Ca2+-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse

Abstract

We report functional and structural evidence for GluA2-lacking Ca²⁺-permeable AMPARs (CP-AMPARs) at the mature hair cell ribbon synapse. By using the methodological advantages of three species (of either sex), we demonstrate that CP-AMPARs are present at the hair cell synapse in an evolutionarily conserved manner. Via a combination of in vivo electrophysiological and Ca²⁺ imaging approaches in the larval zebrafish, we show that hair cell stimulation leads to robust Ca²⁺ influx into afferent terminals. Prolonged application of AMPA caused loss of afferent terminal responsiveness, whereas blocking CP-AMPARs protects terminals from excitotoxic swelling. Immunohistochemical analysis of AMPAR subunits in mature rat cochlea show regions within synapses lacking the GluA2 subunit. Paired recordings from adult bullfrog auditory synapses demonstrate that CP-AMPARs mediate a major component of glutamatergic transmission. Together, our results support the importance of CP-AMPARs in mediating transmission at the hair cell ribbon synapse. Further, excess Ca²⁺ entry via CP-AMPARs may underlie afferent terminal damage following excitotoxic challenge, suggesting that limiting Ca²⁺ levels in the afferent terminal may protect against cochlear synaptopathy associated with hearing loss.SIGNIFICANCE STATEMENT A single incidence of noise overexposure causes damage at the hair cell synapse that later leads to neurodegeneration and exacerbates age-related hearing loss. A first step toward understanding cochlear neurodegeneration is to identify the cause of initial excitotoxic damage to the postsynaptic neuron. Using a combination of immunohistochemical, electrophysiological, and Ca²⁺ imaging approaches in evolutionarily divergent species, we demonstrate that Ca²⁺-permeable AMPARs (CP-AMPARs) mediate glutamatergic transmission at the adult auditory hair cell synapse. Overexcitation of the terminal causes Ca²⁺ accumulation and swelling that can be prevented by blocking CP-AMPARs. We demonstrate that CP-AMPARs mediate transmission at this first-order sensory synapse and that limiting Ca²⁺ accumulation in the terminal may protect against hearing loss.

Keywords: GCaMP; cochlear synaptopathy; excitotoxicity; noise overexposure; zebrafish.

Figure 1.

Waterjet-mediated hair cell stimulation causes afferent terminal Ca²⁺ entry. Tg[elavl3:GCaMP5G] transgenic line was used to measure responsiveness of afferent terminal to waterjet-mediated displacement of hair cell stereocilia. A, B, Frames from a time-lapse video (1 s capture interval) before, during, and after waterjet stimulation are shown in the control condition (A) and following DMSO application (B). Dashed line marks the location of the terminal and thereby the region of interest used for analysis. Scale bar, 10 μm. C, Representative traces showing changes in the fluorescence intensity normalized to baseline. Horizontal line starting at time = 0 marks the duration of the waterjet stimulus. D, E, Summary data of mean peak and integrated area of the response in the control and DMSO condition (n = 6 fish, 1 terminal/fish). Paired t test.

Figure 2.

Perfusion of CTZ enhances terminal responsiveness to hair cell activation. Tg[elavl3:GCaMP5G] transgenic line was used to measure the responsiveness of the afferent terminal to waterjet-mediated displacement of hair cell stereocilia. A, B, Maximum projections of an afferent terminal in the control condition (A) and with CTZ (B). C, D, Frames from a time-lapse video (1 s capture interval) before, during, and after waterjet stimulation are shown in the untreated condition (C) and following CTZ application (D). The dashed line marks the location of the terminal and thereby the region of interest used for analysis. Scale bar, 10 μm. E, Representative traces showing changes in the fluorescence intensity normalized to baseline. F, G, CTZ increases the peak and integrated area of the waterjet response. Paired t test.

Figure 3.

AMPA perfusion diminishes afferent neuron firing in a dose-dependent manner. A, Schematic showing zebrafish head and the location of the pLLG just caudal to the ear. Loose-patch voltage-clamp recordings were obtained from cell bodies in the pLLG (green), which receive glutamatergic inputs from clusters of hair cells (red). B, Representative traces of pLLG neuron firing in ambient conditions (control) and following bath application of varying doses of AMPA. AMPA (100 and 300 μm) caused a statistically significant transient increase in firing frequency in many cells during the first 1–2 min of application (*p < 0.05) followed by a dramatic reduction in firing (#p < 0.001). C, Summary data show that the mean firing frequency decreases dramatically following 100 μm (n = 9 cells) and 300 μm (n = 6 cells) AMPA application but not 30 μm AMPA (n = 8 cells). Two-way ANOVA.

Figure 4.

Perfusion of 100 μm AMPA diminishes terminal responsiveness to hair cell activation. The Tg[elavl3:GCaMP5G] transgenic line was used to measure the responsiveness of afferent terminal to waterjet-mediated displacement of hair cell stereocilia. A–C, Frames from a time-lapse video before, during, and after waterjet stimulation are shown in the control condition (A), following 100 μm AMPA application (B), and 2 h after wash (C). The dashed line marks the location of the terminal and thereby the region of interest used for analysis. Scale bar, 10 μm. D, Response to waterjet is diminished following AMPA application and wash. E, F, Summary data of the mean peak and integrated area of the response in the control, AMPA, and wash conditions. One-way ANOVA, *p < 0.05.

Figure 5.

AMPA does not affect hair cell mechanotransduction. A, Frames from a time-lapse calcium-imaging video acquired from Tg[myo6b:GCaMP3] fish. GCaMP3 fluorescence is shown in the control condition (top) before, during, and after waterjet stimulation (left, middle, and right, respectively). B, Images represent the same sequences during AMPA exposure. C, GCaMP3 responses to waterjet in the control and AMPA condition. Arrowheads mark the time points shown in A and B. D, E, Summary data of the mean peak and integrated area of the waterjet responses. Each point is an average of two to three trials. Control and AMPA data are paired. AMPA does not significantly affect the waterjet response. Scale bar, 10 μm.

Figure 6.

AMPA causes morphological changes to afferent terminals that is prevented by blocking CP-AMPARs. A, Maximum projections of a Tg[neuroD:GFP] afferent terminal in the control condition following bath perfusion of 100 μm AMPA and 90 min of wash. Fish were immobilized with alpha-bungarotoxin (a-BGT). D, Maximum projections in control and following AMPA treatment. A, D, Fish were immobilized with tricaine. In the AMPA images (A, middle; D, right), arrowheads indicate some of the terminal swellings. B, C, E, F, Regardless of the immobilization method, AMPA caused a decrease in the number of branches (B, E), while the mean branch length (C, F) is unchanged. Scale bars, 10 μm. G, Maximum projections of a terminal in the control condition, following IEM application and IEM + AMPA (100 μm). IEM prevented AMPA-mediated changes to terminal morphology. I, However, IEM alone reduced the mean branch length. *p < 0.05, **p < 0.001, ***p < 0.01, #p < 0.0001.

Figure 7.

AMPA does not affect efferent terminal morphology. A, Maximum projection images of efferent terminals from Tg[Islet1:GFP] fish (6 dpf) before and during 100 μm AMPA exposure. Scale bar, 10 μm. B, C, AMPA exposure did not change the number of branches (B) or the mean branch length (C). Each data point represents a measurement taken from a single terminal. Error bars indicate the SD in B and SEM in C.

Figure 8.

Postsynaptic markers following AMPA exposure. A, Representative maximum intensity top-down (x–y) projection of GluA4 (red), afferent PSD (green) and synaptic ribbon (Ribeye b, blue) immunolabeled in a zebrafish neuromast at 5 dpf. Insets correspond to the boxed region of interest in A and highlight overlapping GluA4 and PSD immunolabels. Scale bars: A, 3 μm; insets, 1 μm. B, Representative thumbnail images of ribbon synapses in hair cells exposed to DMSO alone or 100 μm AMPA for 15 min then fixed immediately or allowed to recover for 1 h. Scale bar, 1 μm. C–E, Box plots of integrated presynaptic Ribeye b (C), MAGUK PSD (D), and GluA4 subunit (E) immunolabel intensities. Each plot represents a population of intensity measurements of an individual labeled punctum collected from six to eight individual neuromasts (four to five individual larvae) per condition. Whiskers indicate the minimum and maximum values. Intensities of Ribeye b, GluA4, and PSD immunolabels are not significantly different between conditions (defined by the Dunn's multiple comparison test). F, An aligned dot plot showing the number of intact synapses per individual neuromast hair cell. Bars represent the means; error bars indicate the SEM. An intact synapse was defined as Ribeye-immunolabeled puncta partially overlapping PSD puncta. Number of intact synapses per hair cell are not significantly different between conditions (defined by Dunnett's multiple-comparison test). Hair cell n = 50 (DMSO), n = 61 (AMPA, no recovery), and n = 43 (AMPA, 1 h recovery).

Figure 9.

A–O, High levels of AMPA cause an accumulation of terminal Ca²⁺ that is primarily mediated by Ca²⁺-permeable AMPARs. Tg[elavl3:GCaMP5G] transgenics (A–K) and fish transiently expressing GCaMP3 in pLLG neurons [hsp70l:GCaMP3–2.0cntnap2a] (L–O) were used to measure intracellular Ca²⁺ levels in afferent terminals. A–H, Maximum projections of afferent terminals in the untreated condition (control) and following AMPA application alone or application of a receptor antagonist followed by 100 μm AMPA. I, AMPA has a dose-dependent effect on the accumulation of terminal Ca²⁺. For all data points, the fluorescence intensity of a single terminal was measured in the untreated control condition and following drug application. The 100 and 300 μm concentrations of AMPA significantly increase the fluorescence intensity of terminals. This increase is blocked by DNQX and IEM, but not by the NMDAR blocker APV. J, K, Summary data showing the percentage change in terminal fluorescence intensity relative to the untreated control condition. K, IEM suppresses the accumulation of terminal Ca²⁺ at all doses tested. L, M, Maximum projections of afferent terminals in the untreated condition (control) and following DMSO, AMPA application alone or application of a IEM followed by 100 μm AMPA. O, Summary data showing that AMPA causes significant increases in fluorescence intensity that are blocked by IEM. Scale bars, 10 μm. One-way ANOVA: †p < 0.05; *p < 0.01; **p < 0.001; #p < 0.0001. N values are indicated in all plots above the data points.

Figure 10.

Postsynaptic densities of rat SGNs express the AMPAR subunits GluA2, GluA3, and GluA4. A, Midcochlear region of the organ of Corti of a p25 rat immunolabeled with antibodies to GluA2 (green), GluA3 (red), and GluA4 (blue), imaged with confocal microscopy in the excised whole-mount preparation, shown in merged color and in grayscale. All synapses expressed each of the three AMPAR subunits. B, Immunofluorescence intensity per synapse for each AMPAR subunit scaled approximately linearly with overall volume of GluA_Sum. Intensities and volumes ranged in value over approximately one order of magnitude among synapses (n = 147 from A). Acquisition parameters were adjusted to normalize signals to the maximum pixel intensity in the image on each channel, resulting in synapses with similar intensities on the three channels. C, Frequency distributions of intensity per synapse were positively skewed with similar coefficients of variation: GluA2, 0.60; GluA3, 0.60; GluA4, 0.56; GluA_Sum, 0.58.

Figure 11.

AMPAR subunits appear to occupy partially nonoverlapping domains, suggesting the existence of GluA2-lacking receptor channels in some postsynaptic densities in a mammalian cochlea. A, Confocal immunofluorescence for the three AMPAR subunits, on p25 rat postsynaptic densities oriented en face, shown separately in grayscale and merged in color. GluA2 (green), GluA3 (red), and GluA4 (blue) appear to occupy similar regions in examples 1 and 2. In examples 3 and 4, the subunits appear to reside in partially nonoverlapping domains. B, Line profiles of fluorescence intensity for the four synapses, as shown on the merged color images in A. The mean pixel intensity (y-axis, in arbitrary units) over a 120-nm-wide band perpendicular to the line was plotted as a function of distance along the line (x-axis, in micrometers). On the left, line profiles are shown in absolute scale to illustrate differences between synapses in the same acquired image volume. On the right, line profiles have been normalized to the maximum amplitude of the first peaks in each plot to highlight spatial differences in GluA composition within synapses. Line profiles across postsynaptic densities 3 and 4 suggest the existence of GluA2-lacking regions (arrowheads). C, Images of postsynaptic densities oriented en face with subunit immunoreactivities colored as in A. The subunits appear to occupy partially nonoverlapping domains in some synapses but not in others. D, Frequency distributions of intensity ratios (GluA_subunit/GluA_Sum) compare a representative population of synapses from one image volume (shown in Fig. 1A), where synapses all having identical relative subunit compositions would exhibit ratios of 0.33 for each GluA subunit. Instead, fractional fluorescence ranged from ∼0.25 to 0.4 for GluA2 (left), GluA3 (center), and GluA4 (right). Scale bars: B, 1 μm; C, 2 μm.

Figure 12.

CP-AMPARs mediate a substantial component of the synaptic transmission at auditory hair cell synapses in adult bullfrogs. A, Representative trace of paired whole-cell voltage-clamp recordings of an amphibian papilla hair cell and a postsynaptic afferent fiber, with simultaneous C_m measurements from the hair cell, which was depolarized from −90 to −30 mV for 20 ms. This depolarization elicits an I_Ca in the hair cell that triggers a C_m change produced by the exocytosis of synaptic vesicles. The traces are recorded before (control, black) and after applying 60 μm IEM (at 7 min 20 s after IEM application, red). The inset shows that the initial phasic EPSC was dramatically reduced by IEM. B, The normalized charges of EPSCs are shown as a function of time since the IEM application. The average of normalized EPSC charges are shown in black at each time point, and individual data points are shown in gray (n = 8 pairs). The red dashed line indicates a value of 1. In the continuous presence of IEM, the EPSC charge is progressively blocked by successive 20 ms depolarizing pulses to the hair cell. C, The application of 60 μm IEM reduced the charge of the EPSCs. After 5 min of IEM application, the average charge of the EPSCs was decreased by 31.5 ± 4.2% (n = 16 pairs) compared with control. After 8 min of IEM application, the average charge of the EPSCs was further decreased by 39.3 ± 6.0% (n = 8 pairs).