All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted at the University of Utah in accordance with NIH guidelines. In the present work we used the wild-type strain C57BL/6J and two transgenic mouse lines: a dual Cre-dependent reporter Polr2a-based GCaMP5G-IRES-tdTomato, referred to as PC::G5-tdT (Gee et al., 2014) and a Gad2-IRES-Cre knock-in mouse driver line referred to as Gad2::Cre (Taniguchi et al., 2011). Both breeding pairs were obtained from The Jackson Laboratory [Polr2a^{tm1(CAG−GCaMP5g, −tdTomato)Tvrd} http://jaxmice.jax.org/strain/024477; Gad2^tm2(cre)Zjh http://jaxmice.jax.org/strain/010802]. First generation heterozygous offspring were used in these studies and hereafter referred to as Gad2-G5-tdT. Pups were genotyped using real time PCR (probes: “Polr2a-3”, “GCamp3-1 Tg”, and “tdRFP”; Transnetyx, Inc., Cordova, TN).

Mice of either sex at early postnatal ages (P1-P15) and adult controls (P73-P533) were used in this study. For immunohistological preparations, membranous labyrinths were harvested and immersion fixed in 4% paraformaldehyde overnight at 4°C. Whole mount utricle neuroepithelia were micro dissected, including the removal of otoconia, using fine forceps in 0.1 M phosphate-buffered saline (PBS) (Dumont #5, #55; Leica, M165 FC).

Membranous labyrinths were micro-dissected from Gad2-G5-tdT temporal bones (P1-P15; and adults up to age P533), in cold glycerol-modified Ringer's (in mM: 26 NaHCO₃, 11 glucose, 250 glycerol, 2.5 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂ and 2.4 CaCl_2; pH 7.4) (Rabbitt et al., 2016). The utricle, horizontal and anterior cristae were further dissected to expose the apical surfaces of sensory neuroepithelia (Lim et al., 2011). Tissues were transferred to an imaging chamber (Warner Instruments; RC-22C) and continuously perfused with buffer (5.8 KCl, 144 NaCl, 0.9 MgCl₂, 1.3 CaCl₂, 0.7 NaH₂PO₄, 5.6 glucose, 10 HEPES, 300 mOsm, pH 7.4) at 21°C (Sadeghi et al., 2014). The recording chamber was placed under an upright swept field confocal microscope (Bruker; Prairie SFC), and imaged with a water immersion 60x objective (Olympus, LUMPLFN60XW). Confocal images were collected using a 35 μm slit aperture in linear galvanometer mode, and a 512 × 512 detector (Photometrics, Rolera MGi Plus EMCCD) providing in-plane single pixel size of 0.27 × 0.27 μm. For G5 detection and tdT expression imaging, 488 and 561 nm lasers, respectively, were interleaved for excitation, and a blocking filter (Semrock, R405/488/561/635-25) was used for detection. For G5 fluorescence calcium imaging, excitation was limited to 488 nm and the detection filter was replaced with a band pass filter (Semrock 525/50-25) to block any residual tdT emission at the 488 nm excitation wavelength. A pressure driven perfusion system configured with a micro-manifold (ALA Scientific Instruments, VC3-4PP, uflow-4) was used to continuously perfuse the tissue with control media, or 100 μM-1 mM ACh, 100 μM muscarine, 50 μM of the membrane permeable D-myo-inisitol trisphosphate receptor (IP₃R) antagonist 2-aminoethoxydiphenyl diphenylborinate (2-APB, Sigma), or 5 μM muscarinic cholinergic receptor antagonist atropine (ATR). A computer controlled micro-manifold system was used to rapidly switch between media, ACh, 2-APB or ATR. The SFC confocal, stage position (Scientifica), and perfusion system (ALA, VM4) were controlled and monitored at 10 kHz by custom software (WaveMetrics, Igor) via AD hardware (Heka, ITC-18), IEEE-488 instrumentation interface (National Instruments, GPIB-USB; Sony/Tektronix, AFG-320) and serial interface (USB).

G5 fluorescence was imaged at each “z” focal depth in a time sequence with images collected before, during, and after each ACh puff. Unless otherwise noted, image sequences consisted of 600–1,000 frames collected at 10 frames per second. The puff of each drug was delivered 10 s after initiating the sequence for a duration of 500 ms, unless otherwise noted. The image acquisition time sequence and ACh stimulus was repeated for each z focal depth (x,y,z) to generate 4D XYZT image stacks. Each xy image was smoothed in (x,y) space with a 3 pixel Gaussian filter (WaveMetrics, Igor Pro). To correct motion artifact, time-sequences at each focal plane were registered prior to analysis for full image rotation and translation (Thévenaz et al., 1998). G5 fluorescence modulation was determined pixel-by-pixel using ΔF/F_min, where ΔF = F(t)–F_min, and F_min was the minimum fluorescence intensity pixel-by-pixel over the entire time sequence of images. Image processing and pseudo-color images were generated in Igor Pro (WaveMetrics). Statistical significance of differences in the mean between groups was determined using Student's t-test with p = 0.05. All population data were presented as a mean ± standard error of the mean (SEM).

At the earliest age examined in this study, postnatal day 1 (P1), a unique population of developing HCs and SCs express the transgene tdTomato (tdT) (Figure 1). These tdT expressing cells map to the extrastriolar regions of the otolithic organs in all first generation heterozygous animals. A maximum intensity projection (MIP) from confocal images taken through seventy-microns of the utricle and saccule (1 μm per slice) shows tdT expressing cells primarily in the extrastriolar region of the utricle and saccule in Gad2-G5-tdT mice at P1 (Figure 1A; Olympus FV100, 40XW obj). Immunolabeling was performed with the SC marker, SOX2, and the developmental HC differentiation marker, ATOH1, to further identify the tdT expressing cell types at P1 in the utricle (Figure 1B). One of the distinguishing morphological features of HCs and SCs during early postnatal developmental stages are their mosaics, which coincide with the Delta-Notch signaling (Figures 1B,C). Immunolabeling with SOX2 antibody revealed a mosaic pattern with six SCs surrounding a single HC (white arrows) by age P1 in the utricle (Figure 1B, green). During this early postnatal age, the tdT expressing cells immunolabeled with ATOH1 (Figure 1B, blue), but not SOX2, suggesting that at P1 these cells constitute a more sensory hair cell function (Figure 1B, blue). By postnatal day 15 (P15), tdT expressing cells (Figure 1C, red) remained predominantly sensory HCs (Figure 1C, myosin VIIa blue). SCs immunolabeled with SOX2 also showed tdT fluorescence, but at a low level relative to HCs (Figure 1C). The extrastriolar HCs and SCs expressing tdT by the end of the first two postnatal weeks persisted throughout adulthood. This extrastriolar tdT cell population is illustrated in a low magnification (SFC, 10X air obj) image of live tissue excised from an adult mouse (Figure 1D, P78) (Figure 1D). At the oldest age examined in the present study, postnatal day 533 (P533), tdT expressing cells maintained their extrastriolar location in the utricle. A representative image fixed whole mount utricle is shown from 110 μm confocal z-stack MIP where an ROI is centered on the utricle striola (white outline; Figure 1E Olympus FV1000, 40XW obj). Cells with prevalent tdT expression have hair bundles, although not all hair bundles are visible in the surface preparation shown. Two main cell morphologies among these tdT expressing cells were observed in mature tissue; cells with a long neck and hair bundles typical of type extrastriolar I HCs (arrows; 7 cells in ROI shown; Figure 1E, right panel); and a small number of cells lacking the characteristic long neck. This small subset of cells was not positively identified in the present study, and could be atypical shaped type I HCs, type II HCs, or other cell types.

Spontaneous G5 ΔF/F₀ transients ([Ca²⁺]_i transients) were observed in tdT expressing HCs at P1 (n = 166 cells analyzed; k = 3 P1 mice; see Supplement Video 1). G5 transients were imaged in excised utricles, with the confocal focal plane imaging transversely through the epithelium (Figures 2A–D). The minimum G5 fluorescence over all images in the time sequence was used to define the pixel-by-pixel resting G5 fluorescence, shown within one imaging plane (F₀: Figure 2A; blue). After recording time sequences of G5 fluorescence modulation, tdT was imaged with 60 confocal planes to reconstruct reporter-expressing cells in Figure 3D. Cells expressing tdT in Figure 2A were identified as immature HCs based upon their morphology and immunolabeling (Figure 1). HCs are rendered as orthographic projections (Figure 2A i, ii) viewed perpendicular to the G5 confocal slice. HCs with high resting G5 fluorescence (Figure 2A, blue) also had abundant tdT expression (Figure 2A, red & white), indicating that resting G5 fluorescence in HCs primarily reflects expression of the reporter rather than high resting [Ca²⁺]_i in these cells. Three example HCs (1–3) exhibiting the largest spontaneous [Ca²⁺]_i transients in this tissue are rendered white to highlight their morphology and location (Figure 2A: i,ii). These same three HCs (Figure 2A: G5 F₀, blue) are outlined in the orthogonal cross-section (Figure 2A i,ii) with solid white outlines (Figure 2F; 1–3) to define regions of interest (ROIs) for fluorescence modulation analysis. Two additional modulating HCs are outlined with white dashed outlines in one optical focal plane (Figure 2A; 4–5), but not rendered white in the 3D stacks.

[Ca²⁺]_i transients were tracked in time by computing ΔF/F₀ pixel-by-pixel within outlined ROIs for each frame (1000 frames at 100 ms frame⁻¹). Figure 2B. shows the maximum ΔF/F₀ the over an example 100 s imaging sequence in this tissue. Of 35 HCs exhibiting resting G5 fluorescence in Figure 2 (F₀ blue), an average of 5.4 (±1.46) cells per 138 × 138 μm² focal plane exhibited spontaneous [Ca²⁺]_i transients (ΔF/F₀ >0.5) within each 100 s image acquisition sequence. In the present report, we use the term “spontaneous” in reference to events in the baseline control condition, distinct from stimulus evoked modulation relative to the baseline condition. It is important to acknowledge that baseline spontaneous events observed in excised tissue depend on history and condition of the preparation, which differ from physiological conditions the cell may experiences in vivo. Spontaneous [Ca²⁺]_i transients in utricular HCs excised from P1 mice were not synchronized with each other, and occurred in different cells at different times (see Supplement Video 1). G5 ΔF/F₀ transients were largest in magnitude near the plasma membrane, leading to a ring of increased ΔF/F₀ near the membrane when imaged in cross section (Figure 2B; 1,2,4,5 examples). Although only 16% of 166 P1 HCs examined with prominent tdT expression exhibited spontaneous transients with ΔF/F₀ > 0.5, 87% percent exhibited spontaneous transients at P1 with ΔF/F₀ > 0.2 in at least one image during the sequence, indicating relatively rapid but small [Ca²⁺]_i fluctuations were likely present in the majority of utricular HCs expressing G5 at birth. [Ca²⁺]_i transients lasting only one frame were likely due to events shorter than the 100 ms exposure time and too fast to be resolved by the kinetics of the G5 fluorescent signal used in the present study (Podor et al., 2015). Temporal events with a half width < 1 s (10 frames) were not analyzed in the present report.

Spontaneous calcium transients are shown in Figure 2C for the 5 specific HCs highlighted in Figures 2A,B. Waveforms provide the peak ΔF/F₀ within each outlined cell. Transients at birth consisted of small calcium increases, which we refer to as short “S” transients with characteristic waveforms (Figures 2C–F: 1S, 2S, 5S), large events with complex temporal waveforms (Figure 2C: 1L, 2L, 3L), and multiple medium size events falling between the two extremes. Other time sequences in this same tissue, and sequences from utricles excised from other P1 mice, also revealed 0–7 HCs with large and small spontaneous [Ca²⁺]_i transients within each 138 × 138 μm² image plane. Additional spontaneous transients are shown in Figure 2E, arranged front to back by maximum ΔF/F₀, with cells from panel C shown in the same colors. Spontaneous HC [Ca²⁺]_i transients rising above the background fluorescence fluctuation occurred with an average rate of 0.035 s⁻¹ (±0.026) in P1 mice. Small events had a rise time to peak of 0.659 s (±0.062), a half width of 1.26 s (±0.075), and a decay time of 3.80 s (±0.297). Large transients “L” emerged from the background with a steep slope, consistent with the rise of S events, and had multiple peaks and jagged waveforms consistent with the hypothesis that large events might have resulted from the summation of multiple small S events occurring at different times within each HC. If large transients indeed occurred due to a superposition or burst of S events (Figures 2E,F, 3G), the rate of spontaneous Q events would be predicted to have occurred at an average rate of 2.19 s⁻¹ (±2.74; range 0–4.6). [Ca²⁺]_i transients recorded in P1 HCs appeared to be “spontaneous”, and did not synchronize with each other in time. We were also unable to detect synchronization or modulation of HC [Ca²⁺]_i transients at P1 by ACh. This is illustrated by comparison of spontaneous transients (Figures 2E,F; independent experiments) to transients in after a 500 ms puff application of 100 μM ACh (Figure 2F) in the same tissue, where the time average ΔF/F₀ was not changed by ACh. Data during ACh application was blocked due to image distortion caused by the fluid motion. Although, previous reports suggest METs can be functional as early as embryonic development (Géléoc and Holt, 2003), we did not detect fluid jet evoked G5 fluorescence modulation at P1, suggesting HC MET currents in this preparation were either not functional or insufficient to evoke [Ca²⁺]_i transients detectable by the G5 indicator at P1.

At postnatal day P3, spontaneous [Ca²⁺]_i transients observed in HCs were largely superseded by a period of spontaneous [Ca²⁺]_i activity in cells with low resting G5 fluorescence consisting primarily of SCs (see Supplement Videos 2, 3). Figure 3A shows a single confocal slice cutting through HCs, SCs, and immature afferent calyces (AC). Using the same color format as Figure 2, the minimum fluorescence over the 100 s time sequence of images was used to define the resting fluorescence (Figures 3A,B, min F₀; blue), and the change in fluorescence (ΔF/F₀, green) was used to track changes in [Ca⁺²]_i, with Figures 3A,B showing the superposition of the two. The confocal optical slice was tilted relative to the epithelium in this tissue, cutting through the apical necks of hair cells in zone 1 (Figures 3A,B1), basolateral region in zone 2 (Figures 3A,B2), and below hair cells in zone 3 (Figures 3A,B3). Over 160 cells exhibited detectable G5 fluorescence in each 138 × 138 μm² image and, of these, 97 (±19.6) cells per focal plane exhibited G5 spontaneous calcium transients (874 events per experiment). SCs were easily identified in apical regions of live tissue based on their hexagonal spatial patterning around HCs (Figure 3B, also see Figure 1). Solid white lines in Figure 3B1 show HC ROIs with high resting G5 fluorescence (blue) while dotted white lines show SC ROIs with relatively low resting G5 fluorescence, but high ΔF/F₀ transients (green). Type I HCs were identified based on their relatively high G5 resting fluorescence (blue) and tdT expression (e.g., Figure 1), as well as their characteristic morphology. Although ΔF/F₀ transients were observed near the plasma membrane of some HCs, these events did not extend into HC somata. The spatial resolution was insufficient in the present imaging paradigm to determine if these localized events were in HCs or adjacent cells, Spontaneous G5 ΔF/F₀ transients were present at all focal depths, including below HCs. Although spontaneous G5 transients in the apical zone (A1,B1) were clearly localized in SCs at P3, SCs could not be positively distinguished from other key cell types at deeper focal planes, leaving open the possibility that some spontaneous transients might also be present in afferent neurons or calyces (ACs) at P3 as well.

In utricles excised from P3 mice, spontaneous transients were not synchronized with each other and occurred in different cells at different times (Figure 3C, Supplement Videos 2, 3). Transients were divided into 2 groups for statistical analysis based on kinetics of the G5 ΔF/F₀ temporal waveforms. Group 1 transients were distinguished by their relatively short duration (< 10s) relative to Group 2. A third set of ROIs (3) had mixed [Ca⁺²]_i transients characterized by an inflection point (Figure 3E, arrow), and possibly arising from fluorescence events from ΔF/F₀ reporting a combination of two events with different waveforms occurring within the same ROI or from distinct calcium kinetics (Taheri et al., 2017). Example events are shown in Figures 3D–F (left), aligned in time and arranged in increasing ΔF/F₀ magnitude. Population averages are shown on the right, and population statistics are summarized in Figure 3G. Short duration Group 1 transients (Figure 3C) had an average rise time of 1.96 s, half width of 4.31 s, decay of 6.00 s and peak ΔF/F₀ of 0.44 (data not shown). Long duration Group 2 transients had an average rise time of 2.76 s, half width of 11. 7 s, decay of 25.3 s and peak ΔF/F₀ of 0.40 (Figure 3G). Mixed (3) transients had an average rise time of 2.98 s, half width of 18.8 s, decay of 27.2 s (Figure 3G); and peak ΔF/F₀ of 0.42 consistent with a superposition of events from groups 1 and 2 (data not shown). Transients in each group occurred with similar ΔF/F₀ magnitude and could not be distinguished from each other based on ΔF/F₀ magnitude alone. Occurrence of long transients (56%) modestly outnumbered short transients (44%). There was no detectable correlation between any group and focal depth in tissue at P3 (B1–B3 depths). Group 1 events were more likely to reoccur during the imaging, sequence leading to a second peak in time-domain traces (Figure 3C, ^*). The average rate of spontaneous short events (Group 1) occurring within a single cell was 0.044 s⁻¹ (±0.043) and long events within a single cell was 0.016 s⁻¹ (±0.0035). Since ΔF/F₀ transients at the apical surface were clearly identified as SCs, the lack of correlation with focal depth suggests ΔF/F₀ transients might have been dominated by SCs at all focal depths through the epithelium in P3 aged mice (see Supplement Video 2). However, there were numerous exceptions at P3, based primarily on morphology, where spontaneous [Ca²⁺]_i transients likely occurred in other cell types including HCs, ACs, or other terminals at the base of HCs (see Supplement Videos 3,4).

We applied 50 μM 2-APB to block spontaneous [Ca²⁺]_i transients in SCs of P3-5 aged mice B (k = 3 mice). Example G5 F₀ and ΔF/F₀ are provided in Figure 4 in the control condition (A: resting F₀, gray and B: ΔF/F₀, green), blocked condition (C: ΔF/F₀, red), and after wash (D: ΔF/F₀, blue). G5 ΔF/F₀ transients were reduced more than 5 fold after application of the IP₃R antagonist 2-APB. G5 ΔF/F₀ images were merged (Figure 4E) to reveal specific cells that lost spontaneous [Ca²⁺]_i modulation after application of 2-APB. A vast majority of SCs had a complete cessation of ΔF/F₀ transients in the presence of the antagonist as evidenced by the near absence of overlap between the red and green fluorescent channels (Figure 4E, overlap = yellow). Although washout was incomplete, a subset of SCs clearly recovered spontaneous ΔF/F₀ transients (Figure 4E, recovery = blue-green, e.g., insets i & ii). SCs are arranged in canonical patterns of 6 around the necks of HCs, corresponding to the locations of SC ΔF/F₀ transients (Figure 4E, i). A subset of cells continued to show low-intensity spontaneous ΔF/F₀ transients in the presence of 50μM 2-APB (Figures 4C,E, red). Although the precise identity of these cells is uncertain due to their developmental age and the likelihood that they are actively undergoing differentiation, modulating ΔF/F₀ domains often localized with tdT positive HCs at their base (e.g., Figure 4E i & ii R), suggesting potential origins in other cell types, possibly HCs or developing ACs at this age. On average, the few [Ca²⁺]_i transients that remained in the presence of 50μM 2-APB were relatively small and slow compared with pre-treatment control conditions. Specific examples are shown in Figure 4F, with average transients for all cells in the control condition compared with 2-APB treated cells in Figure 4G. Averages in the control condition are shown for [Ca²⁺]_i transients lasting < 10 s (Group 1) and for transients lasting > than 10 s (Group 2). Population statistics comparing responses in the control condition vs. in the presence of 2-APB are provided in Figure 4H. Group 1 events in the control condition for mice tested with 2-APB (Figure 4, P3-P5) were slightly faster than Group 1 events at P3 (Figure 3), but this study did not determine if this was due differences in maturation or some other unidentified factor. Compared to Group 1 events in the control condition, [Ca²⁺]_i transients in the presence of 2-APB had a reduced peak ΔF/F₀ (0.44±0.21 vs. 0.11±0.023), increased decay time (5.99 ± 0.45 vs. 26.26 ± 2.84 s), with no statistically significant difference in rise time. Compared to Group 2 events in the control condition, [Ca²⁺]_i transients in the presence of 2-APB had a reduced peak of ΔF/F₀ (0.41 ± 0.24 vs. 0.11 ± 0.023), a reduced rise time (3.14 ± 0.43 vs. 1.56 ± 0.26 s), with no significant difference in half width or decay time. These results demonstrate that 2-APB interferes with spontaneous [Ca²⁺]_i transients, significantly reducing the magnitude of all events and reducing the number and kinetics of short duration [Ca²⁺]_i transients in multiple developing cell types. Antagonist action of 2-APB on IP₃R is sufficient to explain these results through disruption of CICR. However, the present experiments did not completely rule out other hypothetical [Ca²⁺]_i mechanisms, which is the focus of future studies.

At postnatal day P5, ΔF/F₀ [Ca²⁺]_i transients were similar to those observed in P3 mice (Figure 3), but significantly fewer in number, occurring in only 4.7 (±1.8) cells per 138 × 138 μm² focal plane. Figure 5 provides example data recorded at a single focal plane (Figures 5A–J), and with population statistics across animals (F-H: n = 75 cells; k = 3 mice). Using the same approach as earlier ages, spontaneous [Ca²⁺]_i ΔF/F₀ transients at P5 were grouped based on duration, with cells having ΔF/F₀ duration < 10 s placed in Group 1 and others placed in Group 2 (Figures 5E–H). Similar to P3 aged mice, these two groups at P5 had similar ΔF/F₀ peak magnitudes (Group 1 0.19 ± 0.04 vs. Group 2 0.20 ± 0.03; Figure 5F), but statistically significant differences in kinetics (Figures 5G–H). Compared to events in Group 1 transients in Group 2 had a slower rise time (1.32 ± 0.17 vs. 3.56 ± 0.49 s; Figure 5G), increased half width (4.59 ± 0.68 vs. 17.7 ± 1.75 s; data not shown), and longer decay times (8.88 ± 2.22 vs. 19.9 ± 1.79 s; Figure 5H). Smaller ΔF/F₀ events approaching the kinetics of G5 resolution occurred in some cells from both groups (C1, D2, arrows). Although the small events sometimes appeared before the large [Ca²⁺]_i transients, most large transients were not preceded by detectable small events suggesting the small [Ca²⁺]_i transients are not likely to be required precursors. Both types of [Ca²⁺]_i transients occurred spontaneously in the control condition, at random times in the imaging sequence. This is illustrated in Figure 5E where individual traces recorded in one focal plane show ΔF/F₀ transients from individual cells. Domains exhibiting spontaneous transients in utricles from P5 mice were often complex in morphology and contacted the basolateral membranes of HCs, similar to domains showing spontaneous transients near the base of HCs at earlier ages.

Although ACh application did not evoke G5 detectable [Ca²⁺]_i (ΔF/F₀) transients in utricles from P1-P3 aged mice, we hypothesized that ACh would begin to evoke G5 detectable [Ca²⁺]_i transients as calyces developed due to the presence of muscarinic AChRs (Holt et al., 2017). In utricular maculae excised from P5 aged mice, puff application of 100 μM ACh indeed synchronized the timing of ΔF/F₀ transients. Figure 5 compares spontaneous ΔF/F₀ [Ca²⁺]_i transients (Figure 5E) to ACh synchronized transients (Figure 5I) in the same cells for repeated application of ACh. Traces were collected in time sequences, repeated following a 5s delay at 4 sequential focal planes. Cells became refractory immediately following a calcium transient, requiring a period of time to recover before a subsequent transient could be evoked by ACh or occurred spontaneously. This is consistent with similar calcium transients observed in astrocytes (Taheri et al., 2017), and is likely due to the long time course of CICR dynamics and signaling. The refractory time during repeated stimuli accounts for the lack of “spontaneous” activity prior to ACh application in Figure 5I (ACh stimuli were repeated in 45s intervals). Synchronization by ACh is illustrated in the form of average response histograms (Figure 5J) where spontaneous transients occurred at random times relative to the onset of the imaging sequence (dotted gray), but became synchronized by application of ACh resulting in a peak probability of a ΔF/F₀ transient occurring at latency of 9.7 s (±7.1) after the ACh puff (Figure 5J, black).

ACh evoked transients became more pronounced in P10-14 mice. Figure 6 shows resting G5 fluorescence (A, blue), superimposed on peak G5 (ΔF/F₀) (C, green) recorded over an 80 s imaging sequence (100 ms frame⁻¹). For quantification, ΔF/F₀ [Ca²⁺]_i transients were recorded in 5 μm diameter ROIs in cells with complex morphological shapes (Figure 6A: “a” closed arrows. Figures 6B,C: 1-red, 2-green, 3-blue) and in simple morphological shapes (Figure 6A: “s” open arrows, Figure 6B: black). Spontaneous transients exhibited similar kinetics in all ROIs and, on average, had a rise time 1.28 s (±0.045), decay time of 1.98 s (±0.083), and half width 1.59 s (±0.075) (Figures 6B,H). A puff application of 100 μM ACh for 1s evoked distinctly longer lasting ΔF/F₀ transients in a subset of ROIs (Figure 6A: “a”. Figures 6C,D: 1-red, 2-green, 3-blue) which, on average, had a rise time 2.80 s (±1.45), decay time of 3.97 s (±1.60), and half width 3.37 s (±0.44). Population kinetics of ΔF/F₀ [Ca²⁺]_i transients in ATR insensitive cells (“s”) vs. ATR-sensitive cells are summarized in Figures 6H,I. The long lasting ACh-evoked transients were blocked by the muscarinic ACh receptor (mAChR) antagonist ATR (5 μM, Figures 6E,F), with residual small transients persisting in some ACh sensitive ROIs (Figure 6F, 1-3). Cells responding to ACh with long lasting calcium transients had morphological shapes consistent with calyceal afferent endings contacting type I hair cells. This is illustrated by comparing the morphology of tubulin labeled afferent endings (cyan) contacting tdT hair cells (red) from the same P14 tissue after fixation (Figure 6G, maximum intensity projection from a 62 μm z-stack). Present data suggest ACh activation of mAChRs on calyceal endings triggers large and long lasting CICR by P14 (Figure 6) that likely plays an important role in responses of mature calyces to ACh and efferent activation (Holt et al., 2017).

Intracellular calcium transients were also observed using G5 in utricles excised from adult mice (Figure 7). [Ca²⁺]_i transients recorded from a mature P533 aged mouse shows persistence of spontaneous ΔF/F₀ transients in control conditions recorded in 18 distinct cells in this tissue at a sampling rate of 100 ms-frame⁻¹. The average of these traces is shown as the solid black curve above the waterfall, and example cells are shown in inset images 1–3 (ΔF/F₀.: green, F₀ blue). Based strictly on morphology and contacts on HCs (blue) these modulating domains are likely to include calyceal endings near the base of HCs (1–2) and bouton terminals (3). The 18 cells in this tissue exhibited spontaneous transients at a rate of 0.37 s⁻¹ (±0.033). When the same cells were exposed to 100 μM ACh for 1s the rate of transients increased to 0.83 s⁻¹ (±0.091), (Figures 7B–D). ACh also evoked a slow increase in [Ca²⁺]_i evidenced by the slow increase of ΔF/F₀ not present in the control condition (Figure 7B: large gray arrows, black trace). In some cells ACh extended the spatial extent of the intracellular [Ca²⁺]_i transient relative to spontaneous events in the same cell in the control condition (Figure 7: A-3 vs. B-3), possibly due to the activation of CICR these cells. Population statistics in this aged utricle confirms that ACh evokes a significant increase in the rate of fast transients in addition to a slow increase in [Ca²⁺]_i (Figure 7C), but there was no detectable influence on kinetics of the fast [Ca²⁺]_i transients as measured by the rise time, half width, or ΔF/F₀ relative to controls (Figure 7D).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.