Presynaptic CaV1.3 channels regulate synaptic ribbon size and are required for synaptic maintenance in sensory hair cells

Abstract

L-type calcium channels (Ca(V)1) are involved in diverse processes, such as neurotransmission, hormone secretion, muscle contraction, and gene expression. In this study, we uncover a role for Ca(V)1.3a in regulating the architecture of a cellular structure, the ribbon synapse, in developing zebrafish sensory hair cells. By combining in vivo calcium imaging with confocal and super-resolution structured illumination microscopy, we found that genetic disruption or acute block of Ca(V)1.3a channels led to enlargement of synaptic ribbons in hair cells. Conversely, activating channels reduced both synaptic-ribbon size and the number of intact synapses. Along with enlarged presynaptic ribbons in ca(V)1.3a mutants, we observed a profound loss of juxtaposition between presynaptic and postsynaptic components. These synaptic defects are not attributable to loss of neurotransmission, because vglut3 mutants lacking neurotransmitter release develop relatively normal hair-cell synapses. Moreover, regulation of synaptic-ribbon size by Ca(2+) influx may be used by other cell types, because we observed similar pharmacological effects on pinealocyte synaptic ribbons. Our results indicate that Ca(2+) influx through Ca(V)1.3 fine tunes synaptic ribbon size during hair-cell maturation and that Ca(V)1.3 is required for synaptic maintenance.

Figure 1.

Characterization of Ca_V1.3a channel localization and activity in cav1.3a mutants. A, Diagrams representing the secondary structure of the α-pore-forming subunit Ca_V1.3a. Each diagram represents the expected protein resulting from the genetic lesions in each cav1.3a mutant. The position of the antibody epitope is denoted by a blue star. B, Mechanically evoked Ca²⁺ responses in WT and cav1.3a mutants at 5 dpf in response to a 2 s, 10 Hz alternating square waveform. Each trace represents the average response of hair cells from four NMs. C, Scatter plot depicts the average Ca²⁺ response per NM in WT and cav1.3a mutants. n ≥ 4 fish and n ≥ 10 NMs per genotype. Error bars are SEM. ***p < 0.001, defined by the Dunnett's multiple comparison test. D–F, Representative labeling of Ca_V1.3a clusters in cross-sections of NMs in 5 dpf WT (B), R1250X (C), and R284C (D) larvae. Dashed lines outline hair cells. Ribeye b label in merged images indicates Ca_V1.3a clusters localized to synaptic ribbons. Scale bar, 3 μm. G, Fraction of Ribeye with localized Ca_V1.3a clusters within NMs (5 dpf). Each data point represents the NM at position 2 along the trunk (NM2) in individual larvae. Error bars are SEM. **p < 0.01, defined by the Dunnett's multiple comparison test. H, Box plots of the fluorescent intensities of presynaptic Ca_V1.3a. These plots show the median value (horizontal bar), the upper and lower quartiles (box), and the range (whiskers). Whiskers indicate the 10th and 90th percentiles. **p < 0.01, defined by the Dunn's multiple comparison test.

Figure 2.

Characterization of ribbon-synapse morphology in cav1.3a mutants at 3 and 5 dpf. A–C, Representative images of Ribeye b (Rib b) and MAGUK immunolabel in NM (position 1) hair cells of a 3 dpf WT (A), R1250X (B), and R284C larva (C). Scale bars: main panels, 3 μm; right panels, 1 μm. D, E, Box plots of puncta intensities in 3 dpf cav1.3a mutant and WT NM1 hair cells. Whiskers indicate the 10th and 90th percentiles. Each plot represents a population of intensity measurements of individual labeled punctum collected from NM1 hair cells of 14–15 individual larvae. D, Intensities of presynaptic Ribeye b. Mann–Whitney U test, ***p < 0.0001 for both mutant alleles. E, Intensities of postsynaptic MAGUK. Mann–Whitney U test, ***p < 0.0001 and **p = 0.0025, respectively. F, Side views of Ribeye b in 3 dpf WT and cav1.3a mutant hair cells. Hair cells are delineated with a dashed outline. White arrows indicate Ribeye aggregates in the cell body; magenta asterisks indicate presynaptic ribbons. Scale bar, 1 μm. G, H, The number of presynaptic and extrasynaptic Ribeye b aggregates per hair cell in 3 dpf WT and cav1.3a mutants. Each circle represents NM1 within a larva. The number of puncta per hair cell was approximated by dividing the number of Ribeye puncta within an NM by the number of hair cells in the NM. Error bars are SEM. G, Number of presynaptic Ribeye b puncta. Unpaired t test, p = 0.6876 and 0.9290, respectively. H, Number of cytoplasmic Ribeye b aggregates. Unpaired t test, ***p < 0.0001 for both mutant alleles. I–K, Representative images of Ribeye b and MAGUK label in NM2 hair cells of a 5 dpf WT (I), R1250X (J), and R284C larva (K). Scale bars: main panels, 3 μm; right panels, 1 μm. L–M, Box plots of puncta intensities in 5 dpf cav1.3a mutant and WT sibling NM2 hair cells. Whiskers indicate the 10th and 90th percentiles. Each plot represents a population of intensity measurements collected from NM2 hair cells of 10–11 individual larvae. L, Intensities of presynaptic Ribeye b. Mann–Whitney U test, p = 0.6138 and ***p < 0.0001, respectively. M, Intensities of postsynaptic MAGUK. Mann–Whitney U test, ***p < 0.0001 for both mutant alleles. N–O, The number of presynaptic and extrasynaptic Ribeye b aggregates per hair cell in 5 dpf WT and cav1.3a mutants. Each circle represents NM2 within an individual larva. Error bars are SEM. N, Number of presynaptic Ribeye b puncta. Unpaired t test, p = 0.0611 and 0. 6214, respectively. O, Number of cytoplasmic Ribeye b aggregates. Unpaired t test, *p = 0.03 and ***p = 0.0001, respectively.

Figure 3.

cav1.3a mutant hair-cell synapses progressively lose juxtaposition of presynaptic and postsynaptic components. A, B, Representative confocal images of Ribeye b (Rib b) and MAGUK in 3 dpf (A) and 5 dpf (B) WT and cav1.3a mutant hair cells. For display purposes, images were resampled (bicubic) in Photoshop to minimize pixilation. Scale bars, 1 μm. C, D, Percentage of MAGUK-label containing pixels overlapping with Ribeye b in 3 dpf (C) and 5 dpf (D) WT and cav1.3a mutant NM hair cells. Each circle represents an NM in an individual larva. Error bars are SEM. C, MAGUK immunolabel overlapped with Ribeye b comparably in 3 dpf mutants and WT. Unpaired t test, p = 0.1811 and 0.1162, respectively. D, MAGUK immunolabel overlapped significantly less with Ribeye b in 5 dpf mutants. Unpaired t test, ***p < 0.0001 for both mutant alleles. E, F, SR-SIM images of ribbon synapses in 3 dpf (E) and 5 dpf (F) cav1.3a mutants and WT. Scale bars, 1 μm. E, Images of 3 dpf ribbon synapses. The synaptic ribbons in cav1.3a mutants appear enlarged and often misshapen. The bottom-view images show MAGUK label beneath the ribbon synapse. F, Images of 5 dpf ribbon synapses. MAGUK appears even less spatially restricted to the synaptic ribbon than in 3 dpf hair cells. G, Fraction of 3 dpf ribbon synapses within individual NMs with PSDs juxtaposing one, two, or three synaptic ribbons. cav1.3a mutant NMs synapses contain two to three synaptic ribbons with much greater frequency than WT (n = 4 NMs per condition, each containing ∼15–25 synapses). H, The shape factor of synaptic ribbons in 3 dpf hair cells. Each spot represents an individual ribbon. The horizontal bars represent the mean values. Synaptic ribbons are significantly less round in cav1.3a mutants than WT siblings (sib). Mann–Whitney U test, *p = 0.0266 and ****p < 0.0001, respectively.

Figure 4.

vglut3 mutant hair cells have normal calcium responses and relatively normal synaptic ribbons. A, Representative immunolabeling of Ca_V1.3a clusters in a cross-section of an NM in vglut3^484+2T>2 larvae at 5 dpf. Merged image includes Ribeye b and DAPI. Scale bar, 3 μm. B, Scatter plot depicts the average calcium response per NM in WT and vglut3 mutants at 3 and 5 dpf. n ≥ 3 fish and n ≥ 7 NMs per genotype. Error bars are SEM. C, D, Representative confocal images of Vglut3, Ribeye b (Rib b), and MAGUK label in NM2 hair cells of a 5 dpf WT (C) and vglut3 ^484+2T>2 larvae (D). Scale bars: mail panels, 3 μm; right panels, 1 μm. E, F, Box plots of immunolabel puncta intensities in 3 and 5 dpf vglut3 ^484+2T>2 and WT sibling NM2 hair cells. Whiskers indicate the 10th and 90th percentiles. Each plot represents a population of intensity measurements collected from NM2 hair cells of 6 (3 dpf) or 10–12 (5 dpf) individual larvae. E, Intensities of presynaptic Ribeye b puncta. Mann–Whitney U test, p = 0.2420 and *p = 0.0156, respectively. F, Intensities of postsynaptic MAGUK puncta. Mann–Whitney U test, p = 0.2249 and ***p < 0.0001, respectively. G, Percentage of MAGUK-label containing pixels overlapping with Ribeye b in 5 dpf WT and vglut3 ^484+2T>2 mutant NM hair cells. MAGUK immunolabel overlapped with Ribeye b comparably in mutants and WT (unpaired t test, p = 0.1811). Each circle represents an NM in an individual larva. Error bars are SEM.

Figure 5.

Pharmacological block of Ca_V1.3a at 3 dpf enlarges synaptic ribbons in WT hair cells. A, Mechanically evoked Ca²⁺ responses in WT untreated NM hair cells (black) and hair cells exposed to 10 μm isradipine (red) for 15 min at 3 dpf. Cells were stimulated with a fluid jet for 2 s, using a 10 Hz alternating square waveform. Each trace represents the average response of hair cells from four NMs. B, Scatter plot depicts the average Ca²⁺ response per NM in WT and cav1.3a mutants at 3 dpf (white circles) and after isradipine treatment (red circles). n ≥ 4 fish and n ≥ 10 NMs per genotype. Error bars are SEM. ****p < 0.0001, defined by a paired t test. C–C″, Representative images of Ribeye a (Rib a), Ribeye b (Rib b), and MAGUK immunolabel in NM1 of 3 dpf larvae exposed to 0.1% DMSO alone (C) or 10 μm isradipine for 15 min (C′) or 1 h (C″). Ribeye a and Ribeye b appear more intense than the control after 15 min exposure to isradipine. Asterisk in C′ indicates nonspecific label of nerve fiber. Scale bars: main panels, 3 μm; right panels, 1 μm. D, Floating bar plot of presynaptic Ribeye b intensities in 3 dpf larvae after exposure to 0.1% DMSO alone or 10 μm isradipine (Israd.) for 15 min, 30 min, and 1 h. The floating bars represent the minimum to maximum intensities, and the horizontal bars indicate the mean intensities. Mann–Whitney U test, ***p = 0.0005 (15′), *p = 0.0021 (30′), and ***p < 0.0001(1h). E, Box plots of the intensities of presynaptic Ribeye a and Ribeye b puncta in 3 dpf larvae exposed to DMSO or 10 μm isradipine for 1 h. Both Ribeye a and Ribeye b are significantly more intense in the isradipine-treated hair cells than the control. Mann–Whitney U test, ****p < 0.0001. Each plot represents a population of intensity measurements collected from NM1 hair cells of 12 individual larvae. F, Cumulative frequency distributions of Ribeye a and Ribeye b puncta intensities in 3 dpf isradipine-treated and control hair cells.

Figure 6.

Pharmacological block of Ca_V1.3a leads to less refined ribbon synapses in hair cells at 3 dpf. A, B, SR-SIM images of ribbon synapses in 3 dpf WT hair cells in larvae exposed to 0.1% DMSO (A) or 10 μm isradipine for 1 h (B). In isradipine-treated larvae, hair-cell synaptic ribbons appear enlarged and often misshapen. Scale bars, 1 μm. C, Average area of synaptic ribbons in control and isradipine (Israd.)-treated hair cells at 3 dpf. DMSO, 233 ± 12 nm²; isradipine, 293 ± 18 nm²; Mann–Whitney U test, *p = 0.0415. Error bars are SEM. D, Fraction of 3 dpf ribbon synapses within individual NMs with PSDs juxtaposing one, two, or three synaptic ribbons. Isradipine-treated NM hair-cell synapses contain two to three synaptic ribbons with slightly greater frequency than control (n = 4 NMs per condition, each containing ∼15–25 synapses). E, The shape factor of synaptic ribbons. Each spot represents an individual ribbon. Synaptic ribbons in hair cells are significantly less round in isradipine-treated larvae than control. Mann–Whitney U test, **p = 0.0051.

Figure 7.

Pharmacological activation of L-type calcium channels reduces synaptic ribbons in hair cells at 3 dpf. A, Mechanically evoked Ca²⁺ responses in untreated NM hair cells (black) and hair cells treated with 10 μm S-(−)Bay K8644 (green) for 15 min at 3 dpf in response to a 2 s, 10 Hz stimulus. Each trace represents the average response of hair cells from four NMs. B, Scatter plot depicts the average Ca²⁺ response per NM in WT and cav1.3a mutants at 3 dpf (white circles) and after S-(−)Bay K8644 treatment (green circles). n ≥ 4 fish and n ≥ 10 NMs per genotype. Error bars are SEM. **p < 0.01, defined by a paired t test. C–E, Representative images of Ribeye a (Rib a), Ribeye b (Rib b), and MAGUK label in NM1 of 3 dpf larvae exposed to 0.1% DMSO alone (C), 10 μm isradipine (Israd.) (D), or 10 μm S-(−)Bay K8644 (E–E′) for 1 h. Scale bars: main panels, 3 μm; right panels, 1 μm. D, Ribeye and MAGUK puncta appear more intense after 1 h exposure to isradipine in hair cells than untreated (data not shown) and DMSO-treated larvae. E, Ribeye and MAGUK puncta appear less intense in the majority (n = 9 of 15 NMs) of S-(−)Bay K8644-treated NMs. In addition, MAGUK puncta often appeared without discernible adjacent Ribeye (white arrowheads; refer to I for quantification). E′, In a subset of S-(−)Bay K8644-treated NMs (n = 6 of 15 NMs) variable Ribeye label was observed. A few hair cells within the NMs showed diffuse Ribeye label with somewhat enlarged puncta (right panels). F, G, Box plots of puncta intensities in 3 dpf NM1 hair cells treated with buffer alone (E3), 0.1% DMSO, 10 μm isradipine, or 10 μm S-(−)Bay K8644 for 1 h. Whiskers indicate the 10th and 90th percentiles (n = 7–15 larvae for each plot). F, Intensity of presynaptically localized Ribeye a. ***p < 0.0001, defined by the Dunn's multiple comparison test. G, Intensity of presynaptically localized Ribeye b. ***p < 0.0001, defined by the Dunn's multiple comparison test. H, Cumulative frequency distribution of presynaptic Ribeye b puncta intensities in 3 dpf hair cells treated with E3 (gray), DMSO (black), isradipine (red), or Bay K8644 (green). I, Fraction of PSDs (MAGUK immunolabel) with adjacent synaptic ribbons within an NM. Each circle represents NM1 in an individual larva. S-(−)Bay K8644-treated NMs have a significantly higher percentage of MAGUK puncta without adjacent Ribeye immunolabel. ***p < 0.0001, defined by the Tukey's multiple comparison test. J, Relative expression level of ribeye b transcripts in larvae exposed to DMSO alone or with drug for 1 h. There was no significant change in ribeye b expression levels in drug-treated larvae (Wilcoxon's signed-rank test). The level of gene expression in DMSO was normalized to one (n = 3 experiments).

Figure 8.

Response and synaptic changes in cav1.3a mutant hair cells to isradipine and S-(−)Bay K8644 exposure. A, Mechanically evoked calcium responses in R1250X hair cells (gray line) and after a 15 min treatment with 10 μm isradipine (Israd.; red line) at 3 dpf. Each trace represents the average response of hair cells from four NMs. B, Box plots of presynaptic Ribeye a and Ribeye b puncta intensities in 3 dpf R1250X and WT sibling (sib) larvae exposed to DMSO or 10 μm isradipine for 1 h. To achieve the greatest dynamic range of intensities, laser settings were adjusted for imaging R1250X hair cells using the brightest R1250X DMSO-treated NM (thus, relative intensities of control R1250X larvae and WT siblings look comparable). Ribeye b intensities in the isradipine-treated R1250X hair cells are comparable with the DMSO-treated mutant hair cells (Mann–Whitney U test, R1250X, p = 0.3374; WT, ****p < 0.0001). Each plot represents a population of intensity measurements collected from NM1 hair cells of seven individual larvae. C, Mechanically evoked calcium responses in R1250X hair cells (gray line) and after a 15 min treatment with 10 μm S-(−)Bay K8644 (green line) at 3 dpf. Each trace represents the average response of hair cells from four NMs. D, Box plots of presynaptic Ribeye a and Ribeye b puncta intensities in 3 dpf R1250X and WT sibling larvae exposed to DMSO or 10 μm S-(−)Bay K8644 for 1 h. Ribeye b intensities in the Bay K8644-treated R1250X hair cells were significantly reduced compared with DMSO-treated hair cells but to a lesser extent than WT siblings (Mann–Whitney U test, R1250X, ****p = 0.0010; WT sibling, ***p < 0.0001). Each plot represents a population of intensity measurements collected from NM1 hair cells of seven individual larvae. E–E′, Fraction of PSDs (MAGUK immunolabel) with adjacent synaptic ribbons within an NM. Each circle represents NM1 in an individual larva. There are no significant differences in the ratio of intact ribbon synapses within hair cells of drug-treated R1250X larvae versus control larvae (E′), but there are significantly fewer intact synapses in S-(−)Bay K8644-treated WT siblings. ***p < 0.0001, defined by the Dunn's multiple comparison test.

Figure 9.

Relatively mature hair-cell synapses in 5 dpf larvae are less susceptible to pharmacological manipulation of L-type calcium channels. A, Representative confocal images of Ribeye a (Rib a), Ribeye b (Rib b), and MAGUK immunolabel in NM2 of 5 dpf larvae exposed to 0.1% DMSO alone (control, A), 10 μm isradipine (B), or 10 μm S-(−)Bay K8644 (C) for 1 h. Scale bars: main panels, 3 μm; right panels, 1 μm. B, Ribeye labeled puncta appear comparable with control-treated larvae after 1 h exposure to isradipine. C, Ribeye labeled puncta appear somewhat less intense in S-(−)Bay K8644-treated NMs compared with controls. D, E, Scatter plots depict the average Ca²⁺ response per NM in WT and cav1.3a mutants at 5 dpf (white squares) and after Bay K8644 (red squares) or isradipine (green squares) treatment. n ≥ 4 fish and n ≥ 10 NMs per genotype. Error bars are SEM. ***p < 0.001, ****p < 0.0001, defined by a paired t test. SIB, Sibling. F, G, Box plots of puncta intensities in 5 dpf NM2 hair cells treated with buffer alone (E3), 0.1% DMSO, 10 μm isradipine, or 10 μm S-(−)Bay K8644 for 1 h. Whiskers indicate the 10th and 90th percentiles. Each plot represents a population of intensity measurements collected from NM1 hair cells of 11–13 individual larvae. *p < 0.05, ***p < 0.0001, defined by the Dunn's multiple comparison test. H, Cumulative frequency distribution of Ribeye b presynaptic puncta intensities in 5 dpf hair cells treated with E3 (gray), DMSO (black), isradipine (red), or Bay K8644 (green). I, Ratio of PSDs (MAGUK immunolabel) with adjacent presynaptic ribbons within an NM. Each circle represents NM2 in an individual larva. There are no significant difference in the ratio of intact ribbon synapses within hair cells of drug-treated larvae versus control (one-way ANOVA, p = 0.3762). J, Representative confocal images of Ribeye a, Ribeye b, and MAGUK label in NM2 after exposure to 10 μm isradipine for ≥12 h. Scale bars: main panels, 3 μm; right panels, 1 μm. K, Intensity of presynaptic Ribeye b puncta (Mann–Whitney U test, ***p < 0.0001). Note the significant increase in Ribeye b label at 5 dpf, indicating that long-term block of Cav1.3a can induce changes in ribbon size at comparatively mature stages. L, Percentage of MAGUK-label containing pixels overlapping with Ribeye b in 5 dpf control and isradipine-treated NM hair cells. Each circle represents an NM in an individual larva. Error bars are SEM.

Figure 10.

Pharmacological manipulation of L-type calcium channels modulates Ribeye immunolabel intensity in zebrafish pinealocytes. A–C, Representative confocal images of Ribeye a immunolabel in the pineal organ of 3 dpf larvae exposed to DMSO (A), 10 μm isradipine (B), or 10 μm S-(−)Bay K8644 (C) for 1 h. Scale bar, 10 μm. D, Average intensities of Ribeye a aggregates in pineal organs at 3 dpf. Each circle represents one pineal organ. Isradipine (Israd.)-treated showed significantly more intense Ribeye a immunolabel, whereas S-(−) Bay K8644-treated showed less intense label than DMSO-treated control larvae. Mann–Whitney U test, **p = 0.0023 and *p = 0.0175, respectively.