harboured larger ribbons than more apically located hair cells. This was true for both normal and Wnt9a-overexposed basilar papillae.

Cnr2 Is Important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors.

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

Differences in Calcium Clearance at Inner Hair Cell Active Zones May Underlie the Difference in Susceptibility to Noise-Induced Cochlea Synaptopathy of C57BL/6J and CBA/CaJ Mice.

Noise exposure of a short period at a moderate level can produce permanent cochlear synaptopathy without seeing lasting changes in audiometric threshold. However, due to the species differences in inner hair cell (IHC) calcium current that we have recently discovered, the susceptibility to noise exposure may vary, thereby impact outcomes of noise exposure. In this study, we investigate the consequences of noise exposure in the two commonly used animal models in hearing research, CBA/CaJ (CBA) and C57BL/6J (B6) mice, focusing on the functional changes of cochlear IHCs. In the CBA mice, moderate noise exposure resulted in a typical fully recovered audiometric threshold but a reduced wave I amplitude of auditory brainstem responses. In contrast, both auditory brainstem response threshold and wave I amplitude fully recovered in B6 mice at 2 weeks after noise exposure. Confocal microscopy observations found that ribbon synapses of IHCs recovered in B6 mice but not in CBA mice. To further characterize the molecular mechanism underlying these different phenotypes in synaptopathy, we compared the ratio of Bax/Bcl-2 with the expression of cytochrome-C and found increased activity in CBA mice after noise exposure. Under whole-cell patch clamped IHCs, we acquired two-photon calcium imaging around the active zone to evaluate the Ca2+ clearance rate and found that CBA mice have a slower calcium clearance rate. Our results indicated that excessive accumulation of calcium due to acoustic overexposure and slow clearance around the presynaptic ribbon might lead to disruption of calcium homeostasis, followed by mitochondrial dysfunction of IHCs that cause susceptibility of noise-induced cochlear synaptopathy in CBA mice.

Electrophysiological assessment and pharmacological treatment of blast-induced tinnitus.

Tinnitus, the phantom perception of sound, often occurs as a clinical sequela of auditory traumas. In an effort to develop an objective test and therapeutic approach for tinnitus, the present study was performed in blast-exposed rats and focused on measurements of auditory brainstem responses (ABRs), prepulse inhibition of the acoustic startle response, and presynaptic ribbon densities on cochlear inner hair cells (IHCs). Although the exact mechanism is unknown, the “central gain theory” posits that tinnitus is a perceptual indicator of abnormal increases in the gain (or neural amplification) of the central auditory system to compensate for peripheral loss of sensory input from the cochlea. Our data from vehicle-treated rats supports this rationale; namely, blast-induced cochlear synaptopathy correlated with imbalanced elevations in the ratio of centrally-derived ABR wave V amplitudes to peripherally-derived wave I amplitudes, resulting in behavioral evidence of tinnitus. Logistic regression modeling demonstrated that the ABR wave V/I amplitude ratio served as a reliable metric for objectively identifying tinnitus. Furthermore, histopathological examinations in blast-exposed rats revealed tinnitus-related changes in the expression patterns of key plasticity factors in the central auditory pathway, including chronic loss of Arc/Arg3.1 mobilization. Using a formulation of N-acetylcysteine (NAC) and disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07) as a therapeutic for addressing blast-induced neurodegeneration, we measured a significant treatment effect on preservation or restoration of IHC ribbon synapses, normalization of ABR wave V/I amplitude ratios, and reduced behavioral evidence of tinnitus in blast-exposed rats, all of which accorded with mitigated histopathological evidence of tinnitus-related neuropathy and maladaptive neuroplasticity.

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea

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

Acoustic Trauma Increases Ribbon Number and Size in Outer Hair Cells of the Mouse Cochlea

Outer hair cells (OHCs) in the mouse cochlea are contacted by up to three type II afferent boutons. On average, only half of these are postsynaptic to presynaptic ribbons. Mice of both sexes were subjected to acoustic trauma that produced a threshold shift of 44.2 ± 9.1 dB 7 days after exposure. Ribbon synapses of OHCs were quantified in post-trauma and littermate controls using immunolabeling of CtBP2. Visualization with virtual reality was used to determine 3-D cytoplasmic localization of CtBP2 puncta to the synaptic pole of OHCs. Acoustic trauma was associated with a statistically significant increase in the number of synaptic ribbons per OHC. Serial section TEM was carried out on similarly treated mice. This also showed a significant increase in the number of ribbons in post-trauma OHCs, as well as a significant increase in ribbon volume compared to ribbons in control OHCs. An increase in OHC ribbon synapses after acoustic trauma is a novel observation that has implications for OHC:type II afferent signaling. A mathematical model showed that the observed increase in OHC ribbons considered alone could produce a significant increase in action potentials among type II afferent neurons during strong acoustic stimulation.