Spiking Pattern of the Mouse Developing Inner Hair Cells Is Mostly Invariant Along the Tonotopic Axis

Abstract

During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This activity at the pre-hearing stage allows the IHCs to autonomously excite the auditory nerve fibers and hence, represents an efficient mechanism to shape the tonotopic organization along the ascending auditory pathway. Using calcium imaging, we show that the activity in the developing cochlea consists of calcium waves that propagate across the supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, consistent with bursts of action potentials recorded in patch-clamp. In addition, adjacent auditory hair cells tend to have a synchronized [Ca²⁺]_i activity, irrespective of their location along the base-to-apex gradient of the cochlea. Finally, we show that the mechanical ablation of the inner phalangeal cells (IPCs), a class of supporting cells, reduces the synchronized [Ca²⁺]_i activity between neighboring sensory cells. These findings support the hypothesis that the tonotopic map refinement in higher auditory centers would depend on the synchronization of a discrete number of auditory sensory cells.

Keywords: action potential; calcium transients; cochlea; sensory cells; spontaneous activity.

Figure 1

Calcium transients in inner hair cells (IHCs) correspond to trains of action potentials. (A) Intracellular calcium dynamic, given by the fura-2 fluorescence ratio (f350/f380), in the apical turn of a developing cochlea (P6). Each frame shows the spontaneous increase in [Ca²⁺]_i within the inner supporting cells (ISCs) of the Kölliker’s organ as well as in the sensory IHCs. The patch-clamped IHC is outlined in dashed white line and the patch-pipette in white lines. The increase in [Ca²⁺]_i within several supporting cells and in the recorded IHC is conspicuous at 160 s. Scale bar: 10 μm. (B) Action potential firing (red, top), spike rate (blue, middle) and fura-2 ratiometric fluorescence measurement (green, bottom) of the corresponding IHC recorded in (A). Each train of action potential is temporally associated with an increase in [Ca²⁺]_i. Yellow background indicates the corresponding time frame shown in (A). (C) Fura-2 fluorescence ratio changes (ΔR) plot against the spike rate. Data were collected from four different recordings (three apical turns between P5 to P8 and one P7 basal turn). Each dot represents the ΔR and spiking rate average over 1 s. Linear regression fit is shown in black. Pearson’s coefficient, R = 0.8.

Figure 2

Calcium transients in IHCs over the first postnatal week. Representative examples of fura-2 f350/f380 ratio in apical (A,B) and basal (C,D) IHCs. (A,C) Time lapse recordings showing spontaneous [Ca²⁺]_i rise in several IHCs from apical (A) and basal (C) cochlear turn. For (A) and (C), scale bar: 10 μm. (B,D) Calcium transients in six adjacent IHCs from (A) and (C) over 25 min of recording. Yellow background indicates the corresponding time frame shown in (A) and (C). Regions of interest (ROIs) are indicated by colored circles onto hair cells. (E–G) Frequency (E), duration (F) and coefficient of variation (CV, G) of the calcium transients in basal and apical IHCs during the first postnatal week. **p < 0.01 and *p < 0.05. The number of cochleas is indicated in white.

Figure 3

Similar pattern of activity in IHCs along the tonotopic axis. (A–C) Frequency (A), duration (B) and coefficient of variation (CV, C) of the calcium transients in apical IHCs plot, respectively, against the frequency, duration and CV in the basal IHCs for each corresponding age (P1–P3, P4–P5, P6–P7). **p < 0.01 and *p < 0.05. Number of cochleas examined: P1–P3 base: 5, P1–P3 apex: 4, P4–P5 base: 8, P4–P5 apex: 4, P6–P7 base: 6, P6–P7 apex: 7.

Figure 4

Synchronous [Ca²⁺]_i activity between neighboring sensory cells and non-sensory cells in the developing cochlea. The synchronous index is plot against the location of the cells. Colored symbols indicate the synchronized activity between neighboring IHCs in the apical (A–C) and basal cochlear turn (D–F). Black and gray symbols indicate synchronous activity between sensory cells and ISCs within the Kölliker’s organ. Lines with white circles show synchronized activity binned over 10 μm distance and dots show individual data set. Stars indicate the significant difference in comparison to SI at 10 μm distance. ***p < 0.001, **p < 0.01 and *p < 0.05. Number of cochleas examined: P1–P3 base: 5, P1–P3 apex: 4, P4–P5 base: 8, P4–P5 apex: 4, P6–P7 base: 6, P6–P7 apex: 7.

Figure 5

Calcium waves activity in the supporting cells within the Kölliker’s organ. Maximal area (A,C) and expansion rate (B,D) from apical (A,B) and basal (C,D) turns over the first postnatal week. **p < 0.01 and *p < 0.05. The number of cochleas is indicated in white.

Figure 6

Synchronous activity between neighboring sensory cells and non-sensory cells along the tonotopic axis. (A) Synchronous index between apical IHCs as a function of the synchronous index between basal IHCs for each corresponding stage of development. (B) Synchronous index between IHCs and ISCs from the apical turn as a function of the synchronous index between IHCs and ISCs from the basal turn for each corresponding age. Black dashed line indicate the y = x regression. *p < 0.05. Number of cochleas examined: P1–P3 base: 5, P1–P3 apex: 4, P4–P5 base: 8, P4–P5 apex: 4, P6–P7 base: 6, P6–P7 apex: 7.

Figure 7

Loss of IPCs reduces the synchronized activity between neighboring cells. (A–C) Frequency (A), duration (B) and CV (C) of the calcium transients in IHCs with IPCs (blue) and after the mechanical removal of IPCs (yellow). (D–F) Frequency (D), maximal area (E) and velocity (F) of the calcium waves within the supporting cells of the Kölliker’s organ. (G–H) Synchronous activity between neighboring sensory IHCs (G) and between IHCs and ISCs (H). **p < 0.01, *p < 0.01. The number of the cochleas (P6–P7, apical turn) is indicated in white.