Acid-sensing ion channel 2 contributes a major component to acid-evoked excitatory responses in spiral ganglion neurons and plays a role in noise susceptibility of mice

Abstract

Ion channels in the degenerin-epithelial sodium channel (DEG-ENaC) family perform diverse functions, including mechanosensation. Here we explored the role of the vertebrate DEG-ENaC protein, acid-sensing ion channel 2 (ASIC2), in auditory transduction. Contributions of ASIC2 to hearing were examined by comparing hearing threshold and noise sensitivity of wild-type and ASIC2 null mice. ASIC2 null mice showed no significant hearing loss, indicating that the ASIC2 was not directly involved in the mechanotransduction of the mammalian cochlea. However, we found that (1) ASIC2 was present in the spiral ganglion (SG) neurons in the adult cochlea and that externally applied protons induced amiloride-sensitive sodium currents and action potentials in SG neurons in vitro, (2) proton-induced responses were greatly reduced in SG neurons obtained from ASIC2 null mice, indicating that activations of ASIC2 contributed a major portion of the proton-induced excitatory response in SG neurons, and (3) ASIC2 null mice were considerably more resistant to noise-induced temporary, but not permanent, threshold shifts. Together, these data suggest that ASIC2 contributes to suprathreshold functions of the cochlea. The presence of ASIC2 in SG neurons could provide sensors to directly convert local acidosis to excitatory responses, therefore offering a cellular mechanism linking hearing losses caused by many enigmatic causes (e.g., ischemia or inflammation of the inner ear) to excitotoxicity.

Figure 8.

Examples of click-evoked ABR waveforms recorded using a series of sound intensities ranging from 80 dB (top data traces) to 25 dB (bottom data traces) changing in a 5 dB step. Measurements were done before (A) and after noise exposures for wild-type (B) and ASIC2 null (C) mice. Hearing thresholds were determined by following amplitude and latency changes of the wave III or IV in the ABRs until a minimal sound level was determined, at which point just a detectable ABR was observed.

Figure 1.

Proton-elicited responses in cultured SG neurons. A, Inward currents elicited by increasing concentrations of protons. The pH values and approximate duration of the application of external solutions to the cell are indicated by the numbers and horizontal bars above the data trace, respectively. Normal external solution was perfused between the proton applications. The currents were recorded from an SG neuron voltage clamped at -70 mV. B, Dose-response curve of the normalized peak current. The data were fitted with a smooth curve using the following equation: %response = 100 × [pH]ⁿ/([pH]_50ⁿ + [pH]ⁿ), in which [pH]₅₀ is the pH value at which half-maximal currents was obtained, and n is the coefficient factor. Black dots and error bars give the average data and SEMs, respectively. C, Action potentials elicited by increasing concentration of protons in an SG neuron. The approximate duration of the application of the pH 6.8 solution is indicated by a horizontal bar underneath the data trace. The neuron was current clamped at its resting membrane potential of -65mV; therefore, no DC was injected.

Figure 3.

Comparison of proton-elicited responses obtained from wild-type and ASIC2 knock-out mice. Inward currents elicited by increasing concentrations of protons in the wild-type (A) and ASIC2 KO (B) mice. pH values and the approximate duration of the external solution applied are indicated by numbers and black bars above the data traces, respectively. Time and amplitude scales are the same for both A and B. C, Ionic fluxes elicited by protons obtained in ASIC2 knock-out mice normalized to that of the wild-type mice. Total ionic fluxes were obtained by integrating inward currents induced by protons. The x-axis gives the pH value of external solutions applied to the SG neurons. Error bars indicate SEM. D, α-DTX (top data trace) and proton-elicited (bottom data trace) voltage responses obtained from the same SG neurons under current clamp. The average resting membrane potential of the SG neuron was approximately -63 mV. The approximate duration of the applications of α-DTX and proton are indicated by horizontal bars under the data traces.

Figure 2.

Ionic specificity and pharmacological block of the proton-elicited responses in SG neurons. A, Comparison of inward currents recorded when Na⁺ ions were substituted by choline⁺ in the external (Ext.) solution. pH values and approximate duration of perfusions are given by the numbers and horizontal bars above the data traces. B, Inhibitory dose-response of amiloride. Black dots and error bars give the average data and SEMs, respectively. The smooth curve was obtained by fitting the data with the following equation: %response = (1 - [amiloride]ⁿ/(IC_50ⁿ + [amiloride]ⁿ)) × 100, in which IC₅₀ is the half-maximal inhibition concentration of amiloride, and n is the coefficient factor. Inset, One example of the pH 4.5-elicited currents blocked by 50 μm amiloride.

Figure 4.

ASIC2 immunolabeling results of a prenatal cochlear sections (E18.5). The negative control (E18.5 ctrl) was obtained by preincubating the primary antibody with antigenic peptide. The enlarged view given in the top right panel shows detailed labeling patterns in areas of the cochlea outlined by a frame. Arrow 1 points to the location of SG neurons, and the arrowhead indicates developing macula of utricle. Scale bar, ∼800 μm. SV, Scala vestibule; SM, scala media; ST, scala tympani.

Figure 5.

Immunoreactivities of postnatal cochlear sections to an antibody against ASIC2. Immunolabeling of cochlear sections obtained at P1, P4, P8, P10, P12, P14, and adult. P1 control (ctrl) shows negative control results obtained by preincubating first antibody with antigenic peptide. ASIC2 null shows labeling results using cochlear sections obtained from adult ASIC2 null mouse (bottom right). The organ of Corti, outlined by a box in P14, is enlarged and shown in Figure 6 for details. Arrows 1 point to the location of spiral ganglion neurons, and arrows 2 point to the location of vestibular (Scarpa's) ganglion neurons. SV, Scala vestibule; SM, scala media; ST, scala tympani.

Figure 6.

ASIC2 immunoreactivities in the peripheral processes near the soma of SG neurons (A) and in the suspected nerve terminals under and near the bottom of the inner hair cell (B). Arrows point to examples of punctuated ASIC2 labeling along the peripheral processes of SG neurons. Scale bars, ∼20 μm. IHC, Inner hair cell; IPC, inner pillar cell.

Figure 7.

Quantification of ASIC2 labeling intensity in cells lining the endolymphatic space and in SG neurons. A, Relative labeling intensity of ASIC2 (y-axis) at various developmental stages (x-axis) was obtained by comparing labeled regions with background regions in the same cochlear sections. B, The specificity of the labels by the ASIC2 antibody is shown by a large reduction in the relative labeling intensity caused by either a peptide block (unfilled bars) or the absence of labeling in cochlear sections obtained from ASIC2 null mice (last unfilled bar on the right). Error bars indicate SEM.

Figure 9.

Comparison of hearing thresholds and noise sensitivities of wild-type and ASIC2^-/- mice. A, Hearing thresholds of wild-type (ASIC^+/+) and ASIC2^-/- mice measured by either click (first pair of columns) or tone bursts of various frequencies (indicated by the numbers under the bars). B, Hearing threshold shifts after exposures to 110 dB white-band noise in wild-type and ASIC2^-/- mice measured by click (first pair of columns) or tone bursts of various frequencies (indicated by the numbers under the bars). C, Hearing threshold shifts after exposures to 125 dB white-band noise in wild-type and ASIC2^-/- mice, measured by click (first pair of columns) or tone bursts of various frequencies (indicated by the numbers under the bars).