ABSTRACT 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

NOISE-INDUCED COCHLEAR SYNAPTOPATHY Article Open access 09 November 2023 SYNAPTIC MIGRATION AND REORGANIZATION AFTER NOISE EXPOSURE SUGGESTS REGENERATION IN A MATURE MAMMALIAN COCHLEA

Article Open access 17 November 2020 EXCESS EXTRACELLULAR K+ CAUSES INNER HAIR CELL RIBBON SYNAPSE DEGENERATION Article Open access 04 January 2021 INTRODUCTION Noise-induced hearing loss

(NIHL) typically involves primary damage to sensory hair cells followed by secondary degeneration of connected auditory neurons. However, moderate levels of noise exposure can result in

degradation of afferent synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs)1,2. This noise-induced cochlear synaptopathy (NICS) leads to the permanent severing of

many surviving IHCs from their sensory innervation3. While hair-cell loss is captured by increases in auditory thresholds—the standard clinical assay of hearing loss—loss of synapses is not,

leading to “hidden” hearing loss (HHL). HHL has been associated with characteristic changes in auditory brainstem response (ABR) waveforms1,4. Behaviorally, HHL is thought to be associated

with significant social, psychological, and cognitive implications, including perceptual difficulties in complex acoustic environments5, tinnitus6,7,8, and hyperacusis6. The presynaptic

region of IHC-SGN synapses is characterized by the presence of electron-dense structures implicated in the modulation of trafficking and fusion of glutamatergic synaptic vesicles. These

presynaptic ribbons are apposed by postsynaptic NMDA, kainate, and AMPA-type receptors on peripheral afferent fibers9,10,11. In the cochlea, release and reuptake of the primary

neurotransmitter glutamate is tightly regulated to optimize fast synaptic transmission12,13,14. The mechanism of NICS is not fully elucidated, but is thought to primarily stem from glutamate

excitotoxicity — increased release of glutamate from overstimulated IHCs15,16,17,18. Noise exposure, anoxia, and treatment with ionotropic glutamate receptor agonists (such as kainic acid

(KA)) cause excitotoxic damage to afferent synapses, prompting a massive influx of water and ions, such as calcium, into auditory fibers1,11,19,20,21. This can lead to swelling, stimulation

of proteolytic enzymes, and an increase of toxic reactive oxygen and nitrogen compounds, which can result in neuronal degeneration and a permanent loss of function22. Much of what is known

on the mechanisms behind NICS in IHC ribbon synapses stems from immunohistochemical studies, by staining against protein markers on pre-synaptic ribbons (anti-c-terminal binding protein 2

(anti-CtBP2)), post-synaptic glutamate receptors (anti-GluR), and SGNs (anti-NF) in fixed cochlear tissue. The reported changes in synapses after acoustic overstimulation or ototoxic drug

treatments include the loss or dysfunction of GluR and postsynaptic density protein 95 on type I afferent terminals23,24, the dissociation or migration of pre-synaptic terminals along the

basolateral membrane of IHCs1,25, and terminal swelling15,26. The damage to synapses caused by noise exposure also appears to worsen the decline of SGNs that occurs with age19,24.

Traditional immunohistochemical staining methods do not reveal real-time dynamics of ribbon synapses under physiologic or pathophysiologic stimuli. Live imaging of perturbed synapses helps

fill in these gaps in knowledge. Work in goldfish retina bipolar cells in which a short peptide with affinity for the CtBP subunit of the RIBEYE protein – the major component of ribbons —

or, later, calcium indicators, were introduced via whole-cell patch pipette, provided critical insight into the development and organization of synaptic ribbons27,28. Subsequent development

of a mouse transgenic line in which RIBEYE is tagged by a red fluorescent protein (RIBEYE-tagRFP), enabled visualization of the dynamic assembly of ribbon synapses during synaptogenesis and

maturation in the mouse retina29. This study showed that the endogenous, genetically encoded RIBEYE-tagRFP signal is an effective tool for tracking the development of synaptic ribbons, and

that while synaptic ribbons can affect the stability of nascent bipolar cell synapses, they are not essential for establishing these synapses in vivo. Similarly, in RIBEYE knockout mice

lacking synaptic ribbons in hair cells, only minor disruptions in the function of hair-cell synapses and minor auditory impairments are present, suggesting the existence of compensatory

mechanisms in this model system30,31,32. However, RIBEYE was shown to play a crucial role in organizing presynaptic Cav1.3 calcium channels, although the localization of these channels at

the hair-cell synapse remained unaffected. In the absence of RIBEYE, numerous small Cav1.3 clusters were observed at each synapse, deviating from the usual single organized structure30,32.

Initial imaging studies of live ribbon synapse activity predominantly relied on dissociated retinal cells to explore structural ribbon dynamics. RIBEYE-tagEGFP transgenic zebrafish have been

used to investigate hair cell ribbons in the lateral line system over extended periods without compromising the structural integrity, osmolarity, or mechanical forces surrounding the cells

– factors that could influence calcium influx and synaptic activity33. Discrepancies with previous studies highlight the potential for advanced imaging technologies to provide deeper

insights into the dynamic nature of ribbon synapses and vesicle interactions in different cell types and experimental conditions. In the present study, we describe the use of a live synaptic

imaging model to observe the behavior of pre-synaptic ribbons in an organotypic tissue culture model of neonatal RIBEYE-tagRFP mouse cochleae, as well as in 7–10 week-old RIBEYE-tagRFP

mature hearing mouse cochleae. Excitotoxic lesioning was induced by KA for neonatal mice, and by noise exposure for mature, hearing mice. We tracked presynaptic ribbon dynamics to visualize

change over time and investigate the mechanism of glutamate excitotoxicity and noise exposure in live cells, enabling direct observation of the initial events underlying NICS. RESULTS

ENDOGENOUS RIBEYE-TAGRFP SIGNAL ACCURATELY REPRESENTS PRESYNAPTIC RIBBONS IN COCHLEAR INNER HAIR CELLS The RIBEYE-tagRFP mouse has previously been shown to accurately label pre-synaptic

ribbons in the retina29. We sought to validate this model in cochlear IHCs from P5 neonatal explant cultures. We first performed immunohistochemistry with antibodies against Neurofilament,

CtBP2, and GluR2 to label neurons, pre-synaptic ribbons, and post-synaptic receptors, respectively. Merged images of RIBEYE-tagRFP and anti-NF signals (Fig. 1a) demonstrate that SGNs and

IHC-SGN connections have been structurally preserved in our in vitro culture model. The IHC-SGN synapses were further confirmed to be intact through visualization of adjacent RIBEYE-tagRFP

and anti-GluR2 labeling, indicating paired synapses (Fig. 1b). Anti-CtBP2 immunohistochemical labeling and endogenous RIBEYE-tagRFP signal overlapped, both qualitatively (Fig. 1c) and

quantitatively: the number of puncta per IHC (Fig. 1d) and the mean volume of puncta (Fig. 1e) identified using RIBEYE-tagRFP signal versus anti-CtBP2 signal alone were not statistically

significantly different, and a cytofluorogram scatter plot shows a strong positive correlation (Pearson’s coefficient: 0.75) between RIBEYE-tagRFP and anti-CtBP2 signals (Fig. 1f). Taken

together, these findings demonstrate that the endogenous, genetically encoded RIBEYE-tagRFP signal accurately represents gold-standard fixed immunohistochemical staining and is an

appropriate tool for live imaging of pre-synaptic ribbons in cochlear IHCs. DYNAMIC THREE-DIMENSIONAL IMAGING AND TRACKING OF PRESYNAPTIC RIBBONS IN COCHLEAR INNER HAIR CELLS We next sought

to establish a method for live imaging, identification, and tracking of RIBEYE-tagRFP puncta, as markers of pre-synaptic ribbons (Fig. 2). Cochlear hair cells were loaded with FM1-43, a live

dye that enters through functioning mechanoelectrical transduction channels34. The FM1-43 signal enabled establishment of a stable three-dimensional reference frame for determination of

RIBEYE-tagRFP punctal position over time. Serial confocal z-stacks were acquired encompassing the entire IHC region within the optical field, and the entire volume of FM1-43 hair cells was

rendered in three dimensions (Fig. 2a). To account for spatial drift over imaging time, the FM1-43 signal was taken as a frame of reference in the three-dimensional reconstruction of the

presynaptic ribbons. The stability of this reference frame was ascertained in each experimental condition by measuring the speed with which the center of mass of the reference frame moved;

this speed did not vary with experimental perturbation (Supplementary Fig. S1). With this technique, we were able to track presynaptic ribbons in three dimensions in multiple IHCs over time,

allowing for visualization and time-lapse observation of dynamic ribbon parameters such as speed, volume, displacement, and directionality (Fig. 2b–d). Acquisition of one complete confocal

Z-stack required 5 min on average; for serial measurement, therefore, the average minimum time interval between successive images was 5 min, which determined the temporal resolution for

dynamic measurements. KAINIC ACID TREATMENT INDUCES MOVEMENT OF RIBBONS IN NEONATAL COCHLEAR CULTURES Exposure to KA to induce glutamate excitotoxicity is a well-described model of cochlear

synaptopathy used in neonatal cochlear explant cultures35,36,37. We first sought to validate this model in our system. 0.4 mM KA, or vehicle, was applied to RIBEYE-tagRFP neonatal cochlear

cultures for 2 h to mimic glutamate excitotoxicity, and cochleae harvested 24 h later for anti-GluR2 immunohistochemistry and visualization of tagRFP signal. The number of ribbon puncta

remained unchanged from the control, but the number of post-synaptic puncta was reduced, making the number of paired synapses reduced following KA treatment consistent with previous

studies35,36,37 (Supplementary Fig. S2). We then performed live imaging of RIBEYE-tagRFP neonatal cochleae and obtained serial confocal z-stacks of IHCs before and 24 h after 2-h application

of 0.4 mM KA. Control cochleae exhibited a decrease in the number of RIBEYE-tagRFP puncta after 24 h in culture. Cochleae treated with KA also showed a decrease in puncta number; the number

of puncta, however, was not statistically significantly different between control and KA-treated cochleae at any timepoint (Fig. 3a). This is consistent with prior findings that the number

of pre-synaptic ribbons is not affected by glutamate excitotoxicity within 24 h25,38. Ribbon volume was also unchanged in control and KA-treated cochleae 24 h after drug exposure (Fig. 3b).

We then assessed movement of synaptic ribbons by measuring the difference in position between a pair of images and dividing this displacement by the time interval between the paired images.

Comparison of speed measurements in live-imaged cochleae and the same cochleae after fixation confirmed that the live-imaged puncta had greater movement, thus excluding imaging or mechanical

artifact (Supplementary Fig. S3). The speed of RIBEYE-tagRFP puncta was not different prior to treatment in control and KA cochleae, and increased in both groups 24 h after treatment.;

however, KA-treated cochleae had significantly greater increase in speed compared with control cochlea (Fig. 3c). These speed measurements were performed using a single pair of images before

and 24 h after a 2-h KA treatment. We then performed serial timecourse imaging during KA exposure. The increase in speed was present immediately after KA exposure (Fig. 3d) and remained

elevated over a 1-hr recording period (Fig. 3e). Though the speed with which the ribbons moved was increased, there was no consistent directionality to ribbon movement. The 95% confidence

interval of the mean velocities for overall movement as well as in each of the relevant cochlear axes spanned the origin, suggesting that the entire population of ribbons, taken as a whole,

does not exhibit any significant directionality in movement. Indeed, the velocity vectors corresponding to punctal movement along the apical-basal and modiolar-pillar axes were evenly

distributed in both control and KA-treated cochleae (Fig. 3f, g). RIBEYE-TAGRFP PUNCTA ARE LESS MOBILE AND LARGER IN VOLUME IN JUVENILE COMPARED WITH NEONATAL COCHLEAE Next, we sought to

perform live ribbon imaging in juvenile, mature-hearing cochleae. The genetically encoded RIBEYE-tagRFP model provided an opportunity to visualize presynaptic ribbons immediately after

euthanasia and cochlear extraction, overcoming the significant difficulty that juvenile cochlear explants rapidly degenerate after extraction, limiting live imaging potential. After rapid

dissection and FM1-43 loading, explant cochleae were imaged within 15 min of euthanasia (Fig. 4a). Hair-cell health was monitored using the FM1-43 signal, and cells determined to be intact

up through 30 min of recording, after which evidence of bleb formation and extrusion of hair cells was noted. Compared with neonatal cochleae, there were fewer RIBEYE-tagRFP puncta in

juvenile inner hair cells (Fig. 4b); however, the punctal volume was significantly higher (Fig. 4c). Both observations are qualitatively consistent with prior reports39,40,41,42,43.

Additionally, RIBEYE-tagRFP puncta moved at significantly lower speed in juvenile compared to neonatal cochleae (Fig. 4d). Though punctal volume was increased in juvenile cochlea, mean

intensity was decreased, suggesting that the puncta become less dense with age (Fig. 4e). These findings are consistent with the known development of ribbon synapses in cochlear inner hair

cells, in which ribbons decrease in number, increase in volume, and become more stable as they form mature, paired synapses39,40,41,42. RIBEYE-TAGRFP PUNCTA ARE MORE MOBILE IN NOISE-EXPOSED

JUVENILE, MATURE-HEARING COCHLEAR CULTURES COMPARED TO UNEXPOSED COUNTERPARTS Exposure to moderately loud noise, sufficient to cause a temporary shift in hearing thresholds that returns

fully to baseline levels, leads to cochlear synaptopathy, in which SGN-IHC synapses are permanently lost. In 7–10 week-old mice, exposure to 8–16 kHz octave-band white noise at 98 dB SPL for

2 h is a well-established model for NICS44. The RIBEYE-tagRFP mouse model provides an opportunity to examine the dynamic behavior of cochlear synaptic ribbons after noise exposure. We

exposed RIBEYE-tagRFP mice to synaptopathic noise, which caused a temporary threshold shift at 8 and 16 kHz (Supplementary Fig. S4). Unexposed, age-matched littermate mice imaged on the same

day were used as controls. Serial live imaging was performed to measure static (number, position, and volume) and dynamic (displacement and speed) characteristics of pre-synaptic ribbons

immediately after completion of the 2-h noise exposure (Fig. 5a). There was no significant difference in the number of RIBEYE-tagRFP puncta between control and noise-exposed mice (Fig. 5b).

However, puncta from noise-exposed mice demonstrated significantly higher ribbon volumes compared with unexposed mice, consistent with prior reports of noise-induced ribbon swelling1,45,46

(Fig. 5c). There was a striking difference in ribbon speed, with ribbons from noise-exposed mice moving significantly more rapidly than those from unexposed mice (Fig. 5d). In a separate

experiment, noise-exposed mice were imaged 2 weeks after noise exposure and compared on the same day to control, unexposed, age-matched littermate mice (Fig. 5e). As was observed immediately

after noise exposure, the number of pre-synaptic ribbons was not different between noise-exposed and unexposed animals, though variance increased significantly, which may reflect long-term

differences between animals in synaptic changes after noise exposure (Fig. 5f). However, there was a large, statistically significant increase in punctal volume (Fig. 5g) and a persistent,

small increase in punctal speed (Fig. 5h) in noise-exposed mice 2 weeks after completion of noise exposure. BASAL-MOST SYNAPTIC RIBBONS MOVE TOWARDS APEX AFTER NOISE EXPOSURE Similar to

neonatal cochleae, synaptic ribbons in control as well as noise-exposed juvenile cochleae did not demonstrate any consistent direction of movement overall (Fig. 6A). However, we separately

analyzed the 25% of ribbons that were closest to the basal pole of the hair cells. Compared with ribbons found more towards the apical aspect of the IHCs, these basal-most ribbons exhibited

consistent movement towards the hair-cell apex and nucleus, as well as a large increase in volume in noise-exposed cochleae immediately after synaptopathic noise exposure (Fig. 6b, c). This

subset of ribbons was examined further over a 20-min recording period. In control cochleae, 127/278 (45.7%) ribbons were able to be followed for the entire recording period; in noise-exposed

cochleae, 93/194 (47.9%) persisted, which was not significantly different. There was no difference in subcellular distribution between puncta that persisted or vanished. Both ribbons that

vanished and those that persisted demonstrated volume increase and initial movement towards the apex in noise-exposed cochleae (Fig. 6d, e). As the persistent ribbons were followed over

time, the initial apical movement in noise-exposed cochleae reversed course, such that towards the end of the recording period, 20 min later, they were moving back towards the base. In

contrast, ribbons in control cochlea exhibited no consistent direction of movement at any point (Fig. 6f). DISCUSSION Pre-synaptic ribbons are electron-dense structures in the retina, inner

ear and pinealocytes that dock synaptic vesicles, facilitating sustained vesicle release in pre-synaptic active zones. In the cochlea, overexposure to moderate levels of sound leads to

cochlear synaptopathy — loss of synaptic connections between sensory IHCs and afferent SGNs — and auditory functional disability. Using a RIBEYE-tagRFP transgenic mouse, we performed live

imaging in cochlear explants to visualize the dynamic behavior of pre-synaptic ribbons in live hair cells in real time and three dimensions, and demonstrate that chemical overstimulation and

noise exposure induce random, as well as directional movement of subpopulations of pre-synaptic ribbons. Prior studies in mammalian cochleae have always used immunohistochemistry to examine

ribbon synapses in fixed tissue at a single timepoint. Although these studies have revealed immense amounts of knowledge about pre- and post- synaptic structures, they are unable to provide

dynamic spatiotemporal information, precluding direct investigation of the effect of noise and other ototoxic insults on ribbon dynamics. In contrast, the genetically encoded model of

RFP-tagged RIBEYE allows monitoring of movement of pre-synaptic ribbons in real time in live cochlear inner hair cells. We identified pre-synaptic ribbons using endogenous RIBEYE-tagRFP

signals, while IHCs were delineated using FM1-43; this enabled us to normalize the reference frame around the stable 3D framework of FM1-43 signal and isolate ribbon movement relative to the

hair cells. Similar to prior studies of live-cell speed in tumor-associated macrophages46, we utilized the Spots function in Imaris to identify and render individual RIBEYE puncta. This

strategy proved to be ideal for detecting the small RIBEYE puncta and assign a globular-like shape to each. As the first measure during image analysis, we used the Quality filter in

Imaris47. If the threshold value is too low, erroneous spots are picked; alternatively, if the value is too high only the brightest puncta are picked. Together with Quality, the Intensity

Threshold parameter needed to be optimized separately for neonatal and adult imaging, and it was necessary to perform contemporaneous control measurements with age-matched mice to ensure

experimental rigor. In the current study, we performed live imaging in neonatal cultures as well as an explant preparation of the mature, hearing cochlea in juvenile, 7–10-week-old mice. In

the case of neonatal cochlear cultures, this imaging enabled investigation of ribbon properties and behavior at multiple timepoints before and after experimental manipulation, including

chemical excitotoxicity using KA, in the same specimen. In juvenile, mature-hearing cochlear explants, inability to maintain healthy tissue ex vivo greater than 30 min precluded similar

prolonged ex vivo investigation; however, this model was uniquely able to provide direct insight into ribbon behavior after physiologically relevant acoustic noise exposure known to cause

cochlear synaptopathy. We first validated our live imaging and processing model against standard immunohistochemical methods, demonstrating our ability to accurately detect pre-synaptic

ribbons in real time. We then evaluated the effect of overstimulation on pre-synaptic ribbons in an established KA model for glutamate excitotoxicity in neonatal cochlear cultures as well as

synaptopathic 98-dB SPL noise exposure in a juvenile, mature-hearing cochlear explant model. At baseline, compared with neonatal cochlea, synaptic ribbons from mature hearing cochlea were

less numerous, larger, and less mobile. The difference in ribbon volume, in particular, was large, with the mean (±SEM) volume of puncta in juvenile cochleae (0.46 ± 0.01 μm3) nearly 20-fold

greater than in neonatal cochleae (0.026 ± 0.001 μm3; Fig. 4c). In a previous study using focused ion beam-scanning electron microscopy (FIB-SEM) to quantity ribbon volume during

development from P9-P34, ribbon size was found to increase approximately 2-fold (0.00344 ± 0.00099 versus 0.00568 ± 0.00045 μm3)40. Measurements in P49 mice (more comparable in age to the

juvenile mice used in our study) were larger still, ranging from 0.008–0.14 μm3 and 0.01–0.15 μm3 in two studies using serial block-face electron microscopy48,49. The absolute volumes

measured in our study, using live fluorescent imaging, are not comparable to the much smaller structural measurements using electron microscopy; however, the trend towards larger ribbons in

older cochleae is consistent. The much larger difference that we observed compared to previous studies may be related to differences in imaging and processing that were necessary between the

neonatal and juvenile cochlear preparations, and is reflected in the significantly lower mean intensity of the punctal signals in juvenile animals (Fig. 4e). After a 2-h exposure of

neonatal cochleae to KA, which induces glutamate excitotoxicity, or exposure of mature hearing mice to 98 dB SPL, which induces cochlear synaptopathy, immediate physiological changes were

observed. Neither chemical overstimulation nor noise exposure affected the number of pre-synaptic ribbons; however, noise exposure was associated with an immediate increase in synaptic

ribbon volume in the mature hearing cochlea that was even more substantial 2 weeks later. The close correlation between the size and mean brightness of pre-synaptic puncta raises the

possibility that observed changes in size may be related to changes in endogenous signal intensity, rather than actual volume increase; however, overall these findings on ribbon number and

size corroborate previous observations from fixed specimens37,38,40. The ability from this live-imaging model to observe dynamic behavior of ribbon synapses revealed that both chemical

overstimulation and noise exposure induces increased movement of pre-synaptic ribbons. Ribbons in KA-stimulated neonatal cochlea and noise-stimulated mature hearing cochlea both exhibited

increased speed of ribbon movement. These initial increases in ribbon speed persisted, albeit to a lesser degree, 2 weeks after noise exposure. Ribbon speed in both neonatal and juvenile

mouse cochlea was significantly slower than that observed in zebrafish using a similar transgenic system. In zebrafish, despite small spatial displacements over extended periods, short-term

instability and high mobility of the ribbon’s underlying structure was observed, challenging traditional views of ribbon physical rigidity and proposing a fluid mosaic model for the ribbon

surface33. Further study in the mammalian system is required, especially improvement in temporal resolution, to further investigate whether these differences truly reflect species-specific

differences in ribbon stability. When the entire aggregate population of ribbons was examined together, there was no consistent directionality to the KA- or noise-induced ribbon

displacement; overall, displacement vectors appeared to be randomly distributed. RIBEYE proteins are dynamic in nature but become stabilized in ribbon synapses50. The increased speed in the

KA-treated and noise-exposed ribbons might be due to induction of instability in the ribbon synapses in the pre-synaptic zone. The lack of overall directionality suggests that ribbons are

becoming unanchored; that is, RIBEYE anchorage is disrupted but without subsequent directed transport. In ribbon synapses, Bassoon anchors RIBEYE at the active zone of the presynaptic

membrane. Although both Bassoon-deficient and wild-type IHCs have large RIBEYE spots at afferent synapses, Bassoon-deficient IHCs were also shown to possess unanchored and floating ribbons

close to the synapses51. Furthermore, physical interaction between Bassoon and RIBEYE is thought to be present at the cytomatrix active zone in retina photoreceptors, maintaining the

integrity of the ribbon complex52. Though, overall, ribbons did not have a concerted vector of displacement, a subgroup of ribbons in the mature hearing cochlea closest to the basal pole of

the hair cells exhibited clear directionality. Compared to all ribbons in control, non-noise exposed animals as well as apical ribbons in noise-exposed animals, which exhibited no

directionality of movement, basal ribbons moved towards the apex immediately after noise exposure. While it is possible that these basal ribbons in juvenile, noise-exposed mice are moving

apically simply because they have no other direction to go after being unanchored by noise exposure, we did not observe this directionality in basal ribbons either in unexposed juvenile

cochlea or in neonatal cochleae after KA exposure, suggesting that this directional movement is specific to noise-exposed mature hearing cochleae. This initial apical movement of the

basal-most ribbons towards the hair-cell nucleus was followed by movement back towards the base of the hair cell, which may reflect re-attachment. These findings provide direct visual

evidence of dynamic, directional movement of basal synaptic ribbons after noise exposure in the mature hearing cochlea. Prior reports showed that synaptopathy occurs rapidly during noise

exposure, with limited subsequent recovery24, though strain-specific differences have been observed53. Synapses around the IHC are arranged based on fiber characteristics; large ribbons are

associated with small receptor patches on the modiolar side, and small ribbons are linked to large receptor patches on the pillar side54. These gradients underlie variation in cochlear nerve

response, reflecting a low-spontaneous discharge rate (SR) to high-SR gradient55. Following noise exposure, synapses are preferentially lost on the modiolar side of the IHC, and more orphan

ribbons are found near the habenular end. These orphan ribbons appeared to recover eventually rather than degenerate. These conclusions, however, were all inferred from fixed imaging; it

could not be determined or observed directly whether ribbons were migrating to and from the receptor patches, or whether they were being dissolved and reformed. Our current study provides

complementary evidence to directly show how ribbons are moving immediately after noise exposure. Though the overall population of ribbons appear to be moving in random directions, suggesting

unanchoring, the basalmost subgroup of puncta migrated apically towards the nucleus and then returned to the cell membrane, suggesting that ribbons are recycled rather than immediately

degraded and then reformed. Further refinement and study of this live imaging model may reveal further subgroups of ribbons that have characteristic behaviors after acoustic overstimulation

that can be targeted for treatment of cochlear synaptopathy. This study was limited by the spatial and temporal resolution of the imaging technique. Adequate 3D imaging of puncta required

high-spatial-resolution laser-scanning confocal microscopy across the entire synaptic region, with each z-stack requiring 5 min to acquire on average, thus limiting temporal resolution. For

large movements or rapidly drifting preparations, this might result in a systemic underestimation or overestimation of the punctal speed even after adjusting for the reference frame, if the

imaged punctum was captured within a significantly different imaging plane from one frame to another. However, the preparations were quite stable, with minimal movement of the reference

frame (Supplementary Fig. S1), and the punctal movements were small; therefore, it is likely that despite the prolonged acquisition time, the measured speeds are likely to be accurate at the

5-min time resolution limit. We are unable to make conclusions, however, about movement occurring faster than this limit; future studies restricting imaging to small volumetric regions of

interest to enable faster image acquisition are needed to evaluate faster synaptic movement. We relied on Imaris software to track puncta from one frame to the next; over a 20-min recording

period with 6 serial images, less than half of puncta could be tracked across all images. These vanished puncta were not interpreted to represent actual loss of puncta over that time period,

and the number of vanished puncta was not different between control and noise-exposed animals; however, it illustrates the challenges inherent to the imaging technique. We observed other

changes that occurred during routine culture and imaging: even in untreated cochleae the number of puncta detected in neonatal cultures decreased after 24 h in culture, and the speed of

punctal movement decreased over the first 20 min of serial imaging, (Fig. 3a, e). The reasons for these changes are unknown, but may reflect either a phototoxic response to the initial

imaging itself or simply evolution of synapses in explant cultures that had previously been undetectable without serial imaging of the same preparation. These finding illustrate the

necessity for consistency of culture duration and inclusion of rigorous control conditions in any study using neonatal cochlear cultures, whether using live or fixed imaging techniques.

While imaging of neonatal cochlear cultures enabled observation of ribbon dynamics during chemical overstimulation with KA, this is not a physiological perturbation; conversely, while we

were able to examine ribbon dynamics after a physiologically relevant stimulus (noise exposure) in the mature hearing cochlea, we could not acoustically stimulate the explanted juvenile

cochlea and directly observe the effect of sound stimulation, as was done previously in a limited fashion in the gerbil cochlear explant56. In future studies, further refinement of the

imaging technique and mature hearing explant model can advance understanding of ribbon dynamics after noise exposure. In summary, our findings suggest that noise exposure induces synapses to

become more loosely anchored, and that a subpopulation of the basalmost synapses migrate apically, towards the nucleus. Within 20 min, some of these ribbons then migrate back towards the

synaptic terminals, likely to re-form paired synapses. These insights suggest that further investigation of ribbon anchoring may uncover molecular targets for intervention to prevent this

initial noise-induced ribbon dissociation or promote re-targeting in the early moments after noise exposure, thereby preventing permanent synaptopathy. METHODS COCHLEAR EXPLANT DISSECTION

AND CULTURE For neonatal cochlear cultures, postnatal day 4 (P4) to P6 C57BL/6 RIBEYE-tagRFP mouse cochleae (gift of Rachel O.L. Wong) of both sexes underwent dissection of the temporal bone

in ice-cold Hanks’ Balanced Salt Solution (HBSS). The membranous labyrinth was extracted from the modiolus, with removal of the stria vascularis and spiral ligament. Reissner’s membrane and

the tectorial membrane were removed. Explants were placed in the center of a 35 mm glass bottom cell culture dish coated with cell-tak and cultured overnight in Dulbecco’s modified Eagle’s

medium (DMEM) with 10% fetal bovine serum (FBS) and 50 ug/mL ampicillin at 37°, 5% CO2. For juvenile, mature-hearing cochlea, 7–10-week-old C57BL/6 RIBEYE-tagRFP mice of both sexes were

euthanized with carbon dioxide and then decapitated. The temporal bone was extracted from the skull by removal of the auditory bulla and then placed in ice-cold Phosphate-buffered saline

(PBS). Soft tissue and ossicles were removed with fine forceps. The bone covering the apical turn of the cochlea was flicked off with a #11 scalpel, preserving the membranous labyrinth and

exposing the helicotrema. Reissner’s membrane was removed (Supplementary Fig. S5) IMMUNOHISTOCHEMISTRY P4-P6 RIBEYE-tagRFP neonatal mouse cochleae were cultured overnight and fixed the next

day with 4% paraformaldehyde at room temperature (RT) for 20 min. After 3 PBS washes, and blocking (94% PBS, 5% normal goat serum, and 1% Triton X-100, 30 min at RT), cultures were incubated

in primary (24 h, RT) and secondary antibodies (1.5 h, RT) in 98.6% PBS, 1% normal horse serum, and 0.4% Triton X-100. Primary antibodies were as follows: 1) mouse (IgG1) anti-CtBP2 (1:200;

#612044, BD Transduction Labs) for pre-synaptic ribbons; 2) mouse (IgG2a) anti-GluA2 (1:2000; #MAB397, Millipore) for post-synaptic receptor patches; 3) rabbit anti-Myosin 7a (1:400;

#25-6790 Proteus Biosciences) for hair cells; and 4) chicken anti-NF-H polyclonal (1:1000; Chemicon). Secondary antibodies: 1) Alexa Fluor 488-conjugated goat anti-mouse (IgG2a; 1:1000;

#A21131, Life Technologies); 2) Alexa Fluor 568-conjugated goat anti-mouse (IgG1; 1:1000; #A21124, Life Technologies); 3) Alexa Fluor 647-conjugated chicken anti-rabbit; (1:200; #A21443,

Life Technologies); and 4) Alexafluor 488-conjugated goat anti-chicken (1:1000). Confocal (Nikon A1R upright line-scanning) microscopic images of the hair-cell region in the mid-portion of

the middle cochlear turn were obtained with 60X oil immersion objective (Plan Apo, 1.40 NA). LIVE IMAGING Neonatal cochlear cultures were incubated for 24 h and then loaded with FM1-43 dye

(5 μM for 15 s) with three 10-sec HBSS washes and incubated for 30 min at 37 degrees for de-esterification to delineate hair cells. Juvenile cochlear explants were dissected as described

above and then immediately loaded with FM1-43 dye (7.5 μM for 10 s) with three 10-s HBSS washes and incubated for 5 min at 37 degrees to delineate hair cells. Specimens were placed on a

custom 3D printed slide and suspended in HBSS in a hole made on a plastic coverslip for live imaging. Time from euthanasia to initiation of imaging averaged 15 min. Volumetric regions of

interest of the organ of Corti with one intact row of IHCs and three intact rows of outer hair cells (OHCs) were visualized. Optical sections in the x–y plane were recorded at 2x averaging,

1.2 AU pinhole, 1.1 μs/pixel dwell time, 0.257 μs frame rate, and 0.4 μm intervals in the z-axis spanning the entire IHC height in the middle cochlear turn (neonates) and apical cochlear

turn (juveniles) on a Nikon A1R upright line-scanning confocal microscope with 60x water-immersion objective (NIR Apo, 1.0 NA) and stagetop incubator (OKOlab). All solutions were prewarmed

to 37 degrees, with incubation and imaging controlled for temperature. CO2, humidity, pressure, and air flow were controlled for neonatal cultures only. Acquisition of the entire confocal

Z-stack took approximately 5 min. For dynamic measures of displacement and speed, a pair of Z-stacks was performed one immediately after the other, thus resulting in an average minimum time

interval between images of 5 min, which determined the temporal resolution of live timecourse imaging. For experiments in which dynamic measurements at multiple timepoints 2–24 h apart,

cochleae were returned to the CO2 tissue-culture incubator in between imaging sessions. For continuous serial measurements, image stacks were acquired continuously with the specimen

maintained in the stagetop incubator. To compare puncta imaged in live and fixed specimens, neonatal cochleae were cultured overnight and two live successive z-stack images were captured.

Cochleae were immediately fixed with 4% PFA and hair cells stained with rabbit anti-Myosin 7a (1:400; #25-6790 Proteus Biosciences) and Alexa Fluor 647-conjugated 126 chicken anti-rabbit;

(1:200; #A21443, Life Technologies). These fixed and stained cochleae were then imaged using identical parameters as for live imaging. IMAGE PROCESSING RIBEYE puncta were rendered using the

Spots function on Imaris (v9.9.1). A volumetric region of interest containing the IHCs was manually segmented. Estimated X-Y Diameter (0.400 μm), Quality (116 for neonates and 85 for adults)

and Region Threshold (300 for neonates and 85 for adults) were determined initially from manual validation; these parameters yielded spot identification that approximated puncta identified

manually in pilot imaging experiments. Parameters were determined separately for neonatal and juvenile experiments, and were kept constant across all age-matched experiments. Auto depth

placement was based on the FM1-43-labeled IHCs in the FITC channel. The Surface function was used to create 3D-rendered hair cells. The Smooth option was selected with the Surface detail set

to 0.4 μm, followed by appropriate thresholding using the Absolute intensity parameter. Next, the filter “Number of voxels img=1” was used to select all the correct hair cell surfaces. This

enabled the establishment of a 3D reference frame with constant IHC placement, upon which movement of the RIBEYE puncta could be ascertained. After punctal identification and tracking, we

extracted, for each timepoint (for static readouts) and successive pairs of timepoints (for dynamic readouts): punctal volume and X, Y, and Z position, from which we calculated displacement

over time (velocity). Workflow for image processing is shown in Supplementary Fig. S6. To quantify the number of RIBEYE puncta per IHC, one blinded investigator performed manual count of the

number of RIBEYE puncta per IHC44. NOISE EXPOSURE AND GLUTAMATE EXCITOTOXICITY As a model for glutamate excitotoxicity, neonatal cochlear explants were dissected, incubated for 24 h, and

exposed to culture media containing 0.4 mM KA for 2 h. Specimens were subjected to live imaging before and during treatment as well as 2 and 24 h post-treatment. As a model for cochlear

synaptopathy, 7–10-week-old RIBEYE-tagRFP mice were exposed to 8–16 kHz octave-band white noise at 98 dB SPL for 2 h44. Noise exposure was performed on awake mice in a custom-built,

calibrated, reverberant sound chamber. Animals were placed freely in an acoustically transparent wire cage on a rotating platform for the duration of the noise exposure. Unexposed

littermates were used as negative controls. Auditory brainstem response (ABR) thresholds were measured in open-field response to pure-tone pips at 8, 16, and 32 kHz (RZ6, Tucker-David

Technologies) in a soundproof chamber44. STATISTICAL ANALYSIS AND QUANTIFICATION Data analysis was performed using Matlab (R2023a, Mathworks) and Prism (v9, GraphPad). Pairwise comparisons

were conducted using Student’s _t_ test (two-tailed, paired or unpaired when appropriate) with data presented as means ± 95% CI. Alpha value of 0.05 was used to determine statistical

significance. Outlying values were removed when indicated as determined by the ROUT method (Prism) with Q value set to 1%. STUDY APPROVAL Experimental protocols were approved by the

University of California, San Francisco Institutional Animal Care and Use Committee, and all methods were carried out in accordance with relevant guidelines and regulations. We have complied

with all relevant ethical regulations for animal use. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

DATA AVAILABILITY All data supporting the findings of this study are available within the paper and its Supplementary Information. The source data behind the graphs in the paper can be

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Download references AUTHOR INFORMATION Author notes * These authors contributed equally: Noura Ismail Mohamad, Peu Santra. AUTHORS AND AFFILIATIONS * Department of Otolaryngology-Head and

Neck Surgery, University of California, San Francisco, San Francisco, CA, USA Noura Ismail Mohamad, Peu Santra, Yesai Park, Ian R. Matthews, Emily Taketa & Dylan K. Chan Authors * Noura

Ismail Mohamad View author publications You can also search for this author inPubMed Google Scholar * Peu Santra View author publications You can also search for this author inPubMed Google

Scholar * Yesai Park View author publications You can also search for this author inPubMed Google Scholar * Ian R. Matthews View author publications You can also search for this author

inPubMed Google Scholar * Emily Taketa View author publications You can also search for this author inPubMed Google Scholar * Dylan K. Chan View author publications You can also search for

this author inPubMed Google Scholar CONTRIBUTIONS Initial design: N.I.M., P.S., D.K.C. Experimental and ethical oversight, and funding: D.K.C. Experimental contributions: N.I.M., P.S., Y.P.,

I.R.M., E.T., D.K.C. Data and statistical analysis: N.I.M., P.S., Y.P., D.K.C. Manuscript drafting: N.I.M., P.S., D.K.C. Manuscript review and approval: N.I.M., P.S., Y.P., I.R.M., E.T.,

D.K.C. Multiple first and corresponding authors: N.I.M. and P.S. share first position. N.I.M. was primarily responsible for experimental design, development of the experimental preparation

and design and conduct of the imaging experiments. P.S. was primarily responsible for development of the analytic process and conducted the majority of the data analysis. Between N.I.M. and

P.S., N.I.M. is listed first due to being the original designer of the project. CORRESPONDING AUTHOR Correspondence to Dylan K. Chan. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Communications Biology_ thanks the anonymous reviewers for their contribution to the peer review of this work. Primary

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THIS ARTICLE CITE THIS ARTICLE Ismail Mohamad, N., Santra, P., Park, Y. _et al._ Synaptic ribbon dynamics after noise exposure in the hearing cochlea. _Commun Biol_ 7, 421 (2024).

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