Fast Ca2+ signals at mouse inner hair cell synapse: a role for Ca2+-induced Ca2+ release

Abstract

Inner hair cells of the mammalian cochlea translate acoustic stimuli into 'phase-locked' nerve impulses with frequencies of up to at least 1 kHz. Little is known about the intracellular Ca2+ signal that links transduction to the release of neurotransmitter at the afferent synapse. Here, we use confocal microscopy to provide evidence that Ca2+-induced Ca2+ release (CICR) may contribute to the mechanism. Line scan images (2 ms repetition rate) of neonatal mouse inner hair cells filled with the fluorescent indicator FLUO-3, revealed a transient increase in intracellular Ca2+ concentration ([Ca2+]i) during brief (5-50 ms) depolarizing commands under voltage clamp. The amplitude of the [Ca2+]i transient depended upon the Ca2+ concentration in the bathing medium in the range 0-1.3 mM. [Ca2+]i transients were confined to a region near the plasma membrane at the base of the cell in the vicinity of the afferent synapses. The change in [Ca2+]i appeared uniform throughout the entire basal sub-membrane space and we were unable to observe hotspots of activity. Both the amplitude and the rate of rise of the [Ca2+]i transient was reduced by external ryanodine (20 microM), an agent that blocks Ca2+ release from the endoplasmic reticulum. Intracellular Cs+, commonly used to record at presynaptic sites, produced a similar effect. We conclude that both ryanodine and intracellular Cs+ block CICR in inner hair cells. We discuss the contribution of CICR to the measured [Ca2+]i transient, the implications for synaptic transmission at the afferent synapse and the significance of its sensitivity to intracellular Cs+.

Figure 2. Effect of Ca²⁺- free bathing solution on the transient increase in [Ca²⁺]_i

Hair cells were depolarized under voltage clamp and confocal images collected in line scan mode with the base of the cell towards the left. A, line scan traces show the [Ca²⁺]_i-dependent increase in fluorescence in response to 20 ms command to −10 mV (white bar at left-hand side). Traces have been corrected for uneven dye distribution (see Methods; 3 × 3 median filter applied). Scale bar for line scan images, 5 μm; colour scale bar, 0 = black, 5 = red. Control responses (upper and lower traces) were recorded in the presence of 1.3 mm[Ca²⁺]_o while a Ca²⁺-free bathing medium with 1 mm BAPTA was used to demonstrate (middle trace) the dependence of the response on [Ca²⁺]_o. Black horizontal bar (upper line scan image) identifies the 1 μm near-membrane band used to estimate the average time course of the [Ca²⁺]_i transient. B, average time course of the [Ca²⁺]_i transient; raw data shows the change in baseline caused by the Ca²⁺-free bathing medium. C, membrane currents recorded during the depolarizing stimulus.

Figure 3. Membrane currents from inner hair cells in whole-cell configuration

A, superimposed whole cell currents recorded using KCl-filled pipette; 50 ms depolarizing commands to different membrane potentials (shown on right-hand side). Holding potential, −84 mV. B, as A but with CsCl-filled pipette containing 20 mm TEA. C, peak inward current-voltage relationship obtained in B. D-F, representative records from inner hair cells under different recording conditions; membrane currents shown on an expanded time scale. Command step to −10 mV imposed at 1 ms; holding potential, −84 mV. Thick traces obtained with different pipette filling solutions; KCl (D), CsCl, no TEA (E), KCl, plus 20 μm external ryanodine (F). Thin traces superimposed on each record obtained with KCl-filled pipette in nominally Ca²⁺-free external solution.

Figure 1. Transient increase in [Ca²⁺]_i in semi-isolated, apical inner hair cells from neonatal mice

FLUO-3 loaded cells in whole-cell configuration were depolarized under voltage clamp and confocal images collected in line scan mode. Left, cartoons show the orientation of the cell; the dashed green line gives the location of the confocal section. Middle, line scan traces show the [Ca²⁺]_i-dependent increase in fluorescence in response to 50 ms command to −10 mV (vertical white bar). Horizontal colour coded bars (top) identify bands used to estimate average time course in different regions of the cell. Traces have been corrected for uneven dye distribution (see Methods; 3 × 3 median filter applied). Scale bar for line scan images shown in A, 5 μm; colour scale bar, 0 = black, 5 = red (A); 0 = black, 3 = red (B,C). Right, change in fluorescence, (F_t - F₀)/F₀), with time in different regions of the cell (see Methods). Black bar on the time axis shows the duration of the depolarizing command. Cells were orientated either for simultaneous fluorescence measurements at the base and towards the apex of the cell (A), or for fluorescence measurements through the base of the cell (B and C).

Figure 4. Effect of internal Cs⁺ or external ryanodine on depolarization-induced [Ca²⁺]_i transient in inner hair cells

Average [Ca²⁺]_i change in 1 μm band adjacent to the plasma membrane. Responses obtained with either K⁺- or Cs⁺-based pipette solutions (A-C) or with a K⁺-based pipette solution ± 20 μM external ryanodine (D-F). Controls with KCl-based pipette solution repeated. A and D, averages of [Ca²⁺]_i transients associated with depolarization to −10 mV (50 ms pulse; see lower trace). KCl-filled pipette, n = 7 cells; CsCl-filled pipette, n = 9 cells; KCl-filled pipette plus 20 μM external ryanodine, n = 9 cells. B and E, mean amplitude of the [Ca²⁺]_i transient plotted against membrane voltage (interpolated data); data normalized to the value obtained at −44 mV (same cells as in A and D). Cs⁺-based pipette solution (×) or K⁺-based pipette solution (•; ○) plus 20 μM extracellular ryanodine (▵). Pulse duration, 8 ms (•) and 50 ms (○, × and ▵). C and F, rise time of averaged responses shown in A and D on an expanded time scale. Data points fitted with continuous lines having one (× and ▵; τ= 14 ms) or two (○; τ= 1.3 and 14 ms) exponential components. Key for symbols as B and E.