Animal work was performed in accordance with a protocol approved by the Italian Ministry of Health (Authorization n.1005/2016-PR, date 21/10/2016, DGSAF Prot. No. 002451-P-25/10/2016 and No. 0001276-P-19/01/2016).

Animals were bread and genotyped in the CNR Monterotondo node of the European Mouse Mutant Archive (EMMA; del Hierro et al., 2016), an ESFRI/INFRAFRONTIER Distributed Research Infrastructure⁷.

Mice tested were P5 pups and adults (1–3 months of age). Male and female homozygous (Panx1−/−), hemizygous (Panx1+/−) and their WT siblings (Panx1+/+) were used for this study. The background strains of these mice was C57BL/6N.

Panx1 mice were genotyped according to published protocols by standard PCR on extracted mouse tail tips (Anselmi et al., 2008; Bargiotas et al., 2011) using the following primers:

The Panx1+/+ allele was targeted by the above f and r primers and identified by a 330 bp band, whereas Panx1−/− was targeted by primers Panx1 f and LacZ, and was identified by a 630 bp band. Panx1+/− was identified by the simultaneous presence of a 330 bp and a 630 bp band.

For some internal control experiments, mice with ubiquitous deletion of connexin 30 (Cx30−/−; International strain name B6.129P2-Gjb6^tm1Kwi/Cnrm; EMMA ID: 00323; MGI ID: 2447863; Teubner et al., 2003; Cohen-Salmon et al., 2007; Schütz et al., 2010; Johnson et al., 2017) were also used. The background strains of these mice was C57BL/6J. Cx30−/− mice were genotyped as indicated in the protocol provided by INFRAFRONTIER:

Cx30+/+ mice were identified by a 544 bp band, whereas Cx30−/− mice were identified by a 460 bp band.

Following euthanasia, cochleae and brains were dissected from adult and P5 Panx1+/+ and Panx1−/− mice and flash-frozen in liquid nitrogen. Total RNA was extracted with RNeasy mini kit (Qiagen, Cat. No. 74106) and retrotranscribed with oligo–dT 12–18 primers (ThermoFisher, Cat. No. 18418012) using Omniscript RT kit (Qiagen, Cat. No. 205111). Subsequently RT-PCR was performed using PCR BIO HS Taq Mix Red (PB10.23–02). The PCR profile was: 95°C for 5 min, 94°C for 15 s, 62°C for 30 s, 72°C for 15 s and 72°C for 2 min for 34 cycles. Primers used were:

The amplified products were run on a 1, 5% agarose gel with SYBR Safe (ThermoFisher, Cat. No. S33102) for visualization of the Panx1 band (336 bp) and the GAPDH band (132 bp) (Figure 1).

QPCR was performed on cDNA to amplify Cx26 and Cx30 and was normalized to GAPDH. Gene expression relative to GAPDH was estimated according to a published method (Pfaffl, 2001). Amplification was carried out using Power SYBR Green (Applied Biosystems, Cat. No. 4367659) on the ABI 7700 sequence detection system equipped with ABI Prism 7700 SDS software (Applied Biosystems) through the following amplification cycles: 50°C for 2 min, 95°C for 10 min, 95°C for 15 min, 60°C for 1 min (40 cycles). Primers used are as follows:

Total proteins were extracted from brains and cochleae of P5 and adult Panx1+/+ (n = 4) and Panx1−/− mice (n = 4). Tissues were dissected, collected on liquid nitrogen, stored at −80°C and homogenized by using ice cold RIPA buffer (Pierce; 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% DOC, 1% Triton X-100, 0.1% SDS, and 1× protease, phosphatase-1, and phosphatase-2 inhibitor cocktails [Sigma]). The lysate was sonicated three times at 10 Hz (Hielscher, Ultrasound Technology UP50H/UP100H), centrifuged (13,000 rpm, 15 min, 4°C), and a 5 μl aliquot of the supernatant was assayed to determine the protein concentration (microBCA kit, Pierce). SDS-PAGE reducing sample buffer was added to the supernatant, and samples were heated to 95°C for 5 min. Protein lysates (70 μg) were loaded onto 12% Tris-glycine polyacrylamide gels for electrophoretic separation. ColorburstTM Electrophoresis markers (Sigma) were used as molecular mass standards. Proteins were then transferred onto nitrocellulose membranes at 100 V for 2 h at 4°C in transfer buffer containing 25 mM Tris, 192 mM glycine, 0.1% SDS and 20% methanol. Membranes were incubated for 1 h with blocking buffer (5% skim milk in TBST), and then incubated overnight at 4°C with primary antibodies directed against Panx1 (1 mg/ml, ThermoFisher, Cat. No. 487900), Cx26 (1 mg/ml, ThermoFisher, Cat. No. 512800), Cx30 (1 mg/ml, ThermoFisher, Cat. No. 712200) and GAPDH (1:2500, Abcam). After three 10 min rinses in TBST, membranes were incubated for 1 h at room temperature (22–25°C) with HRP-conjugated secondary antibodies (Cell Signaling, 1:2500). The membranes were then washed, and the bands were visualized with an enhanced chemiluminescence detection kit (GE Healthcare, UK). Protein expression was evaluated and documented by using a UVItec Cambridge Alliance system.

Cochleae were extracted from P5 and adult mice and processed as previously described (Crispino et al., 2011, 2017). Briefly, samples were fixed in 4% paraformaldehyde and decalcified in ethylenediaminetetraacetic acid (EDTA, 0.3 M). Specimens were included in 3% agarose dissolved in PBS and cut in 100 μm thickness steps using a vibratome (VT 1000 S, Leica). Tissue slices were permeabilized with 0.1% Triton X-100, dissolved in bovine serum albumin 2% solution. Panx1 was immunolabeled by overnight incubation at 4°C with a chicken anti-Panx1 specific antibody (1:500; extracellular loop epitope: VQQKSSLQSES, AvesLab #6358; Hanstein et al., 2013) provided by Prof. Eliana Scemes. Cx30 and Cx26 were immunolabeled by overnight incubation at 4°C respectively with a rabbit polyclonal Cx30 and a mouse monoclonal Cx26 selective antibody (10 μg/ml, ThermoFisher, Cat. No. 712200 for Cx30; Cat. No. 335800 for Cx26). Secondary antibodies (10 μg/ml) were: Alexa Fluor^® 488 goat anti-chicken IgY (H+L), ThermoFisher, Cat. No. A11039; Alexa Fluor^® 488 goat anti-rabbit IgG, ThermoFisher, Cat. No. A11008; Alexa Fluor^® 488 goat anti-mouse IgG, ThermoFisher, Cat. No. A11029), applied at room temperature (22–25°C). F–Actin was stained by incubation with AlexaFluor 568 phalloidin (1 U/ml, ThermoFisher, Cat. No. A12380), and nuclei were stained with 4′,6′diamidino–2′phenylindole (DAPI, ThermoFisher, Cat. No. D1306; 1:200). The same immunostaining procedure was used also for organotypic cultures from P5 pups. All samples were mounted onto glass slides with a mounting medium (FluorSaveTM Reagent, Merk, Cat. No. 345789) and analyzed using a confocal microscope (TCS SP5, Leica) equipped with an oil–immersion objective (40× HCX PL APO 1.25 N.A., Leica).

Auditory function was assessed in a sound-attenuating enclosure (ETS-Lindgren SD Test Enclosure, MDL Technologies Limited, Hitchin, UK) using an ABR Workstation (Tucker-Davis Technologies, Inc., Alachua, FL, USA) comprising: Z-Series 3-DSP Bioacoustic System w/Attenuators and Optic fiber; Medusa 4-Channel Pre-Amp/Digitizer; Medusa 4-Channel Low Imped. Headstage; MF1-M Multi Field Magnetic Speakers—Mono; AEP/OAE Software for RZ6; Experiment Control Workstation. Sound levels were calibrated using a 14 inch Free Field Measure Calibration Microphone Kit (Model 480C02; PCB).

Mice were anesthetized with intraperitoneal injections of ketamine (70 mg/g for males, 100 mg/g for females) and medetomidine (1 mg/g). The depth of anesthesia was periodically verified by the lack of foot-pinch response. Body temperature was maintained at 37°C using a heating pad under feedback control. Corneal drying was prevented by application of ophthalmic gel to the eyes of the animals.

For ABR recordings (Scimemi et al., 2014), acoustic stimuli consisted of clicks (100 μs duration) and tone bursts (1 ms rise–fall time with 3 ms plateau) of 4, 8, 16, 24 and 32 kHz, and were delivered in the free field using a MF1-M speaker. Bioelectrical potentials were collected with gauge 27, 13 mm needle electrodes (Cat. No. S83018-R9, Rochester) inserted subdermally at the vertex (active), ventrolateral to the left ear (reference) and above the tail (ground). Potentials were amplified, filtered (0.3–3 kHz) and averaged over 512 presentations of the same stimulus. Hearing threshold levels were determined offline as the SPL at which a Wave II peak, could be visually identified above the noise floor (0.1 μV).

Otoacoustic emissions (Kemp, 1978) were evoked using a pair of equal intensity primary tones, f₁ = 14,544 Hz, and f₂ = 17,440 kHz delivered at intensities ranging from 20 to 80 dB SPL in 10 dB SPL increments. Each primary tone (20.97 ms duration, 47/s) was emitted by a separate MF1-M speaker, configured for closed field stimulation, and delivered to the mouse ear via a small tube as prescribed by the manufacturer. The cubic distortion product 2f₁ – f₂ = 11,648 kHz, was detected using a small microphone (ER10B+ Low Noise Probe and Microphone, Etymotic Research, IL, USA) coupled to the ear canal.

Cochleae from P5 mouse pups were quickly dissected in ice-cold Hepes buffered (pH 7.2) HBSS (ThermoFisher, Cat. No. 14025050), placed onto 12 mm glass coverslips coated with Cell-Tak (Biocoat, Cat. No. 354240) and incubated overnight at 37°C in DMEM/F12 (ThermoFisher, Cat. No. 11320-074) supplemented with 5% FBS (ThermoFisher, Cat. No. 10270-106) and 100 μg/ml ampicillin (Sigma-Aldrich, Cat. No. A0166).

To visualize gap junction coupling among non-sensory cells of the lesser epithelial ridge (LER), we performed dye-transfer assays using the fluorescent tracer Lucifer Yellow (LY, CH Lithium Salt, Thermofisher, #L12926) for microinjection. Cochlear cultures were transferred on the stage of a spinning disk confocal microscope (DSU, Olympus) and perfused for 5 min at 1 ml/min with EXM, an extracellular solution containing (in mM): NaCl 135, KCl 5.8, CaCl2 1.3, NaH₂PO₄ 0.7, MgCl₂ 0.9, Hepes−NaOH 10, d−glucose 6, pyruvate 2, amino acids and vitamins (pH 7.48, 307 mOsm). For dye delivery, patch pipettes were fabricated from glass capillaries (G85150T-4, Harvard Apparatus, Edenbridge, UK) using a double stage vertical puller (PP-830, Narishige) and were filled with LY dissolved at 220 μM (final concentration) in a 320 mOsm intracellular solution containing (in mM): KCl 134, NaCl 4, MgCl₂ 1, HEPES 20, EGTA 10 (adjusted to pH 7.3 with KOH) and filtered through 0.22 μm pores (Millipore). Pipette resistances were 3–4 MOhm when immersed in the bath. One cell (donor) was patch clamped and maintained in the cell-attach configuration for a few seconds to establish a baseline. The patch of membrane under the pipette sealed to the donor cell was subsequently ruptured, allowing the LY to fill the cell, while leaving the seal intact (whole cell recording conditions). The cell was held at its zero current level using the current clamp configuration of the patch clamp amplifier (Axopatch 200B, Molecular devices). LY diffusion among (first order) cells adjacent to the injected cell was monitored over time by acquiring fluorescence images at a rate of 1 frame per second with a typical exposure time of 70 ms. Fluorescence images were displayed as (F − F_bck)/(F_max − F_bck), where F_max is the maximal value reached in the injected cell at the end of the recordin interval and F_bck is autofluorescence. Experiments were performed at room temperature (22–25°C).

To record spontaneous intercellular Ca²⁺ signal (ICS) activity in nonsensory cells of the mouse cochlea, organotypic cultures of sensory epithelium were incubated for 45 min at 37°C in DMEM/F12 supplemented with the acetoxymethyl (AM) ester of the selective Ca²⁺ sensor Fluo-Forte (16 μM, Enzo Life Science, #ENZ-52014). The incubation medium contains also pluronic F-127 (0.1% w/v, Sigma-Aldrich, #P2443) and sulfinpyrazone (250 μM, Sigma-Aldrich, #S9509) to prevent dye sequestration and secretion. Cultures were then transferred to an upright microscope stage (see below) and continually perfused with EXM (see above) for 15 min at 1 ml/min in a dark environment at 25°C to allow for dye de-esterification. All subsequent imaging experiments were also performed at 25°C.

To record Ca²⁺ signals, we used a two-photon microscope (Bergamo II, Thorlabs) equipped with a resonant scanner and coupled with a mode-locked Ti:Sapphire pulsed laser (Chameleon-Ultra II, Coherent). Fluo-Forte was excited at 940 nm by focusing the Ti:Sapphire beam onto the sample through a water-immersion objective (XLPlan N, 25× 1.05 NA, Olympus). Average power at the sample was ~20 mW. The fluorescence signal, collected by the same objective, was reflected towards the detection arm of the microscope by the 705 nm primary dichroic mirror of the microscope (Semrock, FF705-Di01), placed at 45° above the objective. After traversing a 680 nm short pass filter (FF01-680/SP-25, Semrock) and 495 nm dichroic beam-splitter (T495lpxru, Chroma Technology), the Fluo-Forte signal was selected in the range 435–485 nm by a single band-pass filter (ET460/50m-2p, Chroma Technology) placed in front of a non-descanned GaAsP detector (H7422–50, Hamamatsu). Mechanical ultra-fast shutters were used to limit light exposure to the bear minimum required for image acquisition.

Sequences of 512 × 512 pixels frames were acquired, averaged in lots of nine and presented at a final rate of 5 per second. Illumination intensity, frame average, frame rate and the number of pixels in each frame were adjusted so as to minimize photobleaching and phototoxicity, while achieving sufficient signal to noise ratio and temporal resolution. Image sequences were acquired using ThorImage LS 3.1 software (Thorlabs).

Ca²⁺ signals were quantified as pixel-by-pixel relative changes of fluorescence emission intensity, i.e., ΔF(t)/F₀ where t is time, F(t) is fluorescence at time t, F₀ is the fluorescence at the onset of the recording and ΔF(t) = F(t) − F₀. All data were processed off-line and presented using Vimmaging (F. Mammano and C. Ciubutaru, VIMM, Padova, Italy), a custom-made software routine developed under MATLAB™ environment (The MathWorks Inc., Natick, MA, USA).

Generation and genotyping of Panx1−/− mice were previously described (Anselmi et al., 2008; Bargiotas et al., 2011). Here we report an additional data set based on Panx1 expression analyses by RT-PCT and Western immunoblotting. Panx1 mRNA transcript expression was detected in the cochlea and brain of WT mice at both P5 and adult stage, but not in Panx1−/− mice (Figure 1). Consistent with these results, Western blots failed to reveal Panx1 expression in the cochlea of Panx1−/− mice at both P5 and in the adult stage, whereas bands with the correct molecular weight (~48 kDa) were present in Panx1+/+ (i.e., WT) extracts (Figure 2).

Using a previously validated antibody that targets an extracellular epitope of the Panx1 protein (AvesLab #6358; Hanstein et al., 2013), we detected strong immunoreactivity in epithelial cells lining the endolymphatic surface of the sensory epithelium (inner sulcus, outer sulcus), supporting and epithelial cells of the organ of Corti, Reissner’s membrane, and spiral ganglion neurons of WT mice. Weak immunostaining was also detected in the spiral limbus and spiral ligament of these mice, whereas the AvesLab #6358 antibody failed to label cochlear tissue from Panx1−/− mice (Figure 3). Altogether these results confirm successful ablation of Panx1 in the (brain and) cochlea of Panx1−/− mice.

Next, we sought to corroborate and extend the results obtained by Anselmi et al. (2008) by analyzing in greater detail the hearing performance of Panx1−/− mice. In humans and mice alike, sound-evoked ABR potentials appear as a series of consecutive relative maxima (peaks), termed Waves and labeled with Roman numerals, which arise from the synchronous short-latency synaptic activity of successive nuclei along the peripheral afferent auditory neural pathway (Zheng et al., 1999; Legatt, 2002; Zhou et al., 2006). The first peak (Wave I) arises from the cochlea and/or compound action potential of the auditory nerve ~1 ms after the stimulus onset (latency). Waves from II to V originate from cochlear nuclei, contralateral superior olivary complex, lateral lemniscus and contralateral lateral inferior colliculus. For reference, Table I of Scimemi et al. (2014) presents means and standard deviations (SD) of latency and amplitude values of ABR peaks I–V for C57BL/6 mice.

Hearing threshold estimates from click and pure-tone ABR analysis, as well as latency and amplitude of Wave I and Wave II in Panx1−/− mice aged between P30 and P90, were indistinguishable from those of age-matched Panx1+/− and WT control mice (Figures 4, 5, Tables 1–5).

Sound generated within the mammalian inner ear as a reflection of outer hair cell (OHC) mechanical activity (Nobili and Mammano, 1996; Nobili et al., 2003) can be detected with a sensitive microphone placed in the auditory meatus (Kemp, 1978, 2002). Therefore, as a further non-invasive indicator of cochlear function, we measured the cubic (2f₁ − f₂) DPOAE (see “Materials and Methods” section) and found no significant differences in the DPOAE growth function of Panx1−/− mice and age-matched WT controls (Figure 6).

Altogether, these results indicate absence of detectable defects in auditory function of Panx1−/− mice. This conclusion is based on their normal hearing sensitivity, normal function of the outer hair cell-based “cochlear amplifier” (Frolenkov et al., 1998; Nobili et al., 1998; Ashmore, 2008) and absence of cochlear nerve defects.

Pannexins bear significant sequence homology with the invertebrate gap junction proteins, innexins, and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins (Sosinsky et al., 2011).

Non-sensory cells of the mammalian cochlea express two closely related gap junction proteins, connexin 26 (Cx26) and connexin 30 (Cx30; Lautermann et al., 1998; Ahmad et al., 2003; Forge et al., 2003; Zhao et al., 2006), the expression of which is coordinately regulated (Ortolano et al., 2008). Mouse models indicate that altered expression levels of these connexins in the early postnatal days impacts on organ of Corti development and hair cell maturation (Johnson et al., 2017), preventing normal hearing acquisition (Cohen-Salmon et al., 2002; Teubner et al., 2003; Ahmad et al., 2007; Sun et al., 2009; Crispino et al., 2011, 2017; Qu et al., 2012; Zhu et al., 2013). As regulatory mechanism may potentially be shared between connexins and pannexins, we examined the expression of Cx26 and Cx30 by Western blot analysis and QPCR, and found no significant differences between Panx1−/− mice and age-matched WT controls (Figure 7). The spatial distribution of Cx26 at P5 was investigated also by immunofluorescence and, again, no differences between Panx1−/− mice and age-matched WT controls were detected (Figure 8).

To assess whether the expressed connexins confer cell-to-cell connectivity, we quantified dye transfer in the LER of organotypic cochlear cultures from P5 mice (see “Materials and Methods” section). To gauge transfer rate, we measured the slope of the LY fluorescence growth function at the onset of dye delivery in the donor cell (m₁) and in its nearest neighbors (m₂), and found no significant differences in the m₂/m₁ ratio of Panx1−/− mice and WT controls (Figure 9).

Altogether the results presented in Figures 7–9 indicate that lack of Panx1 in Panx1−/− mice impacts neither on connexins expression, nor on gap-junction coupling in the developing organ of Corti.

Connexins play a crucial development role in the postnatal cochlea (as mentioned above), also by supporting ICS activity both in the LER (Beltramello et al., 2005; Piazza et al., 2007; Anselmi et al., 2008; Majumder et al., 2010; Ceriani et al., 2016) and in the greater epithelial ridge (GER; Tritsch et al., 2007; Schütz et al., 2010; Rodriguez et al., 2012; Wang and Bergles, 2015; Johnson et al., 2017; Mammano and Bortolozzi, 2017). Using focal ATP delivery or photostimulation with caged IP₃, Anselmi et al. (2008) showed ICS failure in cultures with deficient expression of Cx26 and Cx30, whereas ICS in organotypic cultures from Panx1−/− mice was indistinguishable from those of WT controls.

Here, we used multiphoton microscopy to monitor spontaneous ICS activity (Tritsch et al., 2007; Schütz et al., 2010; Rodriguez et al., 2012; Wang and Bergles, 2015; Johnson et al., 2017; Mammano and Bortolozzi, 2017) in organotypic cochlear cultures from P5 mice loaded with the Ca²⁺ indicator Fluo Forte AM (Mammano and Bortolozzi, 2017). Specifically, we examined the frequency of occurrence of spontaneous Ca²⁺ transients (events) in the apical cochlear turn by counting all occurrences within the portion of the GER in the field view from P5 Panx1−/− mice and age–matched WT siblings (Figure 10). We found a similar mean frequencies of occurrence (15.5 ± 6.0 events/min in Panx1+/+ vs. 15.0 ± 3.4 events/min in Panx1−/− cultures). Likewise, amplitude and inter-peak interval distributions of spontaneous Ca²⁺ transients of Panx1−/− cultures overlapped with those of Panx1+/+ cultures. Altogether, these results indicate that spontaneous ICS activity in the GER of the postnatal cochlea is not affected by Panx1 ablation.