Data from: Presynaptic Nrxn3 is essential for ribbon-synapse maturation in hair cells

Description

Notes

Methods

Immunohistochemistry

Zebrafish immunohistochemistry and imaging

Immunohistochemistry was performed on whole larvae at either 3 dpf or 5 dpf. Whole larvae were fixed with paraformaldehyde (PFA 4%; Thermoscientific; 28906) in PBS at 4°C for 3.5 hr. For Ca_V1.3 labeling (Ca_V1.3, Otoferlin, MAGUK or Ca_V1.3, Paravalbumin, CTBP), all wash, block and antibody solutions were prepared with PBS + 0.1% Tween (PBST). For pre- and post-synaptic labeling (MYO7A, CTBP, MAGUK, Calretinin, SOX2, GFP), all wash, block and antibody solutions were prepared with PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). After fixation, larvae were washed 4 × 5 min in PBST or PBDDT. For Ca_V1.3 labeling, prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H₂O in glass vials. The H₂O was removed and replaced with ice-cold acetone and larvae placed at −20°C for 5 min, followed by a 5 min H₂O wash. The larvae were then washed for 4 × 5 min in PBST. For all immunolabels, larvae were blocked overnight at 4°C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST or PBDTT). After block, larvae were incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST or PBDTT) overnight, nutating at 4°C. The next day, the larvae were washed for 4 × 5 min in PBST or PBDTT to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature, with minimal exposure to light. Secondary antibodies were removed by washes with PBST or PBDTT for 4 × 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. Fixed samples were imaged on an upright LSM 780 or 980 laser-scanning confocal microscope with an Airyscan 2 attachment using Zen (Carl Zeiss) and a 63x/1.4 NA Plan Apo oil immersion objective lens. Airyscan z-stacks were acquired every 0.15 µm with a 0.043 µm X-Y pixel size for lateral-line and medial-crista hair cells, and every 0.15 µm with a 0.067 µm X-Y pixel size for hair cells in the anterior macula. The Airyscan z-stacks were autoprocessed in 2D. Experiments were imaged with the same acquisition settings to maintain consistency between comparisons. 

Mouse immunohistochemistry and imaging

Temporal bones were isolated, and an insulin syringe was used to gently flush cold paraformaldehyde (PFA 4%; Electron Microscopy Sciences; 15710) through the cleared oval and round windows after poking a small hole at the cochlear apex. Temporal bones were then immersion-fixed in PFA for 1 hour at 4°C, washed in PBS, and rotated overnight in EDTA 4% for decalcification. The next day, cochleae were dissected in 3 approximate thirds (base, mid and apex) before blocking and permeabilization for 1 hr at room temperature under agitation (1% bovine serum albumin; 0.5% Triton X-100). The following primary antibodies were used: CTBP2, GluR2 and mouse anti-MYO7A. Primary and secondary antibodies were incubated overnight at 4°C in PBS. Samples were washed 3 times in PBS + 0.05% Triton X-100 after each antibody incubation and finally post-fixed in PFA 4% for at least 1 hr at room temperature. Samples were then mounted flat in Mowiol mounting medium (Calbiochem/MilliporeSigma 4759041) using two layers of office tape as a spacers for the coverglass (18x18mm #1.5). Mowiol (10% w/v) was prepared in (25% w/v) glycerol and 0.1M Tris-Cl pH8.5. Mounted samples were imaged on an upright LSM 980 laser-scanning confocal microscope with using Zen Blue 3.4 (Carl Zeiss) and an 63x 1.4 NA oil objective lens. Z-stacks containing 6-9 IHCs were acquired every 0.250 µm with an 0.085 µm X-Y pixel size in confocal mode. 

RNA FISH to detect nrxn3a and nrxn3b mRNA in lateral-line hair cells

To detect mRNA for nrxn3a and nrxn3b in zebrafish, we followed the Molecular Instrument-RNA FISH Zebrafish protocol, Revision Number 10 (https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf), with a few minor changes to the preparation of fixed whole-mount larvae. For our dehydration steps, we dehydrated using the following methanol series: 25, 50, 75, 100, 100% methanol, with 5 min for each step in the series. To permeabilize, we treated larvae with 10 µg/mL proteinase K for 20 min. RNA FISH probes were designed to target the long α form of zebrafish nrxn3a and nrxn3b (Molecular Instrument Probe lot # PRP848, PRP849).After completing the RNA FISH protocol, we mounted the larvae in ProLong Gold Antifade (ThermoFisher, P36930) under 1.5 coverglass. Samples were imaged on an upright LSM 980 laser-scanning confocal microscope with an Airyscan 2 attachment using Zen Blue 3.4 (Carl Zeiss) and a 63x/1.4 NA Plan Apo oil immersion objective lens. Airyscan z-stacks were acquired every 0.160 µm with a 0.043 µm X-Y pixel size. The Airyscan z-stacks were autoprocessed in 2D.

Image processing and quantification of synapses and RNA FISH puncta

To identify and quantify puncta (presynaptic, postsynaptic, Ca_V1.3 cluster or RNA FISH puncta), an automated synapse quantification method using a customized Fiji-based macro, "Complete Synapse Counter v5.2" (https://github.com/KindtLab-NIDCD/CompleteSynapseCounter5.2) was used. Prior to automated puncta quantification, each channel was background subtracted using rolling-ball radius background subtraction. Then each z-stack was max-intensity projected. A mask was generated by manually outlining the region or interest (ex: hair cells) in the reference channel. This mask was then applied to the z-projection of each synaptic component or RNA FISH channel. Each masked image was then analyzed using the automated Fiji macro. In this macro, the images were thresholded using an adaptive thresholding plugin by Qingzong TSENG (https://sites.google.com/site/qingzongtseng/adaptivethreshold) to generate a binary image of the puncta. Individual synaptic or RNA FISH puncta were then segmented using the particle analysis function in Fiji. For particle analysis, the following minimum size thresholds were applied: zebrafish lateral-line images – CTBP: 0.025 μm², MAGUK: 0.04 μm², Ca_V1.3 0.025 μm², nrxn3a, nrxn3b and dapB RNA FISH particles: 0.03 μm² and 0.01 μm², zebrafish inner ear images – CTBP: 0.025 μm², MAGUK: 0.025 μm², mouse IHCs – CTBP: 0.025 μm², GluR2: 0.025 μm². A circularity factor between 0.1-0.5 was also applied to particle analysis. A watershed was applied to the particle analysis result to break apart overlapping synaptic components. After the watershed, the particle analysis was rerun with size and circularity thresholds to generate ROIs and measurements of each synaptic or RNA FISH component. The ROIs were applied to the original z-projection to get the average intensity and area of each punctum.

To recognize paired synaptic components, images were further processed using "Complete Synapse Counter v5.2". Here, the overlap and proximity of ROIs from different channels (ex: pre- and post-synaptic puncta) were calculated. ROIs with positive overlap or ROIs within 2 pixels were counted as paired or partner components. The ROIs were applied to the original z-projection to get the average intensity and area of each paired or unpaired punctum.

Some image datasets required a pre-processing step prior to entry into the "Complete Synapse Counter v5.2". This includes zebrafish samples imaged at 3 dpf and our mouse IHCs datasets. For the pre-processing step, the volumes were segmented in VVDviewer (https://github.com/JaneliaSciComp/VVDViewer). Staining outside of the hair cell was manually segmented or removed using VVDviewer. After this segmentation, the z-stacks were then max-intensity projected and processed using the "Complete Synapse Counter v5.2" macro.

Quantification of lateral-line afferent terminal areas and single afferent selectivity

To quantify the area occupied by the 4-6 afferent terminals beneath lateral-line neuromasts, we examined z-stacks of Calretinin-labeled terminals. Each z-stack was opened in FIJI and max-intensity projected. Projected images were autothresholded to isolate the terminals, and the mean area within the threshold was measured. 

Larvae with positive tdTomato expressions in individual afferent neurons were stained with CTBP and MAGUK to label pre- and post-synapses, as well as phalloidin to visualize hair-bundle orientations. After the immunostaining, single afferent terminals were imaged under using a Zeiss LSM 980 confocal microscope in Airyscan mode as described above. Z-stacks were then loaded in VVDviewer for 3D viewing of afferent terminals and their connections with hair cells. Hair-cells numbers and their orientations were assigned manually based on the weak membrane labeling of MAGUK and phalloidin label respectively. To assign synapses and hair cells to terminals, we manually identified all the complete synapses (paired CTPB and MAGUK label) that colocalized with each tdTomato terminal. This allowed us to determine the number of hair cells innervated and the number of complete synapses formed per terminal.

To analyze the selectivity of individual terminals we used a selectivity index. This index was defined as percentage of the number of hair cells with the domination orientation innervated, divided by the total number of hair cells innervated. Here an index value of 50% indicates that the afferent terminal showed no selectivity when choosing between the two different orientations, while 100% indicates perfect selectivity towards a single orientation.

Calcium imaging of lateral-line hair cells and afferents

For functional imaging, 4-6 dpf larvae were anesthetized in 0.04% Tricaine-S (tricaine methanesulfonate, Western chemical, TRS1), pinned to a Sylgard-filled perfusion chamber at the head and tail, and paralyzed by injection of 125 µM a-bungarotoxin (Tocris, 2133) into the heart cavity, as previously described (Lukasz and Kindt, 2018). Larvae were then rinsed three times in E3 embryo media to remove the tricaine. Next, larvae were rinsed three times with extracellular imaging solution (in mM: 140 NaCl, 2 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES, pH 7.3, OSM 310±10) and allowed to recover. Stimulation was achieved by a fluid jet, which consisted of a pressure clamp (HSPC-1, ALA Scientific) and glass pipette, pulled and broken to an inner diameter 40-50 µm, and filled with extracellular imaging solution. A 500-ms pulse of positive or negative pressure was used to deflect the hair bundles of mechanosensitive hair cells along the anterior-posterior axis of the fish. Hair cells of the two orientations (anterior and posterior) were stimulated separately. Stimuli that deflected kinocilia 5-15 µm were included in the analysis, as these deflections represent saturating stimuli that do not induce damage.

Hair-cell responses to stimuli were imaged with an A1R laser-scanning confocal scan head on an upright Nikon NI-E microscope with a resonant scanner and a 60x/1.0 NA CFI Fluor water immersion objective equipped with a z-piezo. Acquisition was controlled with Nikon Elements Advanced Research v. 5.20.02. GCaMP6s fluorescence was excited with a 488 nm solid-state laser passed through a standard 405/488/561/640 BS20/80 dichroic and collected with a 560 nm low-pass dichroic and 525/50 emission filter. Images were acquired using a GaAsP PMT and 4x averaging. Pixel size for presynaptic imaging was 0.28 µm; pixel size for MET imaging was 0.14 µm. Each neuromast (L2 or L3) was stimulated four times (starting with a posterior-to-anterior stimulus and alternating between the two directions) with an inter-stimulus interval of ~2 min. This enabled us to collect presynaptic responses (collected first) and hair-bundle responses to both stimulus directions for each neuromast. 3 z-slices (1.5 µm step size for presynaptic responses; 0.5 µm step size for hair bundle responses) were collected per timepoint for 110 timepoints at a frame rate of 33 ms for a total of ~100 ms per z-stack and a total acquisition time of ~11 sec. Stimulation began at timepoint 31; timing of the stimulus was triggered by an outgoing voltage signal from Nikon Elements.

Calcium responses in the afferent process were acquired on a Swept-field confocal system built on a Nikon FN1 upright microscope (Bruker) with a 60x/1.0 NA CFI Fluor water-immersion objective. The microscope was equipped with a Rolera EM-C2 EMCCD camera (QImaging), controlled using Prairie view 5.4 (Bruker). GCaMP6s was excited using a 488 nm solid state laser. We used a dual band-pass 488/561 nm filter set (59904-ET, Chroma). Pixel size for postsynaptic imaging was 0.27 µm. Stimuli were delivered as outlined above for hair-cell responses. Each neuromast (L2, L3 or L4) was stimulated two times with an inter-stimulus interval of ~2 min. 5 z-slices (1.0 µm step) were collected per timepoint for 80 timepoints at a frame rate of 20 ms for a total of ~100 ms per Z-stack and a total acquisition time of ~8 sec. Stimulation began at timepoint 31; timing of the stimulus was triggered by an outgoing voltage signal from Prairie view.

Acquired images were converted into TIFF series for processing. Researchers were blind to genotype during analysis. Z-stacks were average projected, registered, and spatially smoothed with a Gaussian filter (size = 3, sigma = 2) in custom-written MatLab software as described previously ((Zhang et al., 2018); https://github.com/KindtLab-NIDCD/ImagePro)). The first 10 timepoints (~1 sec) were removed to reduce the effect of initial photobleaching. Registered average projections were then opened in Fiji for intensity measurements. Using the Time Series Analyzer V3 plugin, circular ROIs (18x18 pixels for presynaptic responses; 8x8 pixels for hair-bundle responses, 12x12 pixels for afferent process) were placed on hair bundles or synaptic sites; average intensity measurements over time were measured for each ROI, as described previously (Lukasz and Kindt, 2018). Neuromasts were excluded in the case of motion artifacts. Hair-bundles responses were excluded if they responded to stimuli of both directions. All other data was included in analyses. Presynaptic responses were defined as >10% ∆F/F0 within the 500 ms stimulus or >20% within 1 sec of stimulus onset. Hair-bundle responses were defined as >15% ∆F/F0 within the 500 ms stimulus and >15% in the 500 ms after the stimulus. Postsynaptic responses were defined as >5% ∆F/F0 and a minimum duration of 500 ms. Square wave responses indicate movement artifacts and were excluded. Calcium imaging data was further processed in Prism 10 (Graphpad). The first 20 timepoints were averaged to generate an F0 value, and all responses were calculated as ∆F/F0. Responses presented in figures represent average responses of synaptically active cells within a neuromast. The max ∆F/F0 was compared between wild-type animals and double mutants. To measure baseline GCaMP6s intensities in hair bundles, the presynaptic region or afferent terminals, the mean GCaMP6s intensity was measured during the prestimulus (2 s) time window. For the images acquired in this time window, the GCaMP6 images were autothresholded, and the mean intensity was measured at each time point. Then all the time points during the prestimulus time window were averaged for each neuromast. 

Mouse auditory brainstem response (ABR) tests

All tests were performed in a sound-attenuating chamber. Animals between P28 and P32 were anesthetized with a mix of ketamine and xylazine (1 mg and 0.8 mg per 10g of body weight, respectively). Body temperature was maintained at 37°C using a heating pad (FHC Inc.). Animals were then tested using the RZ6 Multi-I/O Processor System coupled to the RA4PA 4-channel Medusa Amplifier (Tucker-Davis Technologies). Subdermal needles were used as electrodes, with the active electrode inserted at the cranial vertex, the reference electrode under the left ear, and the ground electrode at the right thigh. ABRs were recorded after binaural stimulation in an open field by tone bursts at 8, 16, 32, and 40 kHz generated at 21 stimuli/second, and a waveform for each frequency/dB level was produced by averaging the responses from 512 stimuli. ABR thresholds were obtained for each frequency by reducing the sound pressure level (SPL) by 5 decibels (dB) between 90 and 20 dB. We compared waveforms by simultaneously displaying 3 or more dB levels on screen at the same time to identify the lowest level at which an ABR waveform could be recognized. Wave I amplitudes were measured by annotating the peak and trough of the first ABR waveform and calculating the difference (nV), and wave I delay was measured at the peak of the first wave (ms). In these experiments, controls consist of a pool of 0 (Cre-negative); Nrxn3^flox/+, 0; Nrxn3^flox/flox, and Atoh1-Cre; Nrxn3^flox/+ animals. Each control genotype was also compared separately (Fig S17C-D).

 Zebrafish startle behavior

A Zantiks MWP behavioral system was used to examine acoustic startle responses. Behavioral trials were performed at 5 dpf, on three independent days. For this behavioral analysis, we compared nrxn3a^+/-; nrxn3b^+/- double heterozygotes to nrxn3a^-/-; nrxn3b^-/- double mutants for an in-clutch, sibling comparison. Nrxn3a^+/-; nrxn3b^+/- double heterozygotes showed a slight (12%) yet significant reduction in complete synapses compared to wild-type controls. We also compared nrxn3a^+/-; nrxn3b^+/- double heterozygotes and nrxn3a^-/-; nrxn3b^-/- double mutants sibling to wild-type animals born the same day; this analysis revealed no difference in startle response between these genotypes.

The Zantiks system tracked and monitored behavioral responses via a built-in infrared camera at 30 frames per second. A 12-well plate was used to house larvae during behavioral analysis. Each well was filled with E3 and 1 larva. All fish were acclimated in the plate within the Zantiks chamber in the dark for 15 min before each test. To induce startle, an integrated stepper motor was used to drive a vibration-induced startle response. A vibrational stimulus that triggered a maximal % of animals startling in controls without any tracking artifacts (due to the vibration), was used for our strongest stimuli. Each larva was presented with each vibrational stimulus 5 times with 100 s between trials. For each animal, the proportion of startle responses out of the 5 trials was plotted. During the tracking and stimulation, a Cisco router connected to the Zantiks system was used to relay x, y coordinates of each larva every frame. To qualify as a startle response, a distance above 4 pixels or ~1.9 mm was required within 2 frames after stimulus onset. Animals were excluded from our analysis if no tracking data was recorded for the animal.