Cacna1d-eGFP^flex mice were generated within the CavNET consortium (EU-CAVNET MRTN-CT-2006-035367) by Katrin Bartels née Kunert, Kai Schönig and Dusan Bartsch, Central Institute of Mental Health, Mannheim, Germany (Satheesh et al., 2012). They were cross-bred with Cacna1d^-/- mice (Platzer et al., 2000) and Pax2::cre mice (Ohyama and Groves, 2004; Zuccotti et al., 2012). To reduce the risk of unwanted effects caused by Cre expression, only mice heterozygous for Pax2::cre were used (Jae Huh et al., 2010; Janbandhu et al., 2014). Animals were housed with free access to food and water at an average temperature of 22°C and a 12 h light-dark cycle. Mice of either sex were sacrificed by decapitation under isoflurane anesthesia and their cochleae were dissected from the temporal bones. All experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and approved by the regional board for scientific animal experiments of the Saarland, Germany. Additional ethics approval was not required according to the local and national guidelines.

The Pax2::cre allele was genotyped using primers detecting Cre: 5′-GCC TGC ATT ACC GGT CGA TGC AAC GA-3′ and 5′-GTG GCA GAT GGC GCG GCA ACA CCA TT-3′ (product size: 726 bp). The Cacna1d-eGFP^flex (“flex”) allele was identified using: 5′-TTC AAG GAC GAC GGC AAC TAC AAG-3′ and 5′-CGG CGG CGG TCA CGA ACT CC-3′ (product size: 380 bp). To exclude accidental occurrence of unwanted embryonal or germline recombination of the flex allele in pups without Cre, we regularly used the following primers: “Flex A” (5′-GGA GTT GTG TAT ATC TGT TAA GCC ATG-3′), “Flex B” (5′-GCT GTT GGG CTG AGA AGT TGG T-3′) and “Flex C” (5′-CCA GAA GAT TCC ACT AAA GGT CAT-3′), detecting wildtype (A-B band, ∼450 bp), intact flex (A-B band, ∼600 bp) and switched flex allele (B-C band, ∼700 bp) (Bartels, 2009). The Cacna1d^- (“Ca_v1.3^-”) allele was genotyped with the primers: “Ca_v1.3 sense (s)” (5′-GCA AAC TAT GCA AGA GGC ACC AGA-3′), “Ca_v1.3 antisense (as)” (5′-TAC TTC CAT TCC ACT ATA CTA ATG CAG GCT-3′) and “Ca_v1.3 neosense (ns)” (5′-TTC CAT TTG TCA CGT CCT GCA CCA-3′) yielding a wildtype (s-as, ∼300 bp) and/or a knockout band (s-ns, ∼450 bp).

Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were recorded in anesthetized mice aged 4–6 weeks as described in Fell et al. (2016). Growth functions of ABR waves I to IV in response to click stimuli were analyzed for peak-to-peak amplitudes and latencies between the click stimulus delivered at t = 0 and the time point of the negative peak of the respective wave.

Apical-turn organs of Corti were acutely dissected from young adult mice (P17–P23) in solution containing (in mM): 70 lactobionate⋅NaOH, 83 NaCl, 10 HEPES, 5.8 KCl, 5.3 glucose, 1.3 CaCl₂, 0.95 MgCl₂, 0.7 NaH₂PO₄. For Ba²⁺ current recordings, the bath solution contained (in mM): 72.5 lactobionate⋅NaOH, 40 NaCl, 35 TEA, 15 4-AP, 10 BaCl₂, 10 HEPES, 5.6 glucose, 0.9 MgCl₂. Both solutions were adjusted to pH 7.35, 320 mosmol kg^-1. Quartz pipettes were used and filled with (in mM): 112 Cs⁺ methane sulfonate, 20 CsCl, 10 Na⁺ phosphocreatine, 5 HEPES, 1 EGTA, 4 MgCl₂, 4 Na₂ATP, 0.3 GTP, 0.1 CaCl₂. Pipette solution was adjusted to pH 7.36, 305 mosmol kg^-1.

Before performing whole-cell patch clamp recordings using an Optopatch (Cairn Research, United Kingdom) or an Axopatch 200B amplifier (Molecular Devices, United States), green fluorescent protein (GFP) fluorescence of the specimen was assessed with an epifluorescence system consisting of a UV lamp and FITC fluorescence filters attached to the patch microscope (Olympus BX51WI with a 40 x water immersion objective, Germany) and a CCD camera (Scientifica, United Kingdom). Ba²⁺ currents were elicited by depolarizing the cells for 8 ms from –98 to +48 mV in 5 mV increments. Uncompensated series resistance was corrected by 70–80%. Analysis, including off-line linear leak subtraction and correction of the currents by subtracting the liquid junction potential of 8 mV, was performed using Igor Pro software (Wavemetrics, United States). I–V relations were calculated as the average current taken from the last ms of the voltage step as a function of the respective voltage.

I–V curves of Ba²⁺ currents were fitted to a second-order Boltzmann function times Goldman-Hodgkin-Katz driving force to determine parameters of activation, the voltage of half-maximum activation, V_h, and the voltage sensitivity of activation determined by the slope factor k, according to

where I is I_Ba at the time point the I–V was calculated (average over 7–8 ms after depolarization); P_max the maximum permeability; ν = zFV/(RT), with z being 2, F the Faraday constant, R the universal gas constant, T the absolute temperature, V the membrane potential. [Ba]_i (set at 50 nM) and [Ba]_o denote the intra- and extracellular Ba²⁺ concentration, respectively.

Immunolabeling was performed on whole-mount organs of Corti of 4–6 week-old mice as described in Fell et al. (2016) using Zamboni’s fixative for 8 min on ice. Specimens were labeled using antibodies against Ca_v1.3 (rabbit polyclonal, Alomone Labs, Israel, 1:500), BKα (rabbit polyclonal, Alomone Labs, Israel, 1:500; mouse monoclonal, antibodies-online, Germany, 1:500), GFP (goat polyclonal, Rockland, United States, 1:500), CtBP2/RIBEYE (mouse monoclonal, BD Transduction Laboratories, Germany, 1:100 – 1:200), SK2 (rabbit polyclonal, Sigma-Aldrich, Germany, 1:400), and calbindin (rabbit polyclonal, Swant Inc., Switzerland, 1:400). Primary antibodies were detected with Cy3-conjugated (donkey anti-rabbit: Jackson Immuno Research Laboratories, United Kingdom, 1:1500; donkey anti-goat: Abcam, United Kingdom, 1:1500) or Alexa 488-conjugated (anti-mouse: Invitrogen, United Kingdom, 1:500; anti-goat: Abcam, United Kingdom, 1:500) secondary antibodies. For immunolabeling experiments, at least three specimens of ≥3 animals were analyzed. z-stacks of fluorescence images were acquired using a confocal laser scanning microscope LSM710 (Carl Zeiss Microscopy GmbH, Germany). Images were analyzed with Fiji (Schindelin et al., 2012).

For quantification of Ca_v1.3 clusters and RIBEYE-positive ribbons, images of 67.5 μm × 38.9 μm size covering eight IHCs were acquired at equal laser and gain settings, and maximum intensity projections (MIPs) were calculated. The channel of interest of a MIP image was background subtracted. A thresholded binary image was created (0 below threshold; 1 above threshold) with thresholds of 10% of the maximum intensity of the green color channel (RIBEYE) and 17% of the red color channel (Ca_v1.3). Fluorescent dots < 0.05 μm² were discarded. Size and number of clusters were analyzed using the particle count routine in Fiji and normalized to one IHC.

Data are provided as mean ± SD, unless otherwise stated. Depending on the distribution of the data, Ba²⁺ current properties, as well as size and number of Ca_v1.3 clusters and ribbons were statistically analyzed using Student’s t-test or Mann–Whitney U test (MWU test; comparison of two groups) or using one-way ANOVA followed by Tukey post hoc test or Kruskal–Wallis test followed by Dunn-Holland-Wolfe post hoc test (comparison of > 2 groups) with Igor Pro software (WaveMetrics, United States) and SPSS statistics (IBM, Germany).

Statistical analysis of hearing measurements was performed with SPSS. Click ABR thresholds were analyzed using a one-way ANOVA, DPOAE amplitudes with a Kruskal–Wallis test and frequency-dependent ABR thresholds with a two-way ANOVA; all tests were followed by a Bonferroni post hoc test. ABR growth functions of amplitudes and latencies could not be tested by a two-way ANOVA due to unequal variances. Instead, a regression analysis was performed, and the parameters of the resulting regression lines (slope and y-axis intercept) were tested for differences using Student’s t-test or MWU test according to Sachs (1999).

In order to assess the phenotype of mice with cochlea-specific ablation of Cacna1d before birth, we analyzed GFP signals and whole-cell Ba²⁺ currents through Ca_v1.3 channels in IHCs of conditional knockout (cKO) Pax2::cre;Cacna1d-eGFP^flex/flex mice, in short cKO-Ca_v1.3^flex/flex. GFP fluorescence of two distinct intensity levels was present in IHCs of the apical cochlear turn acutely dissected from 3-week-old cKO-Ca_v1.3^flex/flex mice (Figure 1B) but not in wildtype IHCs (Figure 1A). Analysis of Ba²⁺ currents (I_Ba) using 10 mM Ba²⁺ as a charge carrier in response to 8 ms step depolarizations revealed lack of I_Ba exclusively in those IHCs with a strong GFP signal (Figure 1D, green trace). In contrast, I_Ba was present in one IHC of cKO-Ca_v1.3^flex/flex mice with weak (blue trace), one IHC without GFP fluorescence (gray trace, Figure 1D) and a wildtype IHC (Figure 1C). Corresponding individual peak I–V relations show that I_Ba was abolished in the IHC with strong GFP fluorescence whereas it was present in the two cKO-Ca_v1.3^flex/flex IHCs with weak or no GFP fluorescence and in the wildtype IHC (Figure 1E). Averaged peak I_Ba from cKO-Ca_v1.3^flex/flex IHCs with strong GFP (–7.9 ± 1.9 pA; n = 4) was significantly reduced compared with IHCs showing weak GFP fluorescence (–101.6 ± 14.2 pA; n = 3; P = 0.0262, MWU test, Figure 1F). We concluded that only those IHCs with strong fluorescence represented true knockout cells with two switched flex alleles whereas IHCs with weak fluorescence represented cells with one switched and one intact flex allele.

In order to increase the ratio of “true knockout” IHCs without remaining intact flex alleles, cKO-Ca_v1.3^flex/flex mice were crossbred with Ca_v1.3^-/- mice resulting in cKO-Ca_v1.3^flex/- mice, in which Cre needed to cut and switch only one flex allele per cell. Here, GFP fluorescence was uniform in IHCs of acutely dissected organs of Corti from 3-week-old cKO-Ca_v1.3^flex/- mice (Figure 2A). Typical I_Ba traces (Figure 2B,C) and corresponding I–V curves (Figure 2D) show that I_Ba was abolished in GFP-positive IHCs of cKO-Ca_v1.3^flex/- mice (Figure 2C) but retained in control IHCs of Ca_v1.3^flex/- mice (Figure 2B) where the flex allele was not switched due to absence of Cre. I–V curves further showed a reduction of I_Ba in control Ca_v1.3^flex/- (gray) compared with wildtype IHCs (black, Figure 2D). The average peak I_Ba from cKO-Ca_v1.3^flex/- IHCs was significantly reduced compared with control Ca_v1.3^flex/- IHCs (Figure 2E, bars; MWU test, P < 0.001). Peak I_Ba from individual cKO-Ca_v1.3^flex/flex IHCs demonstrate that the I_Ba amplitude of IHCs with strong GFP fluorescence (cf. Figure 1) resembled that of GFP-positive IHCs of cKO-Ca_v1.3^flex/- mice whereas I_Ba values of IHCs with weak or no GFP fluorescence were similar to those of Ca_v1.3^flex/- IHCs (Figure 2E, right). The lack of I_Ba was accompanied by a reduction in cell size as evident by a significantly reduced membrane capacitance (P < 0.001, Kruskal–Wallis Test) in cKO-Ca_v1.3^flex/- mice (6.7 ± 0.6 pF; n = 15; P = 0.001, effect of genotype) but not in control Ca_v1.3^flex/- mice (9.5 ± 1.4 pF; n = 10) compared with the wildtype (8.7 ± 1.0 pF; n = 10).

In summary, heterogeneous GFP expression and persistence of I_Ba in IHCs with weak GFP fluorescence of cKO-Ca_v1.3^flex/flex mice show that (i) Cre did not faithfully switch both flex alleles at 3 weeks of age and (ii) GFP fluorescence is no reliable marker for deletion of Ca_v1.3 channels in IHCs of cKO-Ca_v1.3^flex/flex mice. In contrast, in IHCs of cKO-Ca_v1.3^flex/- mice containing only one flex allele, GFP fluorescence unequivocally indicated a cellular knockout genotype.

In Ca_v1.3^-/- mice, mild degeneration of IHCs has been reported in the apical cochlear turn starting after P20 and in the basal cochlear turn after P35 (Platzer et al., 2000; Glueckert et al., 2003; Nemzou et al., 2006). Degeneration of IHCs after cochlea-specific deletion of Ca_v1.3 was analyzed in organs of Corti of 4–5 week-old cKO-Ca_v1.3^flex/flex and cKO-Ca_v1.3^flex/- compared with Ca_v1.3^-/- mice, which were double-immunolabeled for GFP and the hair-cell marker calbindin (Figure 3 and Table 1).

IHCs of all three genotypes showed mild IHC loss of ≤6.3% in the apical turn (Figure 3A–C and Table 1). In contrast, the majority (81.3%) of basal-turn IHCs of cKO-Ca_v1.3^flex/flex mice was missing (Figure 3E and Table 1). This pronounced degeneration was not caused by lack of Ca_v1.3 because basal-turn IHCs of Ca_v1.3^-/- and cKO-Ca_v1.3^flex/- mice did not show any degeneration (<0.5%; Figure 3D,F and Table 1). We conclude that high expression levels of GFP caused by two functional flex alleles in cKO-Ca_v1.3^flex/flex mice (cf. Figure 1A) resulted in a toxic effect of GFP on basal IHCs. The lack of IHC degeneration in the basal cochlea from cKO-Ca_v1.3^flex/- mice suggests a dose-dependent toxicity of GFP that requires more than one functional GFP allele.

The majority of outer hair cells (OHCs) from the apical (Figure 3A–C) but not basal cochlear turn (Figure 3D–F) were degenerated, as described before for Ca_v1.3^-/- mice (Platzer et al., 2000; Glueckert et al., 2003; Engel et al., 2006). Thus, cochlea-specific deletion of Ca_v1.3 channels coupled to GFP expression resulted in a similar degeneration of apical turn OHCs as observed in systemic Ca_v1.3^-/- mice.

The patterns of IHC GFP labeling in either cKO-Ca_v1.3^flex/flex (Figure 3B,E) or cKO-Ca_v1.3^flex/- mice (Figure 3C,F) were similar to the eGFP fluorescence patterns in acutely dissected organs of Corti from these genotypes (Figure 1A, 2A). GFP labeling intensity was heterogeneous between individual IHCs of cKO-Ca_v1.3^flex/flex mice with either intense or weak (arrows) labeling (Figure 3B,E) but uniform in IHCs of cKO-Ca_v1.3^flex/- mice (Figure 3C,F), with few individual IHCs not being labeled (Figure 3C, arrowheads).

The rate of true knockout IHCs was assessed by quantification of apical-turn organs of Corti immunolabeled for GFP, Ca_v1.3 and/or BK channels (Table 2). In cKO-Ca_v1.3^flex/- mice, the knockout rate was 89.2%, which was only slightly higher than the knockout rate of 87.4% in cKO-Ca_v1.3^flex/flex mice at 4–5 weeks of age. Although replacing one flex allele by a knockout (–) allele increased the success rate of Cre in switching one flex allele at 3 weeks of age (cf. Figure 1A,B, 2A), Cre caught up in switching both flex alleles in cKO-Ca_v1.3^flex/flex mice 2 weeks later.

In conclusion, the percentage of knockout IHCs finally was not increased by replacement of one flex allele with a knockout (–) allele to obtain cKO-Ca_v1.3^flex/- mice. However, we found that in cKO-Ca_v1.3^flex/flex mice (i) basal-turn IHCs degenerated, most likely due to dose-dependent GFP toxicity and (ii) GFP expression in IHCs was not unequivocally associated with deletion of Ca_v1.3 channels, thus leading us to further use cKO-Ca_v1.3^flex/- instead of cKO-Ca_v1.3^flex/flex mice.

In wildtype mice, up-regulation of BK K⁺ channels around the onset of hearing (P12) and down-regulation of neonatal SK2 K⁺ channels mark the end of terminal maturation and the onset of the mature function of IHCs (Figure 4A) (Kros et al., 1998; Marcotti et al., 2004). In systemic Ca_v1.3^-/- mice, IHCs maintain an immature-like ion channel composition with persistent expression of SK2 but lack of BK K⁺ channels (Brandt et al., 2003; Engel et al., 2006; Nemzou et al., 2006). The failure of acquiring a mature composition of K⁺ channels may have been caused by lack of Ca_v1.3 currents (i) in the IHC itself or (ii) in brainstem nuclei causing an altered efferent input on the IHC (Hirtz et al., 2011, 2012; Satheesh et al., 2012). Precise timing and patterning of Ca²⁺ action potentials generated by IHCs during a critical period before the onset of hearing are crucial for their maturation (Johnson et al., 2013a). Altered neuronal activity of the efferent input onto neonatal IHCs therefore might also affect their Ca²⁺ action potentials and hence their development.

SK2 immunolabeling was localized at the basolateral pole apart from synaptic ribbons (RIBEYE) of apical turn IHCs from 4 to 5 week-old cKO-Ca_v1.3^flex/- and Ca_v1.3^-/- mice (Figure 4C,D) indicating an immature phenotype. In contrast, no SK2 labeling was found at the basolateral pole of wildtype IHCs (Figure 4B).

BK channel expression was assessed in wildtype and Ca_v1.3^-/- IHCs co-labeled with calbindin (Figure 5A–C) and in Ca_v1.3^flex/- controls, cKO-Ca_v1.3^flex/flex and cKO-Ca_v1.3^flex/- IHCs co-labeled with GFP (Figure 5D–F). BK channels, which are indicators of a mature IHC phenotype, were present at the neck of wildtype IHCs (Figure 5A). In Ca_v1.3^-/- mice, BK labeling was absent from apical-turn IHCs (Figure 5B), whereas sparse and faint labeling was found in basal-turn IHCs (Figure 5C). In Ca_v1.3^flex/- control IHCs, normal BK labeling was found at the neck of IHCs (Figure 5D). In true cKO IHCs i.e., IHCs with strong GFP labeling in cKO-Ca_v1.3^flex/flex mice (Figure 5E) and with GFP labeling in cKO-Ca_v1.3^flex/- mice (Figure 5F), BK labeling was missing. Unexpectedly, BK immunolabeling in heterozygous IHCs of both cKO genotypes, i.e., IHCs with weak (cKO-Ca_v1.3^flex/flex, Figure 5E) or no GFP labeling (cKO-Ca_v1.3^flex/-, Figure 5F), which appeared as small dots at the neck, clearly differed from the large BK patches in control Ca_v1.3^flex/- (Figure 5D) or wildtype IHCs (Figure 5A). The GFP-negative IHCs of cKO-Ca_v1.3^flex/- mice containing a non-switched flex allele should, however, have the same phenotype and thus the same BK labeling pattern as IHCs of control Ca_v1.3^flex/- mice.

We noticed that expression of one or two flex alleles without Cre resulted in smaller IHC Ba²⁺ currents compared with wildtype IHCs (cf. Figure 1D,E, 2B–E). However, in conditional mouse lines, the function of the target gene should remain unaltered unless it is deleted or manipulated by Cre or other recombinases. For generating the conditional Cacna1d construct, loxP sites were inserted in intronic regions flanking exon 2 of the Cacna1d gene, which should not impair its function (Satheesh et al., 2012). To determine the side effect of the construct in the Cacna1d-eGFP^flex allele, we measured I_Ba in IHCs of mice with different combinations of wildtype (+), flex and knockout (-) alleles, i.e., Ca_v1.3^+/-, Ca_v1.3^+/flex, Ca_v1.3^flex/flex and Ca_v1.3^flex/- compared with wildtype mice (Figure 6). Averaged peak I_Ba amplitudes of IHCs from Ca_v1.3^flex/flex (–102.7 ± 16.4 pA; n = 10) and Ca_v1.3^flex/- mice (–89.5 ± 17.2 pA; n = 10) were significantly reduced compared with wildtype (–212.4 ± 48.2 pA; n = 10; P < 0.001, Kruskal–Wallis Test; Figure 6A). I_Ba normalized to the wildtype (100%) was reduced to 48.4% in Ca_v1.3^flex/flex and 42.1% in Ca_v1.3^flex/- IHCs, respectively (Figure 6B). In mice with only one wildtype (+) allele, I_Ba was slightly but not significantly reduced to –171.6 ± 50.9 pA or 80.9% (Ca_v1.3^+/-; n = 10) and –149.5 ± 36.5 pA or 70.4% (Ca_v1.3^+/flex; n = 7; Figure 6A,B), respectively. In contrast, I_Ba was reduced to 5.1% in IHCs of cKO-Ca_v1.3^flex/- mice indicating a complete loss of Ca_v1.3 channels leaving a small residual Ca²⁺ current that has been described before in the systemic knockout (Platzer et al., 2000; Brandt et al., 2003; Dou et al., 2004). Additionally, Cre expression in the cochlea did not affect I_Ba in IHCs of Pax2::cre control mice (–216.4 ± 53.1 pA; n = 10; Figure 6B). Analysis of gating properties by fitting the I–V curves to a second-order Boltzmann function times Goldman-Hodgkin-Katz driving force yielded a small but significant shift of V_h by –2.5 mV in Ca_v1.3^flex/- (–12.6 ± 2.0 mV; n = 10) versus wildtype IHCs (–10.1 ± 2.3 mV; n = 10; P = 0.019, MWU test), whereas the voltage sensitivity of activation determined by the slope factor k was unaffected (Ca_v1.3^flex/-: 11.22 ± 0.97 mV; wildtype: 11.26 ± 0.30 mV; P = 0.762, MWU test).

In summary, reduction of I_Ba amplitude in IHCs of control Ca_v1.3^flex/flex and Ca_v1.3^flex/- mice, as well as altered gating properties in Ca_v1.3^flex/- control IHCs demonstrate that the unswitched Cacna1d flex allele functionally does not fully replace the wildtype allele.

The functional reduction of Ca_v1.3 channels might be caused by a reduced amount of Ca_v1.3 channel protein in the IHC membrane or by a reduced function of Ca_v1.3 channels in Ca_v1.3^flex/- mice. The abundance of Ca_v1.3 channel protein was assessed by co-immunolabeling for Ca_v1.3 (magenta) and synaptic ribbons (RIBEYE, green, Figure 7). Ca_v1.3 clusters were localized at the synaptic ribbons of wildtype IHCs (Figure 7A,a) and at the majority of ribbons of IHCs from Ca_v1.3^flex/- control mice (Figure 7C,c). In contrast, no specific Ca_v1.3 labeling was found at the synapses of Ca_v1.3^-/- IHCs (Figure 7B,b) and most, but not all IHCs of cKO-Ca_v1.3^flex/- mice (Figure 7D,d′). In part of the IHCs from cKO-Ca_v1.3^flex/- mice, Ca_v1.3 labeling was still present at the synaptic ribbons (Figure 7D,d″) indicating that the flex allele was not switched in these cells. Synaptic ribbons (RIBEYE) of Ca_v1.3-deficient IHCs from Ca_v1.3^-/- and cKO-Ca_v1.3^flex/- mice were agglomerated and localized closer to the nucleus (Figure 7B,D) as described before (Nemzou et al., 2006).

To elucidate the cause of the reduced I_Ba amplitude (42% of wildtype, Figure 6A,B) in IHCs of Ca_v1.3^flex/- control mice, a quantitative analysis of the size and number of Ca_v1.3 clusters and synaptic ribbons was performed (Figure 7E–G). Whereas the number of Ca_v1.3 clusters and ribbons was unchanged, the average size of both Ca_v1.3 clusters and synaptic ribbons was significantly reduced to 73 and 89% in Ca_v1.3^flex/- control IHCs compared with wildtype (Figure 7F). This reduction in size also applied to the total area of Ca_v1.3 clusters to 75% and of ribbons to 83% of the total areas in wildtype, respectively (Figure 7G). In conclusion, less Ca_v1.3 protein was produced in IHCs of Ca_v1.3^flex/- mice evident by reduced I_Ba amplitudes and smaller Ca_v1.3 channel clusters, which was accompanied by smaller ribbons.

Next, we assessed how the loss/reduction of Ca_v1.3 channels affected the auditory function of cKO-Ca_v1.3^flex/- and control mice. In 4–6 week-old cKO-Ca_v1.3^flex/- mice, click-evoked ABR thresholds (Figure 8A) were absent (threshold > 100 dB SPL) in 4/3 ears/animals and significantly elevated in the remaining 12/7 out of 16/8 ears/animals (81.3 ± 8.0 dB SPL; P < 0.001) compared with wildtype mice (17.9 ± 6.7 dB SPL, 14/7 ears/animals). In contrast, click ABR thresholds were unaffected in all control groups (Pax2::cre: 13.8 ± 7.6 dB SPL, 16/8 ears/animals; Ca_v1.3^+/flex: 16.3 ± 4.3 dB SPL, 12/6 ears/animals; Ca_v1.3^flex/-: 20.4 ± 4.1 dB SPL; 14/7 ears/animals; P > 0.05; one-way ANOVA with Bonferroni post hoc test, Figure 8A). Frequency-dependent ABR (f-ABR) thresholds (Figure 8B) could only be measured in part of the cKO-Ca_v1.3^flex/- mice analyzed (16/8 ears/animals, single data points) and were thus not included in the statistical analysis. f-ABR thresholds of Pax2::cre (15/8 ears/animals) and Ca_v1.3^flex/- (14/7 ears/animals) significantly differed from wildtype mice (14/7 ears/animals; two-way ANOVA with Bonferroni post hoc test, effect of genotype: P < 0.001). Specifically, thresholds were increased in Ca_v1.3^flex/- control mice (P < 0.001), reaching significance at all frequencies except at 22.6 kHz; and slightly reduced in Pax2::cre mice (P = 0.001), reaching significance at 2 and 8 kHz.

Averaged ABR waveforms of Ca_v1.3^flex/- controls had smaller amplitudes than in wildtype mice for click stimuli 40 dB above threshold (Figure 8C). Growth functions of peak-to-peak amplitudes showed a significant reduction of all waves in Ca_v1.3^flex/- control (14/7 ears/animals) compared with wildtype (wave I, IV: 14/7 ears/animals; wave II, III: 12/6 ears/animals, Figure 8D), revealed by a regression analysis of the smaller slopes of fits to the amplitudes as a function of level above threshold of wave I to IV (wave I, P < 0.001; wave II, P = 0.015; wave III, P < 0.001; wave IV, P = 0.027; MWU test) and a smaller y-axis intercept value of wave I (MWU test, P = 0.002). Growth functions of latencies, calculated as time between stimulus application and the negative peak of the respective wave, were not significantly altered for all waves (I – IV) in Ca_v1.3^flex/- control mice compared with the wildtype (Figure 8E).

Finally, we tested the function of the cochlear amplifier including OHC electromotility by measuring DPOAEs (Figure 8F). Mean 2f1–f2 DPOAE maximum amplitudes averaged between 10 and 18 kHz in 0.5 kHz steps were strongly reduced in cKO-Ca_v1.3^flex/- mice (12/7 ears/animals, P < 0.001, Kruskal–Wallis Test, effect of genotype) but unaffected in all control groups (wildtype: 14/7 ears/animals; Pax2::cre: 16/8 ears/animals; Ca_v1.3^+/flex: 12/6 ears/animals; Ca_v1.3^flex/-: 14/7 ears/animals).

In summary, cochlea-specific deletion of Ca_v1.3 in cKO-Ca_v1.3^flex/- mice resulted in highly elevated ABR thresholds and strongly reduced DPOAEs, reflecting profound hearing loss. Moreover, in Ca_v1.3^flex/- control mice the reduction of mean IHC I_Ba amplitude to 42% and of mean Ca_v1.3 cluster size to 73% is accompanied by increases in f-ABR thresholds up to 10 - 20 dB and strongly reduced amplitudes of ABR waves I to IV. Notably, click-ABR thresholds and DPOAEs were not affected in Ca_v1.3^flex/- control mice.

Satheesh et al. (2012) were the first to analyze a conditional Ca_v1.3 knockout mouse model with deletion in the auditory brainstem, the Egr2::cre;Cacna1d-eGFP^flex/flex mouse. There, eGFP fluorescence was not detectable in unfixed brain tissue (Bartels, 2009), most likely due to the much lower abundance of eGFP/Ca_v1.3 in these brainstem nuclei compared with IHCs. In the present study, the flex allele had the advantage that successful deletion of Ca_v1.3 in IHCs resulted in eGFP fluorescence that could be judged semi-quantitatively at the cellular level. Moreover, the presence of individual IHCs where the flex allele was not switched enabled us to use these cells as positive controls, e.g., for Ca_v1.3 immunolabeling within one specimen.

An unwanted side effect of transgenic animals is unexpected germline expression of Cre recombinase resulting in embryonal recombination of loxP sites that might even occur in Cre-negative offspring carrying a flex or lox allele (Song and Palmiter, 2018). This can be monitored (i) by adapting the genotyping protocol to recognize excised or switched lox or flex alleles and (ii) in the flex switch system as eGFP expression in cells of Cre-negative flex control mice with one or two flex alleles.

Without the GFP reporter function, we might not have detected the incomplete recombination of flex alleles in IHCs. Partial recombination of floxed alleles, resulting in a mixture of cells with recombination of both, one or even no allele, is a frequent problem in conditional knockout mice (Saam and Gordon, 1999; Schulz et al., 2007; Weis et al., 2010). Lack of knowledge about the amount of successful cellular deletion events may lead to wrong conclusions caused by residual functions contributed by non-knockout cells. In this study, about 10% of the IHCs in cKO-Ca_v1.3^flex/- mice carried an unswitched flex allele resulting in residual hearing function compared with complete deafness of Ca_v1.3^-/- mice (Platzer et al., 2000; Dou et al., 2004).

Our attempt to increase the success rate of Cre in switching the flex alleles by replacing one flex by a constitutive knockout (“-”) allele resulted in a higher ratio of true knockout IHCs in cKO-Ca_v1.3^flex/- compared with cKO-Ca_v1.3^flex/flex mice (cf. Figure 1, 2) at 3 weeks of age. However, this difference was no longer present 2 weeks later (Table 2), indicating that Cre managed to switch most flex alleles by this time point. An alternative approach to increase the recombination rate of loxP sites would be to increase expression of Cre recombinase (Schnütgen et al., 2003; Schulz et al., 2007) using Pax2::cre/cre instead of Pax2::cre/+ mice. However, high Cre expression levels on the other hand might increase the risk of possible side effects. High levels of Cre expression in α-myosin heavy chain-Cre mice have for example been demonstrated to be cardiotoxic causing altered cardiac function, DNA damage and inflammation (Bhandary and Robbins, 2015; Pugach et al., 2015). A dose dependence of Cre toxicity has been confirmed in cell culture titration experiments (Loonstra et al., 2001; Baba et al., 2005).

In summary, due to ambiguous eGFP expression in cKO-Ca_v1.3^flex/flex mice when only one flex allele was switched resulting in heterozygous IHCs that still produced Ca_v1.3, we decided to further use cKO-Ca_v1.3^flex/- mice, where eGFP expression was a reliable marker of Ca_v1.3 ablation. Since incomplete recombination of both flex (or lox) alleles is likely to be a general problem in conditional mice, a combination with a systemic knockout allele (flex/– or lox/–) should be used if possible.

Degeneration of IHCs in the basal cochlear turn as early as P25 in cKO-Ca_v1.3^flex/flex but not Ca_v1.3^-/- mice suggests toxicity of excessive eGFP. Furthermore, direct fluorescence of eGFP can be seen in non-fixed IHCs (this study) but not in the auditory brainstem (Bartels, 2009), further indicating particularly high expression of Ca_v1.3 in wildtype and eGFP in cKO-Ca_v1.3^flex/flex IHCs, respectively. GFP toxicity has been demonstrated in cell lines where its expression induced apoptosis (Liu et al., 1999) or inhibited polyubiquitination (Baens et al., 2006). In mice, neuronal expression of yellow fluorescent protein induced multiple dose-dependent stress responses (Comley et al., 2011). A possible cause for these damaging effects is that the pre-mature, colorless form of eGFP, which is present in variable proportions in GFP-expressing cells, produces the free radical O2^●- and hydrogen peroxide (H₂O₂) under consumption of NAD(P)H (Ganini et al., 2017). Such a GFP-induced oxidative stress may explain why only IHCs of cKO-Ca_v1.3^flex/flex mice but not of cKO-Ca_v1.3^flex/- degenerated because of the higher dose of eGFP produced by two flex alleles.

In conditional models, the modifications of the target gene should not affect its function unless recombined by Cre. In the study of Satheesh et al. (2012), who first described the Ca_v1.3-flex model, Ca²⁺ currents were not analyzed. Normal ABR thresholds of Ca_v1.3^flex/flex control mice led the authors to the conclusion that unswitched flex alleles did not affect Ca_v1.3 channel function. Since Ca_v1.3 channels mediate the majority of Ca²⁺ current in IHCs, the present study provided the unique opportunity to analyze potential side effects of the flex construct in detail by measuring Ca²⁺ channel currents and quantitatively analyzing Ca_v1.3 protein clusters. We found reduced I_Ba amplitudes and a lower amount of Ca_v1.3 protein in IHCs of cre-negative Ca_v1.3^flex/flex and Ca_v1.3^flex/- control mice demonstrating that the unswitched flex allele did not fully replace the wildtype function. As has been shown before, a considerable reduction or increase in peak Ca²⁺ current amplitudes of IHCs has only minor effects on click ABR thresholds (Scharinger et al., 2015; Fell et al., 2016), which can be misleading when used as the only method to assess the function of IHCs. Here, frequency-dependent ABR thresholds were increased by 10–20 dB at most frequencies in Ca_v1.3^flex/- control mice upon reduction of I_Ba to 42% of the wildtype value, which is in accordance to threshold increases of 5 – 20 dB in null mutants of the auxiliary α₂δ2 Ca²⁺ channel subunit causing reductions of I_Ba to 60–70% (Fell et al., 2016). The most prominent consequence of I_Ba reduction with respect to hearing function of Ca_v1.3^flex/- control mice are the reduced growth functions of peak-to-peak amplitudes of the ABR waves, especially wave I, indicating strongly reduced IHC output at all levels above threshold. It should be kept in mind that both click and frequency-specific ABR thresholds are determined by only one afferent fiber type, the low threshold, high spontaneous rate fibers, whereas growth functions of ABR amplitudes cover the activity of all (high, medium and low spontaneous rate) afferent fiber types (Kiang et al., 1965; Liberman, 1978, 1982; Petitpré et al., 2018; Shrestha et al., 2018; Sun et al., 2018).

The question arises as to why I_Ba was reduced in IHCs of Cre-negative mice containing at least one flex allele (Figure 6). For generating the conditional Ca_v1.3 mouse, the Cacna1d-eGFP^flex construct was placed outside and a few hundred base pairs up- and downstream of the coding regions of exon 2 in the Cacna1d gene to avoid unintended manipulation of regulatory elements flanking the exon (Bartels, 2009). This insertion might have disrupted unknown regulatory elements and thus reduced the expression level of the channel. Moreover, I_Ba gating properties were altered in Ca_v1.3^flex/- control mice, suggesting that insertion of the flex construct might have affected splicing of Cacna1d mRNA (Bock et al., 2011; Scharinger et al., 2015).

The ablation of Ca_v1.3 channels before birth in cKO-Ca_v1.3^flex/- mice caused an IHC phenotype similar to that of Ca_v1.3^-/- mice, including persistent expression of SK2, lack of BK expression in apical-turn IHCs, and a reduced cell size (Brandt et al., 2003; Glueckert et al., 2003; Nemzou et al., 2006).

Until the onset of hearing, SK2 channels mediate efferent inhibition of IHCs via α9α10 Ca²⁺-permeable nicotinic acetylcholine receptors (nAChRs) (Oliver et al., 2000; Elgoyhen et al., 2001), The origin of these efferent fibers lies in cholinergic neurons in the SOC. Shortly after birth, neurons of the auditory brainstem are spontaneously active and undergo a developmental program including synaptic pruning and establishment of tonotopy (Blankenship and Feller, 2010; Clause et al., 2014). The spiking pattern of SOC neurons is modulated by ascending information from the cochlea, where IHCs produce spontaneous Ca²⁺ action potentials, which are synchronized by Ca²⁺ waves in the transient Kölliker’s organ (Tritsch and Bergles, 2010; Johnson et al., 2011, 2017; Sendin et al., 2014; Eckrich et al., 2018; Mammano and Bortolozzi, 2018). In turn, efferent inhibition from the SOC closes a feedback loop by shaping the spontaneous activity of IHCs (Guinan, 2006; Frank and Goodrich, 2018). In the systemic Ca_v1.3 knockout mouse both SOC neurons and IHCs lack Ca_v1.3 currents (Platzer et al., 2000; Hirtz et al., 2011), the latter of which as a consequence cannot produce action potentials (Brandt et al., 2003). In the SOC of Ca_v1.3^-/- mice, depolarization-induced spiking of lateral superior olive (LSO) neurons was changed from a single to a multiple firing pattern due to a reduction in K_v1.2 channels (Hirtz et al., 2011). This was most likely caused by the specific lack of Ca_v1.3 channels in brainstem neurons despite intact cochlear expression as confirmed in brainstem-specific Ca_v1.3 knockout mice (Satheesh et al., 2012). In the present study with SOC neurons expressing Ca_v1.3 channels, the phenotype of Ca_v1.3-deficient IHCs from cKO-Ca_v1.3^flex/- mice (lack of BK channels, persistence of SK2 channels, smaller cell size) was very similar to that of systemic Ca_v1.3^-/- mice. Therefore a potentially altered feedback signaling by Ca_v1.3-deficient SOC neurons onto IHCs cannot be causative for the IHC phenotype of Ca_v1.3^-/- mice. Nevertheless, the spiking pattern of SOC neurons and, thus, efferent signaling back to immature IHCs might still be altered due to the loss of afferent activation by IHCs. In α9- and α10-nAChR knockout mice, maturation of IHC K⁺ channels was normal despite the complete lack of cholinergic efferent input from SOC neurons (Gomez-Casati et al., 2009; Johnson et al., 2013b). In summary, maturation of the IHC’s K⁺ channel composition is mainly controlled by intrinsic Ca²⁺ signaling within the IHC and does not depend on Ca_v1.3 expression in the SOC exerting efferent feedback.

We found residual BK labeling in IHCs of the basal but not the apical cochlear turn of Ca_v1.3^-/- mice. So far it is unknown why BK protein is missing in IHCs of Ca_v1.3^-/- mice along most of the cochlear length (Brandt et al., 2003) despite expression of the respective Kcnma1 mRNA (Nemzou et al., 2006).

The faint and dot-like BK labeling in GFP-negative IHCs of cKO-Ca_v1.3^flex/- mice clearly differed from the large BK patches found in IHCs of wildtype and control Ca_v1.3^flex/- mice (Figure 5F). Assuming that Cre is not active in these IHCs their phenotype should be the same as that of Ca_v1.3^flex/- controls (Figure 5D) but this was not the case. We can thus exclude that the reduced BK expression was caused by the incomplete wildtype function of the unswitched flex allele. But what are the differences between IHCs of Ca_v1.3^flex/- controls and GFP-negative IHCs of cKO-Ca_v1.3^flex/- mice? Differences intrinsic to the IHCs are: (i) Presence of the Pax2::cre allele at an unknown location in the genome, which might interfere with modulatory sequences affecting BK expression; (ii) Cre might be expressed in these IHCs without switching the flex allele, but it could still interfere with BK expression. Alternatively, a factor extrinsic to the IHC might be causing the reduced BK expression. GFP-negative IHCs of cKO-Ca_v1.3^flex/- mice are surrounded by Ca_v1.3-deficient, electrically silent IHCs, whereas neonatal IHCs produce Ca²⁺ action potentials in wildtype and presumably Ca_v1.3^flex/- mice (Brandt et al., 2003). This activity causes periodic efflux of K⁺ ions from the IHCs, which depolarizes neighboring phalangeal cells and IHCs, thereby amplifying and synchronizing Ca²⁺ AP activity (Wang et al., 2015; Eckrich et al., 2018). In summary, impaired expression of BK channels in solitary GFP-negative IHCs surrounded by true Ca_v1.3 knockout IHCs of cKO-Ca_v1.3^flex/- mice may result from a lack of mutual activation and synchronization of Ca²⁺ AP activity among IHCs during the critical developmental period. It would be interesting to analyze whether the Ca²⁺ action potential activity in GFP-negative IHCs of cKO-Ca_v1.3^flex/- is altered compared to Ca_v1.3^flex/- controls.