Fast Ca²⁺ Transients of Inner Hair Cells Arise Coupled and Uncoupled to Ca²⁺ Waves of Inner Supporting Cells in the Developing Mouse Cochlea

Abstract

Before the onset of hearing, which occurs around postnatal day 12 (P12) in mice, inner hair cells (IHCs) of the immature cochlea generate sound-independent Ca²⁺ action potentials (APs), which stimulate the auditory pathway and guide maturation of neuronal circuits. During these early postnatal days, intercellular propagating Ca²⁺ waves elicited by ATP-induced ATP release are found in inner supporting cells (ISCs). It is debated whether IHCs are able to fire Ca²⁺ APs independently or require a trigger by an ISC Ca²⁺ wave. To identify the Ca²⁺ transients of IHCs underlying Ca²⁺ APs and to analyze their dependence on ISC Ca²⁺ waves, we performed fast Ca²⁺ imaging of Fluo-8 AM-loaded organs of Corti at P4/P5. Fast Ca²⁺ transients (fCaTs) generated by IHCs were simultaneously imaged with Ca²⁺ waves in ISCs. ISC Ca²⁺ waves frequently evoked bursts consisting of >5 fCaTs in multiple adjacent IHCs. Although Ca²⁺ elevations of small amplitude appeared to be triggered by ISC Ca²⁺ waves in IHCs of Ca_v1.3 knockout mice we never observed fCaTs, indicating their requirement for Ca²⁺ influx through Ca_v1.3 channels. The Ca²⁺ wave-triggered Ca²⁺ upstroke in wildtype IHCs occurred 0.52 ± 0.27 s later than the rise of the Ca²⁺ signal in the adjacent ISCs. In comparison, superfusion of 1 μM ATP elicited bursts of fCaTs in IHCs starting 0.99 ± 0.34 s prior to Ca²⁺ elevations in adjacent ISCs. PPADS irreversibly abolished Ca²⁺ waves in ISCs and reversibly reduced fCaTs in IHCs indicating differential involvement of P2 receptors. IHC and ISC Ca²⁺ signals were however unaltered in P2X2R/P2X3R double knockout or in P2X7R knockout mice. Together, our data revealed a fairly similar occurrence of fCaTs within a burst (56.5%) compared with 43.5% as isolated single fCaTs or in groups of 2–5 fCaTs (minibursts). We provide evidence that IHCs autonomously generate single fCaTs and minibursts whereas bursts synchronized between neighboring IHCs were mostly triggered by ISC Ca²⁺ waves. Neonatal IHCs thus spontaneously generate electrical and Ca²⁺ activity, which is enhanced and largely synchronized by activity of ISCs of Kölliker’s organ indicating two sources of spontaneous activity in the developing auditory system.

Introduction

Inner Hair Cells (IHCs) generate Ca²⁺ action potentials (APs) in the absence of sound before the onset of hearing, which in mice occurs around postnatal day 12 (P12). These APs translate into release of glutamate (Kros et al., 1998; Brandt et al., 2003, 2007; Marcotti et al., 2003b; Johnson et al., 2007, 2011, 2013; Sendin et al., 2014), initiate excitatory postsynaptic potentials and APs in spiral ganglion neurons (Glowatzki and Fuchs, 2002) and activate the afferent auditory pathway (Tritsch et al., 2010; Clause et al., 2014). Experience-independent structured electrical activity is a common phenomenon in developing sensory systems, thought to contribute to maturation of synapses, ion channel expression and refinement of neuronal networks (Shatz, 1996; Stellwagen and Shatz, 2002; Moody and Bosma, 2005; Blankenship and Feller, 2010; Leighton and Lohmann, 2016).

It is debated whether IHCs generate Ca²⁺ APs autonomously (Kros et al., 1998; Brandt et al., 2003, 2007; Marcotti et al., 2003b; Johnson et al., 2007, 2011, 2013, 2017; Sendin et al., 2014) or require Ca²⁺ waves of inner supporting cells (ISCs) as a trigger (Tritsch et al., 2007, 2010; Tritsch and Bergles, 2010; Wang et al., 2015). The immature cochlea contains Kölliker’s organ, which harbors tall cylindrical cells that spontaneously generate intercellular Ca²⁺ waves (Tritsch et al., 2007; Anselmi et al., 2008; Majumder et al., 2010; Mammano and Bortolozzi, 2018). Wave propagation is sustained by ATP-dependent ATP release: extracellular ATP releases intracellular Ca²⁺ through a P2Y receptor-IP₃-dependent mechanism. In turn, the rise in [Ca²⁺]_i increases the open probability of connexin hemichannels for ATP, which are located at the endolymphatic surface of the ISCs (Tritsch et al., 2007; Anselmi et al., 2008; Majumder et al., 2010; Mammano and Bortolozzi, 2018). IP₃ may also contribute to wave propagation by diffusing through gap junctions to ISC neighbors (Anselmi et al., 2008; Majumder et al., 2010; Ceriani et al., 2016b).

ISC Ca²⁺ waves may trigger Ca²⁺ APs in IHCs by two potential mechanisms: (i) ATP-induced depolarization to the threshold for regenerative APs (Tritsch et al., 2007; Johnson et al., 2011) mediated by P2X2R, P2X3R and P2X7R (Brändle et al., 1999; Nikolic et al., 2003; Housley et al., 2006; Huang et al., 2010) or (ii) [Ca²⁺]_i-dependent activation of TMEM-16A channels in ISCs that allow Cl⁻ efflux followed by efflux of K⁺ and water (Wang et al., 2015). The resulting increase in extracellular K⁺ may sufficiently depolarize IHCs to evoke Ca²⁺ APs. Accordingly, a Ca²⁺ wave in the ISC compartment would elicit synchronized bursts of Ca²⁺ APs in neighboring IHCs (Wang et al., 2015).

Although IHC Ca²⁺ APs, which are based on regenerative opening of Ca_v1.3 Ca²⁺ channels, K_v and SK2 K⁺ channels, have been analyzed in depth by patch-clamp recordings (Brandt et al., 2003; Marcotti et al., 2003b; Johnson et al., 2007, 2011, 2013, 2017; Sendin et al., 2014) the accompanying Ca²⁺ transients in IHCs have not been studied so far. Moreover, we still lack simultaneous imaging data resolving the temporal and spatial correlation between fast Ca²⁺ transients in IHCs and Ca²⁺ waves in ISCs. We therefore conducted Ca²⁺ imaging in the immature mouse organ of Corti to determine whether Ca²⁺ transients in IHCs occur independent of ISC Ca²⁺ waves.

Materials and Methods

Animals

All experimental procedures were conducted in agreement with the European Communities Council Directive (2010/63/EU) in accordance with the German law on the use of laboratory animals and were approved by the Saxonian District Government, Leipzig (TVV59/16 and T34/16), and the regional board for scientific animal experiments of the Saarland. Prehearing mice (P4–P6) of either sex were studied. NMRI mice (Charles River, Sulzfeld, Germany and bred in-house; 64 animals) were used unless stated otherwise and are designated as controls. Ca_v1.3^−/− mice (Platzer et al., 2000), which were bred to either C57BL6/N (seven animals) or NMRI background (13 animals) for >10 generations, mice deficient for the purinergic receptor P2X7 (P2X7R^−/− mice; The Jackson Laboratories, Bar Harbor, ME, USA; 21 animals) and P2X2R/P2X3R double knockout mice (P2X2R^−/−/P2X3R^−/−; 20 animals; Cockayne et al., 2005) both on C57BL6/N background were used. Every experimental observation was confirmed in at least three experiments using different animals.

Tissue Preparation

Neonatal mice were sacrificed by decapitation. Cochleae were immediately dissected in ice-cold physiological solution mimicking perilymph (in mM): 143 NaCl, 5.8 KCl, 1.3 CaCl₂, 0.9 MgCl₂, 5.6 glucose, 10 HEPES and 0.9 NaH₂PO₄, 305 mOsm/l, pH 7.35. The organ of Corti was dissected out of the cochlea with great care to preserve the integrity of the tissue. The apical turn was cut and placed in a recording chamber underneath a soft thread of a nylon mesh, which was attached to a steel ring (Figure 1). Nylon threads had an average distance to the recording window of 253 ± 47 μm (determined for 26 recordings). After cautiously removing the tectorial membrane, the specimen was incubated in the perilymph-like solution containing 10 μM of the cell-permeable Ca²⁺ indicator Fluo-8^® AM (AAT Bioquest Inc., Sunnyvale, CA, USA) that had been dissolved in a Pluronic-DMSO mixture (100 mg/ml; Thermo Fisher Scientific Inc., Waltham, MA, USA). To allow for uptake of Fluo-8 AM and hydrolysis, the recording chamber with the tissue was kept in a dark, humid plastic box for 40 min at room temperature. The chamber was mounted on the stage of the upright laser scanning microscope LSM 710 (Zeiss Microscopy GmbH, Göttingen, Germany) and perfused with perilymph-like solution for at least 7 min to remove any Fluo-8 AM prior to the experiment. Only healthy looking epithelia with smooth surfaces, absence of vacuoles and low background fluorescence in both IHCs and ISCs were used for imaging (Figure 2A). IHCs were recorded within a fractional distance of 10–25% from the apex, which corresponds to a frequency range of about 7–10 kHz in the adult animal (Müller et al., 2005). Agonists and antagonists (ATP; 1 or 10 μM; PPADS; 100 μM; both Sigma-Aldrich, St. Louis, MO, USA) were diluted in perilymph-like solution (see above) and applied by a gravity-driven custom-made application system with a multi-barreled pipette (Figure 1), which led to a change of the solution above ISCs and IHCs within 3–6 s. Unless stated otherwise, the specimen was constantly superfused with either perilymph-like solution, the same solution containing the respective agonist/antagonist, or with Ca²⁺-free solution, respectively, via the application system at 0.29 ml/min. To avoid movement artifacts caused by changes in fluid pressure, the switch between two solutions was performed by simultaneously closing respectively opening the valves of the respective reservoirs. The Ca²⁺-free solution containing the following (in mM): 143 NaCl, 5.8 KCl, 2.2 MgCl₂, 5.6 glucose, 10 HEPES, 0.9 NaH₂PO₄ and 0.5 EGTA acid, 305 mOsm/l, pH 7.35. The distance between the scan field and the lateral edges of the tissue combined with the application of perilymph-like solution without/with agonist/antagonists from the pillar side prevented spillover of ATP from potentially damaged cells and ISCs. The delay in solution flow from reaching the IHCs to reaching the ISCs was estimated to be <20 ms.

Figure 1

Figure 2

Ca²⁺ Imaging, Analysis and Data Handling

Data were recorded using a LSM 710 based on an upright AxioExaminer microscope with 40×/1.0 NA or 20×/1.0 NA Plan Apochromat water immersion objectives and Zen-2012 SP1 (Black edition) software (all Zeiss). Fluo-8 fluorescence was excited with the 488 nm line of an argon laser. Laser intensity was set to very low levels to allow for long continuous recordings (>10 min, up to 30 min) while avoiding photobleaching and strong phototoxicity. We opened up the pinhole to maximal seven airy units accepting a reduced spatial resolution in favor of increased fluorescence intensity at the chosen low laser power and a higher scan speed. Recordings were performed at room temperature (21°C ± 1°C). Because Fluo-8 based on Fluo-4 (Gee et al., 2000) is a non-ratiometric Ca²⁺ indicator with a high quantum yield and a K_d of 389 nM we determined the relative fluorescence changes corresponding to [Ca²⁺]_i changes in ISCs and IHCs off-line:

where F(t) is the fluorescence intensity of a pixel at a given time t and F₀ the fluorescence of this pixel averaged over the first 10 images without obvious Ca²⁺ changes.

For imaging the spread of ISC Ca²⁺ waves in Kölliker’s organ, scan fields of about 200 μm × 100 μm were chosen resulting in frame rates of ≤5 Hz (scan times >200 ms). To resolve the much faster fCaTs in IHCs, we increased the frame rate by: (i) imaging smaller scan fields; and (ii) increasing the pixel size to 0.4 μm × 0.4 μm (up to 0.6 μm × 0.6 μm). Scan fields were 140 μm ± 21 μm in width (axis parallel to the IHCs) and 27 μm ± 5 μm in height (Figures 1, 2A) and comprised the basolateral poles of 7–9 (up to 23) IHCs with a stripe of ISCs extending up to 36 μm in the modiolar direction. This led to frame rates of >31 Hz and to corresponding shorter scan times of ≤32 ms (except Figure 6, 36.2 ms), which allowed simultaneous imaging of Ca²⁺ waves in ISCs and fCaTs in IHCs. For achieving an even better temporal resolution of fCaTs, a subset of control IHCs was imaged at 175 Hz (scan time of a single frame: 5.73 ms). With these settings, photobleaching amounted to 0.5 ΔF/F₀ at maximum during periods of ≥6 min. Offline analysis and graphical representation was performed using FIJI (Schindelin et al., 2012), IGOR 6.12 (Wavemetrics, Inc., Lake Oswego, OR, USA) and Inkscape™ 0.91. Regions of interest (ROIs) in a time series (t-series) of fluorescence images were defined by activity patterns within IHCs and ISCs and were validated using a high-resolution transmitted light image acquired prior to the t-series with a 100-fold smaller pixel area. Transmitted light images were also taken after a t-series to check for a potential tissue drift. Data are reported as mean ± standard deviation (SD) except data in Figure 10B, where mean ± SEM is given.

Figure 3

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Figure 6

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Figure 9

Figure 10

Results

Using Fluo-8 AM-incubated explants of the apical organ of Corti, which were dissected with greatest care, we observed spontaneously occurring Ca²⁺ waves and corresponding waves of light refraction (crenation waves) in the region of ISCs. This is consistent with previous studies of ISCs of Kölliker’s organ in the immature organ of Corti (Tritsch et al., 2007; Anselmi et al., 2008; Dayaratne et al., 2015; Wang et al., 2015). ISCs generated diverse Ca²⁺ phenomena ranging from Ca²⁺ elevations confined to a single ISC (Ca²⁺ transients) to Ca²⁺ waves extending over various distances. Small Ca²⁺ waves propagated radially to maximum two neighboring ISC circles comprising about 200–600 μm², whereas large waves covered areas from 600 μm² upto >2200 μm² within Kölliker’s organ involving about 75 ISCs. Sources and directions of the waves were random, indicating that the tissue had not been impaled in or near the field of view and that ATP potentially leaking out from injured or dying cells at the outermost edges of the tissue piece did not affect ISCs and IHCs in the field of view.

Simultaneous Imaging of Ca²⁺ Waves in ISCs and Fast Ca²⁺ Transients in IHCs

Figure 2 shows a typical recording of concomitant ISC and IHC Ca²⁺ signals. Two rows of ISCs and the basolateral poles of eight IHCs are visible in the transmitted light image with the focus set at the maximum width of IHC nuclei (Figure 2A). IHCs are enwrapped by specialized ISCs, the inner phalangeal cells, which stay throughout maturity. We do not differentiate here between transient ISCs of Kölliker’s organ and inner phalangeal cells because they cannot be unambiguously discriminated in DIC images. IHCs numbered 6–8 within the Fluo-8 AM-loaded specimen showed a slightly elevated initial raw fluorescence at t = 0 s (green; Figure 2B). Usually care was taken to eliminate frames in which IHCs showed transient fluorescence elevations for determining the baseline (the first 10 frames) to avoid ΔF/F₀ values <0 at later time points. Here, we show such an example on purpose to demonstrate that the calculation of relative fluorescence changes (ΔF/F₀) eliminates different raw fluorescence values (Figures 2B,C). Figures 2C–H depict relative changes in fluorescence intensity, ΔF/F₀ (see Eq. 1 in “Materials and Methods” section) starting at t = 0 s (Figure 2C) and at subsequent time points. The basolateral pole of IHC 3 showed elevated ΔF/F₀ (Figures 2D,E, arrowheads) reflecting an autonomous single fCaT. About 1 s later, a Ca²⁺ wave originating from ISC III (Figure 2F, arrow) spread in all directions, reaching the IHCs (Figures 2G,H). fCaTs occurred in IHCs 1, 3, 4, 5, 7. Averaging ΔF/F₀ in ROIs indicated in Figure 2A as a function of time (Figure 2I) revealed fCaTs in IHCs 3, 4 and 7, and a large and long-lasting Ca²⁺ transient in ISC III triggering the ISC Ca²⁺ wave seen at maximum spread in Figure 2H. IHC 3 was active before its adjacent ISC III generated a Ca²⁺ wave (Figure 2I, inset i), whereas the fCaTs in IHCs 4 and 7 started after Ca²⁺ had risen in the nearby ISC III. While IHC 7 produced 4 fCaTs shown at the onset of the recording, no elevation in Ca²⁺ could be observed in ISCs (Figure 2I, left).

Properties and Incidence of Fast Ca²⁺ Transients in IHCs

To study the temporal properties of fCaTs in detail, IHCs were imaged at 175 Hz (scan time of a single frame: 5.73 ms), which required a smaller scan field not allowing for simultaneous imaging of ISCs. During a 573 s-long recording of eight IHCs, seven of them generated fCaTs. Single fCaTs, which were not generated within a burst of activity, had an average half-width of 32.5 ± 9.8 ms (n = 18) and decayed to baseline after 895.9 ± 381.2 ms (n = 17; Figure 3A). Time to peak, measured from 10% of the peak amplitude to the maximum fluorescence, amounted to 16.3 ± 5.5 ms (n = 19). Decay time defined as time from peak back to 10% above baseline was 879.7 ± 379.1 ms (n = 17), thus revealing a much slower decay in fluorescence compared to the rise (Figure 3A, Table 1). Besides single isolated fCaTs, IHCs generated fCaTs within events we designate as minibursts (2–5 fCaTs in close sequence, Figure 3B) and within bursts with more than five consecutive fCaTs (Figure 3C, top). The mean frequency of fCaTs was 0.32 ± 0.11 Hz, but they were not evenly distributed. Within bursts, frequencies varied from 4 Hz to 10 Hz (Figure 3C, bottom) similar to previously reported frequencies in bursts of IHC Ca²⁺ APs (Johnson et al., 2011, 2017; Sendin et al., 2014). A total of 49 bursts and 54 minibursts were generated in the seven active IHCs during 573 s recording time. Of those, 42 bursts and 18 minibursts occurred simultaneously in adjacent IHCs with at least two IHCs involved (mean number of simultaneously active IHCs: 4.62 ± 1.85). Notably, ΔF/F₀ continuously exceeded values of 1.5 only within bursts of fCaTs (Figure 3C, top).

To study the incidence of the three types of IHC fCaTs (single events, minibursts and bursts) perfusion was switched off in a set of experiments to exclude the possibility that the bath flow may have caused an interaction between ISCs and IHCs. Fifty IHCs from four control mice were monitored in seven recordings lasting between 544 s and 1120 s. The incidence of fCaTs was analyzed during a total observation time of 598 min (total number of IHCs multiplied by the length of respective recordings), irrespective of Ca²⁺ elevations in ISCs. IHCs generated 9668 fCaTs with 18.3% being single fCaTs, 25.2% occurring within minibursts of 2–5 fCaTs and 56.5% occurring within bursts of 6–67 fCaTs (Figure 3D). A detailed analysis showed that bursts consisted on average of 15.3 ± 9.5 fCaTs (n = 353) whereas minibursts contained an average number of 2.8 ± 1.0 fCaTs (n = 867; Table 1). During bursts of fCaTs, ΔF/F₀ generally increased to >2 with maximal values <4 and declined slowly within 10 s. In contrast, ΔF/F₀ reached the baseline within 1–2 s in events with ≤5 fCaTs (see Figures 3B,C). These data indicate that although the majority of fCaTs (56.5%) were embedded in a burst, a comparable number (43.5%) appeared outside of bursts as single fCaTs or as part of a miniburst (Figure 3D).

fCaTs of IHCs Depended on External Ca²⁺ and on the Expression of Voltage-Gated Ca_v1.3 Channels

In order to test the Ca²⁺ dependance of fCaTs, nominally Ca²⁺-free solution was applied. To achieve perfusion of the cell bodies under the reticular lamina, the application pipette here was positioned at a lateral edge of the epithelium, in contrast to the setting shown in Figure 1. When IHCs were bathed in Ca²⁺-free solution for >1 min, single fCaTs, minibursts and bursts were reversibly abolished (Figures 4A–C), indicating that influx of extracellular Ca²⁺ is required to generate fCaTs. Ca²⁺ signals of ISCs, however, did not vanish in 0 mM Ca²⁺ (Figure 4A; black trace). The decrease in the baseline of ΔF/F₀ in both IHCs and ISCs was likely caused by a reduction in the intracellular Ca²⁺ concentration in the Ca²⁺-free solution.

Because Ca²⁺ APs of IHCs crucially depend on Ca_v1.3 channels (Brandt et al., 2003), which account for more than 90% of voltage-gated Ca²⁺ currents in neonatal IHCs (Platzer et al., 2000), Ca²⁺ measurements were conducted in IHCs and ISCs of Ca_v1.3^−/− mice in standard (1.3 mM) extracellular Ca²⁺ concentration (Figures 4D,E). In 103 IHCs from three Ca_v1.3^−/− mice we did not observe a single fCaT (Figure 4E) during a total recording time of 787 min (recording length multiplied by number of IHCs). However, those IHCs contacted by an ISC Ca²⁺ wave typically showed slowly rising Ca²⁺ elevations of small amplitude (ΔF/F₀ <1) temporally matching the Ca²⁺ signals of the ISC (Figure 4E, left). This slow IHC signal may have resulted from: (i) potential bleedthrough of the fluorescence signal of the ISCs; (ii) Ca²⁺ influx through P2X receptors; or (iii) Ca²⁺ influx through the small percentage of Ca²⁺ channels other than Ca_v1.3 (mostly Ca_v1.2 and some Ca_v2.3-mediated currents; Brandt et al., 2003). We never saw this kind of small slow Ca²⁺ elevation in IHCs in the absence of an invading ISC Ca²⁺ wave. On rare occasions, IHCs of Ca_v1.3^−/− mice did not respond to an invading Ca²⁺ wave with small and long-lasting Ca²⁺ elevations (Figure 4E, left). Summarizing the results from control mice in 0 mM Ca²⁺ solution and those from Ca_v1.3^−/− mice in 1.3 mM extracellular Ca²⁺ we conclude that fCaTs in IHCs reflect the Ca²⁺ increases mediated by Ca_v1.3 channels.

Synchronized Bursts of fCaTs in Adjacent IHCs Were Triggered by ISC Ca²⁺ Waves or by Extracellular ATP

IHCs were able to generate fCaTs autonomously without being triggered by ISC Ca²⁺ waves (see Figure 3 and below). However, when an ISC Ca²⁺ wave approached the IHC region it typically evoked bursts of fCaTs in a number of adjacent IHCs (Figures 5A–C). The spontaneous Ca²⁺ wave involving ISC I (at t ~70 s) triggered bursts in IHCs 1 and 2, and although ISC IV was silent, IHCs 3–5 were active at the same time. The Ca²⁺ elevation of ISC I preceded that of the IHCs (Figure 5C). Analyzing 12 waves causing burst events in IHCs from a larger data set, the Ca²⁺ wave-evoked fluorescence elevation in ISCs lining the IHC row preceded the onset of fCaTs within bursts in the closest IHC by 0.52 ± 0.27 s (mean ± SD). Though less frequent, synchronized IHC bursts were also observed without any ISC activity within the recording frame as e.g., in IHCs 1, 2, 4, 5, 6, 8 at t ~90 s (Figure 5B and see below).

To validate previous results showing ATP-induced inward currents in neonatal IHCs (Tritsch and Bergles, 2010) in our preparation, we tested the effect of ATP on IHC fCaTs. Application of 1 μM ATP for 5 s or 10 s from the pillar side (Figure 5B) strongly increased ΔF/F₀ in all cells of the sensory epithelium (cells of the inner and outer sulcus, interdental cells (IDCs), spiral ganglion tissue and IHCs), indicating an overall expression of purinergic receptors at the age of P4/P5. The ATP-evoked increase in ΔF/F₀ in ISCs resembled that induced by a spontaneous Ca²⁺ wave, lasting for 10–15 s (Figure 5B; black traces). In IHCs, ATP-evoked bursts of fCaTs strongly increased ΔF/F₀ up to 4. Repetitive ATP stimulation (five times at intervals >30 s) did not deplete the burst responses in IHCs (see Figure 5B). However, fCaTs within ATP-induced bursts were smaller in amplitude compared with fCaTs triggered by a Ca²⁺ wave and were challenging to detect (Figure 5D). Together, these experiments corroborate earlier studies showing that ATP generates Ca²⁺ waves in ISCs (Tritsch et al., 2007, 2010). To test the hypothesis if ATP can activate IHCs independently of ISC Ca²⁺ elevations, the ATP had been applied from the pillar side (Figures 5B,D,E). Such a direct stimulation of purinergic receptors on IHCs evoked bursts of fCaTs that preceded Ca²⁺ elevations in ISCs by 0.99 ± 0.34 s (n = 23 recordings). When taking into account that the solution flow reached the IHCs ~20 ms earlier than the ISCs, these results still indicate that ATP can elicit bursts in IHCs independent of ISC Ca²⁺ elevations.

Block of P2 Receptors by PPADS Irreversibly Suppressed Ca²⁺ Waves in ISCs and Reversibly Reduced Fast Ca²⁺ Transients in IHCs

To further analyze the importance of purinergic signaling on fCaTs, we tested the effect of 100 μM PPADS, a broad spectrum blocker of P2Y and P2X receptors (Abbracchio et al., 2006; Coddou et al., 2011). Before PPADS application, control measurements were conducted for 180 s in order to simultaneously record Ca²⁺ waves in ISCs and single fCaTs, minibursts and bursts in IHCs (Figures 6B–D). PPADS superfusion (100 μM) completely abolished fCaTs in 33 of 44 IHCs. The remaining 11 IHCs still generated single fCaTs, but neither minibursts nor bursts. Single fCaTs, minibursts or bursts were recorded after washout in 27 IHCs, albeit with strongly reduced amplitude (Figures 6C,E). Notably, blocking P2 receptors had an even stronger effect on ISCs (Figures 6B,E): the incidence of ISC Ca²⁺ waves was reduced from 5.4 waves/100 s (35 waves in 645 s total recording time, three control mice) to 1.0 waves/100 s (five waves during 509 s in total). After removal of PPADS, the ability of ISCs to generate Ca²⁺ waves was very weak, as only three Ca²⁺ waves occurred during 1910 s washout (0.2 Ca²⁺ waves/100 s; Figures 6B–E).

IHC fCaTs and ISC Ca²⁺ Waves Were Not Affected by the Lack of P2X2R/P2X3R or P2X7R

Because: (i) ATP strongly promoted generation of IHC bursts (Figure 5); (ii) IHCs express P2X2, P2X3 and P2X7 receptors (Brändle et al., 1999; Nikolic et al., 2003; Huang et al., 2005; Housley et al., 2006); and (iii) P2X2 and P2X3 receptors were proposed to mediate an increase in Ca²⁺ AP activity (Tritsch et al., 2007; Johnson et al., 2011), we tested the generation of fCaTs and their interplay with spontaneous ISC Ca²⁺ waves in P2X2R/P2X3R double knockout (P2X2R^−/−/P2X3R^−/−) and P2X7R knockout (P2X7R^−/−) mice. Both, ISC waves and IHC fCaTs were unaffected in P2X2R^−/−/P2X3R^−/− mice (Figures 7A–E). As in control mice, burst-like activity of IHCs was mostly triggered by Ca²⁺ waves and was synchronized between adjacent IHCs (Figures 7C,D). Overall, 30 IHCs from five recordings obtained from three animals, each lasting 628 s, regularly generated fCaTs. Of the 4526 fCaTs during the 314 min recording time, 665 (14.7%) were single fCaTs, 721 (15.9%) occurred in minibursts and 69.4% fCaTs were found in bursts (n = 3140). Examples of IHC Ca²⁺ signals from P2X2R^−/−/P2X3R^−/− mice are shown in Figure 7E. Experiments in P2X7R^−/− mice involving 48 IHCs (five recordings, four animals) revealed similar IHCs fCaTs and ISC Ca²⁺ waves as in control mice (Figures 7F–J). Burst-like activity of neighboring IHCs in P2X7R^−/− mice was mostly triggered and synchronized by Ca²⁺ wave activity in close vicinity (Figures 7H,I). Of the 4648 fCaTs recorded during the total of 503 min, 1135 (24.4%) were single fCaTs, 1122 (24.1%) occurred in minibursts and 2389 (51.4%) fCaTs in bursts. Examples of IHC Ca²⁺ signals from P2X7R^−/− mice are shown in Figure 7J. In summary, neither P2X2, P2X3 nor P2X7 receptors were essential for the generation of IHC fCaTs nor the coupling of IHC bursts to ISC Ca²⁺ waves.

Fast Ca²⁺ Transients Can Occur Independently of ISC Ca²⁺ Waves

It has been suggested previously that IHCs can generate Ca²⁺ APs independently of Ca²⁺ wave activity in adjacent ISCs (Johnson et al., 2011, 2017; Sendin et al., 2014). These studies are based on electrical recordings where patch pipettes could have potentially liberated ATP from injured cells at the recording site, making the interpretation of “spontaneous activity” challenging. Performing imaging only, we were able to simultaneously observe IHC fCaTs and ISC Ca²⁺ waves and their potential coupling (Figure 8). Though autonomously generated fCaTs were mostly solitary fCaTs or minibursts, spontaneous bursts were observed, too. An example is shown in Figures 8A,B where bursts of fCaTs appeared in IHC 2 without Ca²⁺ elevation in any ISC as indicated by asterisks referring to the IHC 2-trace in red. Yet, bursts with >5 fCaTs were more frequently triggered by a Ca²⁺ wave reaching the IHC region (Figure 8B, indicated by triangles, and Figure 8C) with an overall delay of 0.52 ± 0.27 s (n = 12 waves from four animals, see above). However, not every ISC Ca²⁺ wave elicited burst-like activity in the adjacent IHC 2 (Figure 8D). This lack of activity of IHC 2 was transient because it autonomously generated bursts (Figure 8B**) or single fCaTs and minibursts (Figure 8E) as well as ISC-evoked activity (Figure 8B, single and double triangle) thereafter. Together, our data support the hypothesis that isolated fCaTs and minibursts, but also bursts can be generated in IHCs irrespective of ISC Ca²⁺ waves.

Simultaneous Bursts in Neighboring IHCs Were Mostly Triggered by ISC Ca²⁺ Waves

Due to their spatial spread, most Ca²⁺ waves that invaded the IHC area evoked bursts of fCaTs in more than one IHC, causing synchronous Ca²⁺ activity in two to eight IHCs. The upper number may be larger, but was limited by the size of the scan field. An example in Figure 9 shows the spatiotemporal coupling of a wave starting from ISC II, which elicited bursts in IHCs 1, 2, 3, 4 (Figures 9A–C,F). Notably, IHCs also generated simultaneous bursts without being triggered by an ISC Ca²⁺ wave. IHCs 4–8 responded with bursts to a wave starting in ISC VIII (Figure 9D), but IHCs 1–3 at 10–30 μm distance from the ISC Ca²⁺ signal also responded with bursts (Figures 9E,F) suggesting direct coupling of activity between IHCs. This means that simultaneous Ca²⁺ activity in adjacent IHCs can occur without a Ca²⁺ elevation in the ISCs contacting these IHCs or their direct IHC neighbors.

We analyzed burst activity synchronized between IHCs in periods of 15 s, the approximate duration of a large Ca²⁺ wave at a given ISC ROI, for data from four control mice (seven recordings comprising a total of 50 IHCs). The relative incidence of synchronous miniburst activity in at least two adjacent IHCs without being triggered by an ISC Ca²⁺ was 40.3% (149 cases) compared with 59.7% (221 cases) with a Ca²⁺ wave as trigger (Figure 9G). Spontaneous synchronous bursts in at least two adjacent IHCs had a relative incidence of 32.1% (92 cases), which increased to 67.9% (195 cases) when triggered by a Ca²⁺ wave (Figure 9G). A possible explanation for the coupling of Ca²⁺ activities between IHCs in the absence of Ca²⁺ waves might be an activity-driven K⁺ efflux from IHCs through their voltage-gated K⁺ channels. If an increase in the extracellular K⁺ concentration around IHCs was not effectively prevented by inner phalangeal cells/ISCs, neighboring IHCs would become depolarized, which in turn would trigger Ca²⁺ APs and hence fCaTs. A similar K⁺ accumulation-based activation of IHCs was proposed as coupling mechanism between Ca²⁺ waves and IHC activity, although the latter originates from K⁺ efflux of ISCs including inner phalangeal cells (Wang et al., 2015).

Efficacy of ISC Ca²⁺ Waves in Triggering fCaTs in IHCs

To quantify the efficacy of a Ca²⁺ wave invading the IHC area in generating fCaTs, ISC Ca²⁺ signals were classified into four groups (Figure 10A). Wave class 0 reflects the lack of ISC activity during a 15 s period, which is the approximate duration of a large ISC Ca²⁺ wave. Class 1 defines a non-propagating Ca²⁺ transient restricted to a single ISC characterized by a mean duration of 8.5 ± 3.7 s (n = 12 Ca²⁺ transients). Class 2 denotes a small ISC Ca²⁺ wave that radially propagated ≤2 cells from its origin (mean duration of 10.1 ± 1.9 s, n = 9 waves), whereas class 3 defines a large ISC Ca²⁺ wave propagating >2 cells from its origin (mean duration of 12.5 s ± 2.0 s, n = 19 waves; Figure 10A). Small and large Ca²⁺ waves were only included into the analysis if they originated within the scan field. A rank order of IHC activities was defined as follows: burst > miniburst > single fCaT > inactive IHC. For a given wave class, the IHC event type with the highest rank was determined for each IHC in the scan field during the respective time window. These event numbers were added, normalized to the total number of IHCs in the scan field and averaged between recordings, yielding the relative incidence of IHC events for the respective wave class (Figure 10B).

Relating the class of ISC activity to the highest rank of the simultaneous IHC activity revealed striking differences (Figure 10B): bursts were extremely rare when ISCs were silent (4% ± 0.8%; mean ± SEM, n = 196 time windows without ISC activity during 15 s) or showed class 1 events (2% ± 1.5%, n = 13 non-propagating ISC transients). However, bursts predominated when IHCs were activated by class 3 waves. When ISCs were silent (class 0), the incidence of inactive IHCs was 57% ± 1.8% (n = 196) and most of the autonomous activity observed were single fCaTs (22% ± 1.4%) or minibursts (18% ± 1.3%). During a non-propagating Ca²⁺ transient of a single ISC (class 1), a similar picture emerged as with silent ISCs (Figure 10B). However, during class 2 Ca²⁺ waves (n = 11) the incidence of bursts increased to 17% ± 6.8% and of minibursts to 23% ± 4.5%, whereas the proportion of inactive IHCs was reduced to 40% ± 10.8%. In the presence of class 3 waves (n = 19), the fraction of bursts increased to 64% ± 7% and that of minibursts to 25% ± 5.7%. In contrast, the incidence of single fCaTs or inactive IHCs was reduced to 5% ± 2.8% and 5% ± 2%, respectively. These data indicate that most bursts of fCaTs reflecting burst-like action potentials of IHCs require ISC Ca²⁺ waves. On the other hand, IHCs can generate single fCaTs and minibursts without wave activity of ISCs.

Discussion

Spontaneous activity is a hallmark of developing sensory systems including the auditory system (Blankenship and Feller, 2010; Leighton and Lohmann, 2016). Spontaneously occurring Ca²⁺ signals in IHC and their dependance on ISC Ca²⁺ waves was presently studied with Fluo-8 AM Ca²⁺ imaging on freshly dissected explants of organs of Corti. Since ATP can trigger Ca²⁺ waves in Kölliker’s organ of the immature organ of Corti (Tritsch et al., 2007; Anselmi et al., 2008; Majumder et al., 2010) and also Ca²⁺ APs in IHCs (Tritsch et al., 2007; Tritsch and Bergles, 2010; Johnson et al., 2011) it was necessary to avoid unintended release of ATP and putative ATP-dependent signaling. Great care was taken to keep the epithelium as intact as possible. Moreover, the recordings were performed from free-running tissue in the middle of the explant far from the lateral edges. By using small scan fields and a pixel size of ~0.17 μm² it was possible to temporally resolve fCaTs in IHCs and classify them into single fCaTs, mini-bursts (2–5 fCaTs) and bursts of fCaTs. Our ΔF/F₀ values of fCaTs in Fluo-8 AM-loaded IHCs varied from 0.5 up to 5 (in bursts), providing a much higher amplitude resolution than previously reported using Fluo-4 AM (Ceriani et al., 2016a).

IHC fCaTs Are Consistent With Ca²⁺ APs

In the first postnatal week, mouse and rat IHCs generate Ca²⁺ APs that vanish shortly before the onset of hearing (Kros et al., 1998; Brandt et al., 2003, 2007; Marcotti et al., 2003b; Johnson et al., 2005, 2007, 2011, 2012, 2013; Sendin et al., 2014; Iosub et al., 2015). Ca²⁺ APs appear as single events, but more often in bursts of 10–20 single APs. They are reversibly abolished in nominally Ca²⁺-free solution (Marcotti et al., 2003b) and missing in Ca_v1.3^−/− mice (Brandt et al., 2003). Similarly, the IHC fCaTs recorded in this study were reversibly suppressed in nominally Ca²⁺-free solution and absent from IHCs of Ca_v1.3^−/− mice. Time-to-peak of fCaTs measured at the highest scan rate was 16 ms, which is in the range of the half-width of 15 ms for a Ca²⁺ AP at room temperature (Marcotti et al., 2003b). It is not surprising that a single fCaT lasts longer than the electrical AP because (i) Ca²⁺ influx into an IHC extends to the repolarizing phase of the AP due to the large driving force for Ca²⁺ and (ii) AP repolarization by delayed rectifier K⁺ and SK2 channels (Marcotti et al., 2003a; Mammano and Bortolozzi, 2018) is, particularly in minibursts and bursts, faster than clearance of Ca²⁺ from the cytosol by Ca²⁺ ATPases (Grati et al., 2006) or by uptake into intracellular stores and mitochondria (Kennedy, 2002), see Figure 3C. Ca²⁺-induced Ca²⁺ release from ryanodine-sensitive stores (Iosub et al., 2015) may additionally increase and prolong the Ca²⁺ signal in IHCs. Further, Fluo-8 acts as a Ca²⁺ buffer itself and could slow the Ca²⁺ decay kinetics. The dissociation time constant of the chemically related Fluo-3 is ~5 ms (Eberhard and Erne, 1989), much faster than the average decay time of fCaTs of 880 ms. Further, our fCaTs resemble the events measured with simultaneous imaging of Fluo-4FF-filled IHCs and Ca²⁺ APs recordings in current clamp (Iosub et al., 2015). Taken together, IHC fCaTs imaged under our conditions reflect the Ca²⁺ signals resulting from IHC APs.

It might be argued that due to the limited z-resolution of a confocal LSM compared with its resolution in x and y, we recorded signals from inner phalangeal cells rather than from IHCs, especially when IHCs were oriented steeply within the organ of Corti. However, Ca²⁺ signals of ROIs taken well inside those IHCs that had a shallow position clearly showed a different temporal pattern including fCaTs compared with adjacent ROIs likely involving inner phalangeal cells, which always lacked fCaTs (e.g., Figures 2A,I, ISC III). However, some bleed-through of fluorescence from inner phalangeal cell signals to IHCs in the z-direction cannot be excluded.

ATP Independently Triggers Ca²⁺ Elevations in Both ISCs and IHCs

Applying 1 μM ATP from the pillar side towards the modiolus elicited bursts of fCaTs that were highly synchronized between neighboring IHCs and appeared before Ca²⁺ had risen in adjacent ISCs (Figure 5). This indicates that ATP can trigger IHC Ca²⁺ APs independently of ISC Ca²⁺ waves. Moreover, the large reduction of Ca²⁺ wave-independent IHC fCaTs under the broad spectrum P2R blocker PPADS (Figure 6) favors a role of P2 receptors in increasing the IHC’s susceptibility to generate Ca²⁺ APs. These results are consistent with ATP-induced inward currents through P2X receptors in IHCs (Tritsch and Bergles, 2010), which according to mRNA/protein expression data or PPADS sensitivity suggests contribution of P2X2, P2X3, P2X4, P2X7 before the onset of hearing (Nikolic et al., 2003; Huang et al., 2005; Coddou et al., 2011), yet contribution of P2Y receptors cannot be ruled out (Abbracchio et al., 2006; Huang et al., 2010). Regarding PPADS-sensitive ATP-mediated Ca²⁺ signals in ISCs, P2Y receptors such as P2Y1, P2Y2, P2Y4 and P2Y6 may be involved (Abbracchio et al., 2006; Huang et al., 2010) but P2X receptors may additionally contribute (Tritsch et al., 2007; Coddou et al., 2011).

Electrical recordings of IHC Ca²⁺ APs, which require access of the patch pipette to the body of the IHC, inevitably cause tissue damage and release of ATP, either during “cleaning” of an IHC from ISCs at one side before establishing the pipette seal, or when approaching the IHC under pressure similar as in a tissue slice. Due to local ATP release in patch-clamp experiments, the interpretation of IHC AP activity as spontaneously occurring should be taken with caution. Release of ATP also occurs when recording from ISCs (Tritsch et al., 2007; Wang et al., 2015), which makes the interpretation of the players in the interaction between Ca²⁺ waves and IHC Ca²⁺ APs and a differentiation of putative roles of ATP rather challenging. Our free-running whole-mount preparation has the advantage that the epithelium was left intact within a region much larger than the field of view, which allowed imaging the interaction of ISC Ca²⁺ waves and IHC fCaTs without local tissue damage and contamination with ATP. The fact that ISC Ca²⁺ waves did not repeatedly originate from the same foci suggests that the tissue in the field of view was not injured.

Electrical Properties of IHCs Sustain Autonomous Ca²⁺ AP Activity

Frequently, single fCaTs and minibursts but sometimes also bursts were observed in IHCs in the absence of Ca²⁺ waves in adjacent ISCs (Figures 5, 8, 9; summarized in Figure 10), which were termed here spontaneous or autonomous IHC events. These events reflecting Ca²⁺ APs were therefore neither the consequence of extracellular K⁺ accumulation through K⁺ efflux nor of ATP release from the ISCs (see below). It is rather likely that sizes and properties of IHC currents, such as Ca_v1.3 currents, various K⁺ currents including SK2, transducer currents at rest, purinergic currents, cholinergic currents (Brandt et al., 2003; Marcotti et al., 2004; Housley et al., 2006; Johnson et al., 2007, 2012) favor to the ability of the IHC to produce regenerative Ca²⁺ APs.

Coupling of ISC Ca²⁺ Waves to IHC Ca²⁺ Signals

When an ISC Ca²⁺ wave invaded the IHC region it typically elicited bursts of fCaTs in IHCs. The sizes of ISC Ca²⁺ waves correlated with the number of activated IHCs; large waves activated more IHCs that responded by synchronously generating bursts with an average delay of 0.52 s (Figure 10). The mechanism of coupling between ISCs and IHC APs is not entirely clear. One proposed mechanism is the regenerative efflux of ATP through connexin hemichannels into the endolymph and activation of P2X2 and P2X3 receptors (perhaps also P2X7) at the IHC (Anselmi et al., 2008; Majumder et al., 2010; Johnson et al., 2011; Mammano and Bortolozzi, 2018). As the ATP superfused from the pillar side evoked bursts in IHCs before eliciting Ca²⁺ transients in the ISCs (Figures 5D,E), a direct ATP activation of IHCs could possibly occur in vivo. However, the fact that IHC fCaT activity was unaffected in P2X2R^−/−/P2X3R^−/− and in P2X7^−/− mice indicates that neither of the receptors was essential for activating IHCs by incoming Ca²⁺ waves. Because IHCs from P2X4R^−/− mice produced normal Ca²⁺ APs (Sendin et al., 2014), an essential role of P2X4 receptors for IHC Ca²⁺ AP activity can be excluded, too. Recently, an alternative, ATP-independent mechanism of IHC activation has been proposed, in which the Ca²⁺ elevation in ISCs opens Ca²⁺-dependent Cl⁻ channels resulting in a coupled efflux of Cl⁻, K⁺ and water (Wang et al., 2015). The accumulation of K⁺ ions around the IHCs in turn depolarizes the IHC over the threshold of AP generation (Wang et al., 2015).

Does AP-Induced Extracellular K⁺ Accumulation Synchronize Neighboring IHCs?

We repeatedly observed that neighboring IHCs synchronously generated Ca²⁺ signals (from single fCaTs to bursts) in the absence of ISC Ca²⁺ waves (Figure 5A,B, 9E). Because AP activity of an IHC itself causes efflux of K⁺ ions (Marcotti et al., 2003a, 2004), a sizeable amount of K⁺ ions may have been able to depolarize neighboring IHCs over the threshold for generating Ca²⁺ APs. The basolateral part of an IHC is surrounded by specialized ISCs, the inner phalangeal cells, leaving a small extracellular volume in which K⁺ efflux from the IHC may lead to a substantial increase in the extracellular K⁺ concentration. Inner phalangeal cells around the IHCs normally remove K⁺ ions by means of Kir4.1 channels (Eckhard et al., 2012), but their uptake rate might be insufficient at times, leading to a series of fCaTs in an IHC and its neighbors.

Spontaneous Activity in the Immature Cochlea

The limited number of fCaTs in a burst (mean: 15.3 ± 9.5) and its limited duration suggest that a rising Ca²⁺ level probably activated SK2 channels, which in turn hyperpolarized the membrane potential of the IHCs and prevented further Ca²⁺ APs (Johnson et al., 2007), shaping a temporally structured pattern of Ca²⁺ APs (Johnson et al., 2011; Sendin et al., 2014). In vivo, AP patterning in the central auditory system is additionally shaped by inhibition of IHC Ca²⁺ AP activity through efferent cholinergic fibers acting via α9/α10 acetylcholine receptors coupling to SK2 channels (Glowatzki and Fuchs, 2000; Clause et al., 2014). Our data support the existence of two independent types of spontaneous activity in the developing auditory system, autonomous generation of Ca²⁺ APs in IHCs independent of ISC Ca²⁺ waves and the stochastic generation of Ca²⁺ transients in ISCs that elicit Ca²⁺ waves inducing synchronized bursts of IHC Ca²⁺ APs. The role of these different types of spontaneous activity for the firing patterns of spiral ganglion neurons with a functioning inhibitory efferent system in vivo needs to be established.

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