L-type voltage-gated calcium channel agonists mitigate hearing loss and modify ribbon synapse morphology in the zebrafish model of Usher syndrome type 1

Abstract

The mariner (myo7aa^-/- ) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa^-/- mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa^-/- zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa^-/- hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa^-/- mutants have fewer postsynaptic densities - as assessed by MAGUK immunolabeling - compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa^-/- mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa^-/- mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa^-/- mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse - in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta - shift swimming behavior and improve acoustic startle response.

Keywords: Hair cell; Hearing loss; Ribbon synapse; Zebrafish; myo7aa.

Fig. 1.

L-type voltage-gated calcium channel agonists restore function in hair cells. (A) In a normal hair cell, sound causes stereocilia to deflect towards the tallest stereocilium and induces the mechanotransduction channels (METs) at the top of the stereocilia to open in response, allowing cations such as calcium (Ca²⁺ ) and potassium (K⁺) to flow into the cell. This causes a change in membrane potential, which leads to the opening of L-type voltage-gated calcium channels at the basolateral sides of the cell. Calcium enters the cell and increases intracellular calcium concentrations, thereby mediating neurotransmitter release from synaptic vesicles within the ribbon synapse into the synaptic cleft, thus, stimulating afferent neurons. (B) In cells that lack MYO7A, correct MET channel gating does not occur. Therefore, the appropriate membrane potential is not reached to allow L-type voltage-gated calcium channels to open, and there is insufficient synaptic transmission to the auditory nerve to create meaningful interactions. (C) We hypothesize that, by augmenting the downstream signal in myo7aa^−/− mutant hair cells, a new functional response to sound can be reconstituted when the sensitivity of the calcium channel is increased through treatment with L-type voltage-gated calcium channel agonists.

Fig. 2.

myo7aa^−/− larvae have altered mechanotransduction activity, stereocilia structure and ribbon synapse structure. (A,B) Representative light sheet fluorescence microscopy images showing the lateral view of 5 dpf wild-type (A) and myo7aa^−/− (B) larvae after brief exposure to FM1-43. FM1-43 staining indicates functional mechanotransduction. Arrowheads point to the swim bladder; myo7aa^−/− larvae do not have an inflated swim bladder. (C,D) Representative confocal microscopy images showing the stereocilia of the lateral crista of wild-type (C) and myo7aa^−/− (D) larvae at 5 dpf, stained with Alexa-Fluor-488 tagged to phalloidin, a high-affinity filamentous actin probe. Arrows indicate the organized and smooth stereocilia in wild-type hair cells, and disorganized and splayed stereocilia in myo7aa^−/− hair cells. Notice that not all stereocilia are splayed in myo7aa^−/− hair cells. (E,F) Representative TEM images showing the ribbon synapse structures of 5 dpf wild-type (E) and myo7aa^−/− (F) larvae. Arrows point to the ribbon density, stars indicate a halo of tethered vesicles in the inner ear hair cells. Scale bars: 200 nm. (G) Wild-type ribbon synapses have a comparable ribbon area to myo7aa^−/− ribbon synapses (two-tailed t-test). (H) Wild-type ribbon synapses have an increased number of tethered vesicles compared to myo7aa^−/− ribbon synapses (two-tailed t-test). The bold black lines represent the mean of the data set and error bars are 95% confidence intervals. Electron microscopy experiments were replicated four times for wild type and three times for myo7aa^−/− mutants.

Fig. 3.

myo7aa^−/− Ctbp2 and MAGUK synaptic elements are different from those of wild-type larvae. (A,B) Representative maximum-intensity projection (z-stack top-down image) of MI1 neuromasts from 5 dpf wild-type larvae (A) and myo7aa^−/− larvae (B). Nuclei were stained with DAPI (blue), Ctbp2 is shown in red. Dotted circles outline one hair cell. Scale bars: 10 µm. (C,D) Representative maximum-intensity projection (z-stack top-down image) of MI1 neuromasts from 5 dpf wild type (C) and myo7aa^−/− (D). Nuclei were stained with DAPI (blue), MAGUK is shown in green. Dotted circles outline one cell. Scale bars: 10 µm. (E) Distribution of Ctbp2 puncta across a collection of ribbon-containing cells reveals that most 5 dpf wild-type ribbon-containing cells have two Ctbp2 puncta compared to three Ctbp2 puncta in myo7aa^−/− hair cells. All error bars are 95% confidence intervals. (F) Distribution of MAGUK puncta across a collection of postsynaptic densities reveals that the proportion of postsynaptic densities is comparable between wild-type and myo7aa^−/− larvae. (G,I) 5 dpf wild-type MI1 neuromasts have a greater number of ribbon-containing cells and greater total number of Ctbp2 puncta compared to myo7aa^−/− MI1 neuromasts (two-tailed t-test). Bold black lines represent the mean of the data set and error bars are 95% confidence intervals. (H,J) 5 dpf wild-type MI1 neuromasts have a greater number of postsynaptic densities and a greater number of total MAGUK puncta compared to myo7aa^−/− MI1 neuromasts (two-tailed t-test). Bold black lines represent the mean of the data set and error bars are 95% confidence intervals. (K) Depiction of the basal end of a hair cell innervated by the auditory nerve. Ctbp2 is alternatively spliced to produce Ribeye protein, the main component of the synaptic ribbon. Ribeye has a halo of tethered vesicles that form the synaptic ribbon (red). Members of the membrane associated guanylate kinase (MAGUK) superfamily are a part of the postsynaptic density (PSD), targeting and anchoring glutamate receptors to the synaptic terminals on the postsynaptic cell (green). Experiments were replicated five times for wild type for both Ctbp2 and MAGUK, and five times for myo7aa^−/− mutants for Ctbp2 and two times for MAGUK.

Fig. 4.

myo7aa^−/− larvae exhibit larger turning angles. Movement tracking of 5 dpf wild-type and myo7aa^−/− larvae at 2.5-min intervals, with 5 ms electric stimuli (50 mV) administered every 20 s. Ctrax software was used for video processing and MATLAB 2012b for video analysis. (A,B) myo7aa^−/− larvae have larger absolute smooth orientations (global change in body orientation) compared to wild-type larvae. Individual turning angles from a population of wild-type and myo7aa^−/− larvae were used to construct the lines (two-tailed t-test). Bold black lines in B represent the mean of the data set and error bars are 95% confidence intervals. Experiments were replicated five times for both wild-type and myo7aa^−/− mutants.

Fig. 5.

myo7aa^−/− mutant ribbon synapse abnormalities are diminished after exposure to (R)-Baclofen. (A-E) Representative TEM images showing ribbon synapses of untreated wild-type (A) and untreated myo7aa^−/− mutant (B) larvae, as well as myo7aa^−/− mutants treated with 5 µM (±)-Bay K 8644 (C), 250 µM Nefiracetam (D) or 125 µM (R)-Baclofen (E). All images were taken at 5 dpf. Scale bars: 1000 nm. (F) The ribbon area in untreated myo7aa^−/− is smaller compared to that in myo7aa^−/− treated with 125 µM (R)-Baclofen. Ribbon synapses of untreated wild-type larvae have a ribbon area comparable to that of myo7aa^−/− mutants treated with 125 µM (R)-Baclofen (two-tailed t-test). (G) Ribbons of untreated myo7aa^−/− mutants have fewer tethered vesicles compared to those of myo7aa^−/− mutants treated with 125 µM (R)-Baclofen. Ribbons of untreated wild-type larvae have a number of tethered vesicles comparable to those of myo7aa^−/− larvae treated with 125 µM (R)-Baclofen (two-tailed t-test). Bold black lines represent the mean of the data set and error bars are 95% confidence intervals. Experiments were replicated three times for myo7aa^−/− mutants treated with each drug.

Fig. 6.

myo7aa^−/− neuromasts exposed to L-type voltage-gated calcium channel agonists show a Ctbp2 distribution which more closely resembles that in wild-type larvae. (A-D,K-N) Representative maximum-intensity projection (z-stack top-down image) of MI1 neuromasts from untreated and treated 5 dpf wild-type larvae (A-D) and untreated and treated myo7aa^−/− larvae (K-N). Nuclei were labeled with DAPI (blue), staining for Ctbp2 is shown in red. Scale bars: 10 µm. (I) The majority of 5 dpf untreated wild-type ribbon-containing cells show three Ctbp2 puncta, with a statistical significance of wild-type larvae treated with 5 µM (±)-Bay K 8644 showing one, two, three or four puncta and those treated with 125 µM (R)-Baclofen showing two puncta. There was no statistically significant change upon incubation with 250 µM Nefiracetam. (S) The majority of 5 dpf untreated myo7aa^−/− ribbon-containing cells have two Ctbp2 puncta. Upon incubation with 5 µM (±)-Bay K 8644, 250 µM Nefiracetam or 125 µM (R)-Baclofen the majority of hair cells have three Ctbp2 puncta, with a statistical significance of three puncta in all treatment groups. (E-H,O-R) Representative maximum intensity projection (z-stack top-down image) of MI1 neuromasts from 5 dpf wild-type larvae untreated and treated (E-H) and myo7aa^−/− larvae untreated and treated (O-R). Nuclei were labeled with DAPI (blue), staining for MAGUK is shown in green. Scale bars: 10 µm. (J) There is no statistical significance in the proportion of one, two, three, four, five or six+ MAGUK puncta between untreated wild-type larvae and any of the treatment groups – except the occurrence of four puncta in response to treatment with 5 µM (±)-Bay K 8644. (T) There is no statistical significance in the proportion of one, two, three, four, five or six+ MAGUK puncta between untreated myo7aa^−/− and any of the treatment groups. All error bars are 95% confidence intervals. Experiments for which Ctbp2 staining was carried out in wild-type animals were replicated twice for 5 µM (±)-Bay K 8644, three times for 250 µM Nefiracetam and twice for 125 µM (R)-Baclofen; MAGUK-labeling experiments were conducted twice for 5 µM (±)-Bay K 8644, three times for 250 µM Nefiracetam and three times for 125 µM (R)-Baclofen. Experiments for which Ctbp2 staining was carried out in myo7aa^−/− mutants were replicated twice for 5 µM (±)-Bay K 8644, twice for 250 µM Nefiracetam and three times for 125 µM (R)-Baclofen; MAGUK-labeling experiments were conducted twice for 5 µM (±)-Bay K 8644, three times for 250 µM Nefiracetam and three times for 125 µM (R)-Baclofen.

Fig. 7.

myo7aa^−/− swimming behavior and acoustic startle response improved during treatment with L-type voltage-gated calcium channel agonists. Movement tracking of 5 dpf wild-type and myo7aa^−/− larvae over a 2.5-min interval with a 5 ms electric stimulus (50 mV) administered every 20 s. Ctrax software was used for video processing and MATLAB 2012b for video analysis. (A,B) 250 µM Nefiracetam and 125 µM (R)-Baclofen did not affect the absolute smooth orientation (global change in body orientation) of wild-type larvae; however, 5 µM (±)-Bay K 8644 resulted in a decreased absolute smooth orientation compared to that of untreated wild-type larvae. When myo7aa^−/− larvae were individually treated with 5 µM (±)-Bay K 8644, 250 µM Nefiracetam or 125 µM (R)-Baclofen, either treatment decreased absolute smooth orientations, with the most robust response observed during incubation with 5 µM (±)-Bay K 8644. Individual turning angles from a population of wild-type and myo7aa^−/− larvae were used to construct the lines (two-tailed t-test). Bold black lines represent the mean of the data sets and error bars are 95% confidence intervals. Experiments were conducted twice for each drug treatment. (C) Sample movement tracing of wild-type and myo7aa^−/− larvae within individual treatment groups indicates that in the presence of 5 µM (±)-Bay K 8644, 250 µM Nefiracetam or 125 µM (R)-Baclofen induces changes to the swimming of myo7aa^−/− larvae that include smoother trajectories with more zig-zag-like swimming and fewer circling episodes. Diameter of the wells was 20 mm. (D,E) Acoustic startle response was captured by administering three stimuli at each frequency per experiment. Videos were scored blindly and the mean number of responses per total stimuli (mean response rate) was determined. myo7aa^−/− larvae showed little to no response at all frequencies. Upon incubation with 5 µM (±)-Bay K 8644 acoustic startle response in wild-type larvae decreased at all frequencies; however, myo7aa^−/− larvae showed a significant increase, with a response rate of >20% at 200 and 600 Hz. Incubation in 250 µM Nefiracetam increased acoustic startle response in wild-type and myo7aa^−/− larvae at all frequencies, although the increase in the myo7aa^−/− larvae is modest. 125 µM (R)-Baclofen decreased acoustic startle response significantly in wild-type larvae and had little to no effect on acoustic startle in the myo7aa^−/− larvae. Bold black lines represent the mean of the data set and error bars are the mean variance. Experiments were conducted eight times for wild-type and myo7aa^−/− mutant controls, twice for wild-type and myo7aa^−/− mutants incubated in 5 µM (±)-Bay K 8644 or 125 µM (R)-Baclofen, three times for wild type incubated in 250 µM Nefiracetam, and twice for myo7aa^−/− larvae incubated in 250 µM Nefiracetam.