Compartmentalization of antagonistic Ca2+ signals in developing cochlear hair cells

Abstract

During a critical developmental period, cochlear inner hair cells (IHCs) exhibit sensory-independent activity, featuring action potentials in which Ca²⁺ ions play a fundamental role in driving both spiking and glutamate release onto synapses with afferent auditory neurons. This spontaneous activity is controlled by a cholinergic input to the IHC, activating a specialized nicotinic receptor with high Ca²⁺ permeability, and coupled to the activation of hyperpolarizing SK channels. The mechanisms underlying distinct excitatory and inhibitory Ca²⁺ roles within a small, compact IHC are unknown. Making use of Ca²⁺ imaging, afferent auditory bouton recordings, and electron microscopy, the present work shows that unusually high intracellular Ca²⁺ buffering and "subsynaptic" cisterns provide efficient compartmentalization and tight control of cholinergic Ca²⁺ signals. Thus, synaptic efferent Ca²⁺ spillover and cross-talk are prevented, and the cholinergic input preserves its inhibitory signature to ensure normal development of the auditory system.

Keywords: calcium; cochlea; hair cells; synaptic transmission.

Fig. 1.

Ca²⁺ signals in IHCs following efferent fiber electrical stimulation. (A) Schematic representation of an IHC in the postnatal developmental period previous to the onset of hearing. Both efferent cholinergic and afferent glutamatergic contacts are present at the basal pole of the IHC. (B) Sequence of wide-field microscopy images of an IHC filled with Fluo-4 and illuminated with 488-nm LED light during efferent electrical stimulation. “Stim” indicates when the electric stimulus was applied. The IHC base contour is outlined with a dashed line. The ROI is defined by the red shape depicted in the first image. (C) Representative whole-cell current traces during efferent electrical stimulation (V_h = −120 mV). Black traces represent those trials where an eIPSC was detected after the stimulus artifact (failures are shown in gray). (D) Fluorescence signals measured at the ROI indicated in B during the same trials shown in C. Red traces correspond to trials in C where an IPSC was detected, and failures are shown in pink. (E, i–iii) Representative responses activating fluorescence changes in three different portions of the IHC cytoplasm. Each panel represents a different single synaptic event. (Top) Black traces show the electrophysiological signal. (Bottom) Traces show the spatially diverse Ca²⁺ fluorescence signals measured in the radial ROIs, indicated by colors in the scheme. Images represent the peak of the Ca²⁺ signal measured for the corresponding synaptic event.

Fig. 2.

Three-dimensional reconstruction of functional IHC Ca²⁺ domains using swept-field confocal microscopy. (A, Left) Scheme depicting the stimulation protocol used for imaging efferent Ca²⁺ entry sites (50 ms, 80-Hz electrical stimulation of efferent axons). (A, Right) Single Z-plane of an IHC illuminated at 488 nm and loaded with Fluo-5F and 10 mM EGTA. Cell fluorescence at rest is represented in blue to indicate location. Green hotspots represent Ca²⁺ domains at the peak of efferent stimulation. (Scale bar: 5 μm.) (B, Left) Scheme depicting the depolarization protocol to detect Ca²⁺ entry hotspots through VGCCs. (B, Right) Similar to A, but Ca²⁺ hotspots due to VGCC activation are represented in red. (Scale bar: 5 μm.) (C) Montage of peak Ca²⁺ signals in different Z-planes obtained during the protocols described in A and B. (D) Three-dimensional reconstruction performed with the Z-stack of images in C. (E) Representation of the same IHC illustrated in C. Red and green circles pinpoint the center of the afferent and efferent hotspots, respectively. (F) Frequency histogram of distances from each efferent hotspot in a given cell to its closest afferent hotspot.

Fig. 3.

Synaptic contacts on a P9 rat IHC. (A) Example of neighboring afferent and efferent synaptic contacts. The afferent bouton is shaded in brown, the ribbon is colored red, the efferent bouton shaded in purple, and the synaptic cistern is colored green. (Scale bar: 500 nm.) (B–D) Reconstruction of the IHC from a P9 rat cochlea. This cell had 19 afferent and 37 efferent contacts, associated with 25 ribbons and 37 cisterns. (B) Hair cell (gray) with afferents (brown), efferents (purple), ribbons (red), and cisterns (green). (Scale bar: 5 μm.) (C) Hair cell with efferent and cisterns only. (Scale bar: 2.5 mm.) (D) Hair cell with cisterns and ribbons only. (Scale bar: 2.5 μm.)

Fig. 4.

Ca²⁺ influx produced by trains of efferent stimuli. Cells loaded with Fluo-4 (400 μM) and EGTA (500 μM) were imaged on a wide-field microscope using 488-nm LED illumination during efferent electrical stimulation protocols. (A) Representative synaptic responses (red traces) and changes in fluorescence [black traces, expressed as ∆F/F₀ (%)] produced in an IHC during a 3-s electrical stimulation of efferent fibers at 5 Hz (i), 20 Hz (ii), and 80 Hz (iii). (B) Three-dimensional representation of the fluorescence signal [∆F/F₀ (%)] as a function of ROI number and time during electrical stimulation of efferent axons at 5 Hz (i), 20 Hz (ii), and 80 Hz (iii). (C) Maximal ∆F/F₀ (%) reached during 5-, 20-, and 80-Hz stimulation trains. Friedman’s test, *P < 0.01. (D) Correlation between the maximal fluorescence signal and the charge (Q) accumulated at the end of the stimulation protocol (integral of synaptic currents) Spearman correlation, P < 0.0001. (E) Fluorescence CV across ROIs at the time point when the Ca²⁺ peak was reached. Friedman’s test, *P < 0.01.

Fig. 5.

Local ACh application produces an increase in the afferent EPSC rate. (A) Representative whole-cell recording of an afferent bouton with spontaneous EPSCs (5.8 mM K⁺). (Inset) Detail of superimposed EPSC traces. (B) Bouton recording upon application of 25 mM K⁺ in the extracellular solution. (Inset) Voltage-clamp recording from the IHC that lays presynaptic to the bouton. Representative bouton recordings during local application of 300 μM ACh (C) and 300 μM ACh + 1 μM strychnine (D) are shown. (Insets) IHC cholinergic currents upon application of 300 μM ACh in the absence (C) or presence (D) of 1 μM strychnine (note: IHC recordings were obtained from the cells presynaptic to the bouton shown in each panel). (Scale bars: Insets, 100 pA, 10 s.) (E) Average (Avg.) EPSC activation rate recorded in boutons during drug applications (calculated up to the end of the perfusion). Strych, strychnine. Kruskal–Wallis test, *P < 0.05. (F) Amplitude of ACh responses in IHCs. (G) IHC charge (Q) accumulated after 15 s of ACh application. Kruskal–Wallis test, *P < 0.05.

Fig. 6.

Efferent electrical stimulation does not produce a rise in afferent synaptic activity. (A) Representative intracellular recordings of an afferent bouton in the presence of 10 μM isradipine. (B and C) Same as in A but during electrical stimulation of efferent fibers at 5 Hz (B) and 80 Hz (C). (Inset) Voltage-clamp recordings from the IHC presynaptic to the bouton. Stimulation of efferent fibers produced cholinergic synaptic currents. (D) Average (Avg.) EPSC rate before, during, and after efferent train stimulations at 5, 20, and 80 Hz. Each symbol represents a single bouton-IHC pair experiment. Friedman’s test, P > 0.05. (E) Maximal synaptic current amplitude measured in the IHC during train stimulation. Friedman’s test: *P < 0.05; **P < 0.01. (F) Charge (Q) accumulated at the end of the 3-s efferent train protocol at each stimulation frequency. Friedman’s test: *P < 0.05; **P < 0.01.

Fig. 7.

Efferent train stimulation inhibits IHC AP firing. (A) Representative current-clamp recordings from an IHC during a short burst of APs. A 10-pA steady current was applied to trigger firing. Efferent fibers were electrically stimulated at different frequencies during the time indicated by dashed lines. (Scale bars: 20 mV, 1 s.) (B) Average rate of AP firing during efferent stimulation (AP Freq_stim). Values were normalized to the rate obtained during the prestimulation period (AP Freq_pre). Different uppercase letters (A–C) represent statistical differences between frequencies, Friedman’s test, P < 0.05. (C) Representative voltage-clamp recordings from the same IHC in A during efferent stimulation (Eff. Stim.) at different frequencies. The timing of efferent stimulation is indicated by dashed lines. (Scale bars: 100 pA, 1 s.) (D) Charge (Q) at the end of the 3-s efferent train protocol at each stimulation frequency. Different letters (A–E) represent statistical differences between frequencies. Friedman’s test, P < 0.05.

Fig. 8.

Cisternal Ca²⁺-ATPases and high intracellular Ca²⁺ buffering shape electrically evoked IPSCs. (A) IHCs were loaded with the fluorescent Ca²⁺ indicator Fluo-4 (400 μM) and EGTA (500 μM), and 10-stimuli trains at 5, 20, and 80 Hz were applied to efferent fibers. Ca²⁺ transients were imaged during stimulation before (black traces) and after incubation with the cisternal Ca²⁺-ATPase inhibitor CPA (5 μM) (red traces). (Scale bars: 1 s, 0.2 normalized ∆F.) (B) Decay kinetics of Ca²⁺ transients in A, computed as the τ_decay between 20% and 80% of the response peak. Kruskal–Wallis test, P > 0.05. Ctrl, control. (C) Representative traces of eIPSCs recorded at V_h = −40 mV in IHCs in whole-cell mode, using different Ca²⁺ buffering conditions, and in the perforated-patch (PP) configuration. Black traces represent the average of multiple repetitions of stimulation in a given cell. (Scale bars: 50 ms, 20 pA.) The accumulated charge (Q) (D), amplitude (E), and HW (F) in each buffering condition are shown. (D) Q: 2.2 ± 0.1 pC in 0.5 mM EGTA (n = 7), 2.1 ± 0.2 pC in 5 mM EGTA (n = 8), 1.9 ± 0.2 pC in 0.2 mM BAPTA (n = 7), 1.2 ± 0.1 pC in 2 mM BAPTA (n = 7), and 1.3 ± 0.1 pC (n = 5) in PP. (E) Amplitude: 21.1 ± 2.6 pA (n = 7) in 0.5 mM EGTA, 22.4 ± 1.3 pA (n = 8) in 5 mM EGTA, 18.3 ± 1.8 pA (n = 7) in 0.2 mM BAPTA, 13.0 ± 1.7 pA (n = 7) in 2 mM BAPTA, and 16.6 ± 2.2 pA (n = 5) in PP. (F) HW: 47 ± 3 ms (n = 7) in 0.5 mM EGTA, 44 ± 4 ms (n = 8) in 5 mM EGTA, 42 ± 2 ms (n = 7) in 0.2 mM BAPTA, 28 ± 2 ms (n = 7) in 2 mM BAPTA, and 31 ± 3 ms (n = 5) in PP. Different letters indicate statistically significant differences between groups. Kruskal–Wallis test, P < 0.05. (G) m determined by the failures method: 0.83 ± 0.11 in 0.5 mM EGTA (n = 7), 0.98 ± 0. 08 in 5 mM EGTA (n = 8 cells), 0.74 ± 0.04 in 0.2 mM BAPTA (n = 7), 0.69 ± 0.10 in 2 mM BAPTA (n = 7), and 0.79 ± 0.09 in PP conditions (n = 5). No significant differences were found.