Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.Lyme disease, caused by  B. burgdorferi, the major cause of Lyme disease in the U.S., joint disease. The basis for these differences is unknown, but likely involve strain-specific interactions with host components in the target tissue. The sequence of the outer surface lipoprotein OspC varies with the strains, and we found that this variation influences the spectrum of host extracellular matrix components recognized. Infection of mice with strains that are identical except for ospC revealed that OspC variants that differ in binding spectrum promote infection of different tissues. A strain producing OspC invaded and colonized the joint in living animals, but an altered OspC protein incapable of binding tissue components did not. Thus, tissue-binding by OspC is critical for infection and joint invasion, and OspC variation directly influences tissue tropism.Infection by different Lyme disease bacteria is associated with different manifestations, such as cardiac, neurologic, or, in the case of  Borrelia  that arS. alone . An initectively –7.Borrelia spp. are highly adhesive and produce at least 19 outer surface proteins that recognize host cells or extracellular matrix (ECM) ..N40-D10/ studies , 43, enhB. burgdorferi adhesins in the tissue invasion process, an emerging model is that movement of the spirochete from the bloodstream into tissue is a multistep process mediated by a series of adhesive events [B. burgdorferi outer membrane protein P66 is essential for joint invasion [Combined with previous work defining the role of several e events , 12. Thee events , 76, 77.invasion . Here weInvasion of mammalian leukocytes into tissues during the inflammatory process features several parallels, as it is well-documented to occur via a multi-step process involving counterreceptors for both lectins and integrins , 79. GivAll mouse experiments were performed in strict accordance with all provisions of the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the PHS Policy on Humane Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Medical College of Wisconsin, Tufts University School of Medicine, Wadsworth Center-New York State Department of Health, or University of Calgary. All efforts were made to minimize animal suffering.Cd1d-/- mice in a BALB/c background (Jax #2962) were bred in-house at the Clara Christie Centre for Mouse Genomics at the University of Calgary. Mice of both genders between 6–8 weeks of age were used.Female C3H/HeN or C3H/HeN-SCID mice were acquired from Charles River Laboratories  and used at 4 wks of age for long term intradermal inoculation studies, or 8 wks of age or older for short term intravenous (i.v) inoculation experiments. For intravital microscopy B. burgdorferi strains were grown in Barbour-Stoenner-Kelly (BSKII) medium made in-house [8 spirochetes/ml. When antibiotic selection was required, kanamycin was used at 200 μg/ml and gentamicin was used at 40 μg/ml. Presence of genomic plasmids and shuttle vectors was confirmed in each culture by PCR prior to inoculation into mice , generated previously [spC alleles from various strains and species of Borrelia from the ospC promoter and ribosome binding site from strain B31-A3, or constitutively from the flagellar promoter (flaB) and ribosome binding site native to each ospC allele [ospCB31 under control of the OspCB31-A3 promoter, 200bp upstream of the start codon of ospCB31-A3 with the coding sequences of ospCB31 were cloned into pBSV2G, resulting in pOspCB31 [ospCN40-D10/E9, ospCPBr, or ospCB31-ECM- under control of the ospCB31-A3 promoter, ospC alleles were amplified using primers . Amplified DNA fragments were inserted into the TA cloning vector, pGEM-T Easy (Promega corp.). To generate pGEM-T Easy encoding OspCB31-ECM-, site-directed, ligase-independent mutagenesis (SLIM) was used to replace lysines-116, 121, 123, 128, and 129 of OspC from B. burgdorferi strain B31-A3 with methionines on pCR2.1 TOPO encoding OspCB31-A3 as described [ospCN40-D10/E9 or ospCPBr, ospCB31-ECM-, were then digested with SalI and BamHI and ospC alleles were subcloned into pBSV2G at the SalI and BamHI sites [ospC from B. burgdorferi B31-A3, 184 bp to 9 bp upstream of the start codon of ospC [N40-D10/E9, pOspCPBr, and pOspCB31-ECM-. Note that pOspCB31 differs from pOspCB31-ECM- (as well as from pOspCN40-D10/E9 and pOspCPBr) not only in the ospC coding sequence, but also in the eight nucleotides just 5’ to the ospC ATG start codon. We thus also generated pOspCB31 #2 with the same nucleotides 5’ to the ospC ATG start codon . ospC alleles containing the native ribosome binding sites  were amplified from genomic DNA using the oligonucleotides described in ospC alleles were cloned in pGEM-T Easy . Plasmid pTM61spc-MCS was generated by annealing oMCS3 and oMCS4 and ligating the fragment into pGEM-T Easy. The MCS was excised from pGEM-T Easy  using BspHI and EarI, and ligated in pTM61spc [B. burgdorferi strain B31-A3 genomic DNA using P-FlaB oligonucleotides , and cloned downstream of the flaB promoter in pTM61spc-MCS  [ospC alleles under the control of the constitutive flagellar promoter (PflaB) in pTM61spc-MCS as previously described [ospC promoter in pBSV2G (ospC/pTM61-OspCB31(PospC)) and GCB4452 (B31-A3ΔospC/pTM61-OspCB31-ECM-(PospC)) with ospC under the control of the ospC promoter in pTM61, the wild type (ospCB31) and mutated (ospCB31-ECM-) genes were first sub-cloned in pJET1.2 . To do so, both genes were amplified by using OspCB31fp/pTM61 and OspCB31rp/pTM61, including restriction sites for SbfI and AvrII respectively (ospCB31 under the control of the ospC promoter was pOspCB31(PospC), while ospCB31-ECM- controlled by the ospC promoter was amplified from pOspCB31-ECM-(PospC). Each insert was ligated into pJET1.2.Plasmids pTX4 (pJET::ospCB31), pTX5 (pJET::ospCB31-ECM-) and pTM61 were double digested with the restriction enzymes SbfI and AvrII, acquired from NEB. Digestions were run in a 1% agarose gel and the bands corresponding to pTM61 (6.7 Kb), ospCB31 (817bp) and ospCB31-ECM- (817bp) were gel purified. Ligations were performed to obtain plasmids pMC114 (pTM61::ospCB31) and pMC115 (pTM61::ospCB31-ECM-). The resulting constructs were sequenced with oligos OspCB31fp/pTM61 and OspCB31rp/pTM61 to confirm their integrity. Strains GCE3815 (ospCB31) and GCE3817 (ospCB31-ECM-) were grown overnight in LB to purify the respective plasmids pMC114 and pMC115 by midiprep . 150 μg of plasmids pMC114 and pMC115 were incubated individually with the GpC methyltransferase M.CviPI according to NEB protocols. After precipitation with isopropanol followed by 70% ethanol, 50 μg of each plasmid was transformed into electrocompetent strain B31-A3ΔospC (GCB3022). Candidate transformants were screened for the presence of the gentamicin cassette and the ospC insert, either wild type (ospCB31) or ECM mutant (ospCB31-ECM-). They were also tested for green fluorescence (GFP harbored in pTM61). Finally, the plasmid profiles were analyzed as reported [ospC version lost lp28-4 and lp56. However, these plasmids have not been shownto affect infectivity [ospC/pTM61-OspCB31(PospC)) and B31-A3ΔospC/pTM61-OspCB31-ECM-(PospC) (GCB4452).To generate GFP-producing (OspCK1)  was engiescribed  or undern pBSV2G . To builectively . The DNA ospCB31 7bp and oectivity . The finE. coli were isolated using the QIAGEN Plasmid Maxi Kit  and transformed into B. burgdorferi by electroporation of 50 μg of CpG methylated DNA and plating into semi-solid agar or in liquid in 96-well plates as previously described [ospC alleles were found to have the correct sequence when carried on pBSV2G or pTM61 in B. burgdorferi with the exception of the ospC allele from B. burgdorferi strain N40-D10/E9, which was found to encode the single missense mutation N46S after multiple independent PCR amplifications [Plasmid constructs in escribed , 86. Allications . The quaospC open reading frames lacking the putative cleaved signal sequences from B. burgdorferi strains B31-A3, 297, and N40-D10/E9, B. garinii strain PBr, B. afzelii strain VS461, and an altered open reading frame encoding OspCB31-ECM-  were amplified (E. coli strain BL21(DE3) and the plasmid inserts were sequenced (Tufts core sequencing facility). The GST-tagged OspC variants were produced and purified by glutathione chromatography according to the manufacturer’s instructions .To generate recombinant GST-tagged OspC proteins, the mplified  and clon2CO3, pH 9.0). One hundred microliters of increasing concentrations  of GST (negative control) or GST-tagged OspC variants, including OspCB31, OspC297, OspCN40-D10/E9, OspCVS461, OspCPBr, or OspCB31-ECM- were added to the coated wells and incubated for 1 hr at ambient temperature. To detect the binding of GST-tagged proteins, goat anti-GST  and HRP-conjugated donkey anti-goat IgG  were used as primary and secondary antibodies, respectively. The plates were washed three times with PBST (0.05% Tween20 in PBS buffer), and 100 μl of tetramethyl benzidine (TMB) solution  was added to each well. The reaction was stopped by adding 100 μl of 0.5% hydrosulfuric acid to each well and plates were read at 405 nm using a Synergy HT ELISA plate reader . To determine the dissociation constant (KD), the data were fit to the following equation using KaleidaGraph software .Quantitative ELISA for fibronectin and dermatan sulfate binding by OspC proteins was performed as previously described  with the3COONa, pH 4.0) was flowed through a CM5 chip  whereas 10 μg of dermatan sulfate in 10 μl of PBS buffer was biotinylated as described [B31, OspC297, OspCN40-D10/E9, OspCVS461, OspCPBr, or OspCB31-ECM-, were injected into the control cell followed by the flow cell with immobilized fibronectin or dermatan sulfate at 10 μl/min, 25°C. To obtain the kinetic parameters of the interaction, mean sensogram data were fit to a curve using BIAevaluation software version 3.0  with the one step biomolecular association reaction model (1:1 Langmuir model), resulting in optimum mathematical fit with the lowest Chi square values.Interactions of OspC with fibronectin or dermatan sulfate were analyzed by SPR using the Biacore 3000 . To conjugate SPR chips with fibronectin or dermatan sulfate, 10 μg of fibronectin in 10 μl of acetate buffer . After 1 hr at 37°C, unbound bacteria were removed by washing with PBS containing 0.2% BSA. Plates were air-dried, and bound bacteria in each well were quantified by liquid scintillation. The percent of bound bacteria was calculated by normalizing the counts of each well to the counts in the inoculum.Binding of escribed . Briefly4B. burgdorferi strain B31-A3/pBSV2G, an ospC-deficient B. burgdorferi strain B31-A3ΔospC/pBSV2G, or B31-A3ΔospC carrying a plasmid encoding ospCB31, ospCN40-D10/E9, ospCPBr, or ospCB31-ECM- driven by the B31-A3 ospC promoter . At 24 hrs post spirochete injection, vascular transmigration was scored in the knee to a depth of about 150 microns in the living mice by intravital microscopy using a spinning disk laser confocal microscope. The area exposed was over the patellar ligament and to the medial side of the right hind limb. We examined post-capillary venules that drain blood from the anterior tibialis muscle and joint with the anterior tibial vein that drains into the great saphenous vein (see [4 pixels). For spirochete clearance assays on mice used for intravital microscopy, blood was withdrawn from mice at 5 and 60 minutes post-inoculation. Blood cells were allowed to settle overnight in a heparinized capillary tube and spirochetes in the plasma were directly counted by dark-field microscopy [For vascular transmigration assays, the deeply anesthetized eviously . A GFP-pein  strain B31 (“Bb B31”), 297 (“Bb 297”), and N40-D10/E9 (“Bb N40-D10/E9”), B. afzelii strain VS461 (“Ba VS461”), and B. garinii strain PBr (“Bg PBr”) using BioEditor Sequence Alignment Editor [B31 lysines that were mutated to methionines to abrogate ECM binding, resulting in OspCB31-ECM-. The fragment of OspC from N40-D10/E9 selected for binding to joint and heart by phage display [ClustalW alignment performed on protein sequences of OspC from t Editor . At each display  is under(TIF)Click here for additional data file.S2 FigECM- mapped onto the crystal structure of OspCB31 [ospC alleles. “*” indicates residues mutated in OspCB31-ECM-. (C) Far-UV CD analysis of OspCB31 and OspCB31-ECM-. The molar ellipticity, Φ, was measured from 190 to 250 nm for 10 μM of each protein in Tris buffer (pH7.5). (D) The indicated concentrations of GST, GST-OspCB31, or GST-OspCB31-ECM- were added to quadruplicate wells coated with human C4b, and binding (± standard deviation) was measured by ELISA . Shown is a representative of three independent experiments. The KD values obtained from the average of three independent experiments were calculated and shown in the inset.(A) The location of the quintuple basic residues  mutated in OspC OspCB31 , 92. Hel(TIF)Click here for additional data file.S3 Fig(A) Flow cytometry analysis of OspC localized to the surface of parental strain B. burgdorferi B31-A3/pBSV2G , ospC deletion strain B31-A3ΔospC/pBSV2G , and the ospC deletion strain bearing a plasmid encoding OspCB31 . (B) OspC (top) or flagellin (bottom) on the surface of the indicated untreated (solid bars) or methanol-permeabilized (open bars) strains was quantitated by flow cytometry after staining with anti-OspC or anti-flagellin, respectively (see B. burgdorferi strain B31-A3 harboring the empty vector. Each bar represents the mean of four independent determinations ± SEM. (*): indicates that surface production of the indicated proteins was significantly lower  than the detected production of OspC or Flagellin by B. burgdorferi strain B31-A3 harboring the vector.vely see . Values (TIF)Click here for additional data file.S4 Fig(A) C3H/HeN mice infected with 104B. burgdorferi strain B31-A3/pBSV2G (“B31-A3/Vector”), ospC deletion strain B31-A3ΔospC/pBSV2G (“Vector”), or the deletion strain bearing a plasmid encoding either OspCB31-ECM- or OspCB31 #2, which differ only in the ospC coding sequence, were sacrificed at 21 days post infection. The bacterial loads at the inoculation site, ear, bladder, heart, knee, and ankle joint were determined by qPCR. Data shown are the geometric mean of bacterial loads ± 95% confidence interval of 10 mice per group. Statistical significance was determined using ANOVA with the Kruskal-Wallis test followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli. Significant (p < 0.05) differences in spirochete number relative to the ospC deletion strain (“*”) and between two strains relative to each other (“+”) are indicated. (B) Joint diameter of the mice infected with 104 of B. burgdorferi strain B31-A3/pBSV2G (“B31-A3/Vector”), ospC deletion strain B31-A3ΔospC/pBSV2G (“Vector”), or the deletion strain bearing a plasmid encoding either OspCB31-ECM- or OspCB31 #2 was measured by caliper on day 21 post infection. Data shown are the median of the diameter of ankle joints ± the range of these values in five mice per group. Statistical significance was determined using ANOVA with the Kruskal-Wallis test followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli. Significant (p < 0.05) differences in the average diameter of ankle joint relative to the ospC deletion strain (“*”) and between two strains relative to each other (“+”) are indicated.(TIF)Click here for additional data file.S5 Fig4B. burgdorferi strain B31-A3/pBSV2G (“B31-A3/Vector”), ospC deletion strain B31-A3ΔospC/pBSV2G (“Vector”), or the ospC deletion strain bearing a plasmid encoding the indicated OspC variants sacrificed at 21 days post infection. Shown are the geometric mean of antibody titers ± 95% confidence interval of 10 mice per group. Statistical significance was determined using ANOVA with the Kruskal-Wallis test followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli. Significant (p < 0.05) differences in the antibody titers relative to the ospC deletion strain (“*”) and between two strains relative to each other (“+”) are indicated.Serum titers of IgG (top panel) and IgM (bottom panel) of C3H/HeN mice infected with 10(TIF)Click here for additional data file.S6 Fig4 WT B. burgdorferi strain B31-A3 carrying the empty vector ("B31-A3/Vector"), B31-A3ΔospC carrying the empty vector ("Vector"), B31-A3ΔospC exogenously producing OspC from B. burgdorferi strain B31-A3 ("B31"), N40 clone D10/E9 ("N40-D10/E9"), or B. garinii strain PBr ("PBr") under control of the ospC promoter from B31-A3. Tissues were harvested at 21 days post infection, and bacterial genomes were quantified in each tissue by qPCR. Shown are the geometric mean of bacterial loads ± 95% confidence interval of 10 mice per group. Statistical significance was determined using ANOVA with the Kruskal-Wallis test followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli. Significant (p < 0.05) differences in spirochete number relative to the ospC deletion strain (“*”) and between two strains relative to each other (“+”) are indicated.C3H/HeN-SCID mice were intradermally inoculated with 1x10(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Cochlear implants (CI) are the treatment of choice in profoundly deaf patients. Measuring the electrically evoked compound action potential (ECAP) has become an important tool for verifying the function of the spiral ganglion neurons (SGN), which are the target cells of the CI stimulation. ECAP measurement is only possible after electrode insertion. No information about the neuronal health status is available before cochlear implantation. We investigated possible correlations between the ECAP amplitude growth function (AGF) slope and anamnestic parameters to identify possible predictors for SGN health status and therefore for CI outcome. The study included patients being implanted with various electrode array lengths. Correlation analysis was performed for the mean AGF slope of the whole array, for separate electrodes as well as for grouped electrodes of the apical, medial, and basal region, with duration of deafness, age at implantation, residual hearing (grouped for electrode length), and etiology. The mean ECAP AGF slopes decreased from apical to basal. They were not correlated to the length of the electrode array or any etiology. For the mean of the full array or when grouped for the apical, middle, and basal part, the ECAP AGF slope was negatively correlated to the duration of hearing loss and the age at implantation. Since a significant negative correlation of the ECAP AGF slope and age at cochlear implantation and duration of deafness was observed, this study supports the statement that early implantation of a CI is recommended for sensorineural hearing loss. Additional factors such as the cochlear coverage and insertion angle influence the ECAP AGF slope and performance of the patient and should be included in future multifactorial analysis to study predictive parameters for the CI outcome. Worldwide, 466 million people suffer from disabling hearing loss with negative effects in social, emotional, and economic capacity due to a limited ability of communication . PatientIn contrast to animal models, histological information on SGN number and electrophysiological data in humans are rare. It is known that, as in animal models ,12, in hIn CI users, it became possible to record the ECAP via the inserted electrode array , and thiAt our clinic, ECAP measurements are routinely performed intra- as well as postoperatively to receive information on the electrode–nerve interface. In this retrospective analysis, the postop measurements are used, which were recorded via the clinical software MAESTRO  using the ART (Auditory Nerve Response Telemetry) task. Within the ART task, “Alternating Polarity” is used as artifact reduction paradigm per default. Two stimuli are applied consecutively, one with the anodic leading phase and another with the cathodic leading phase. By averaging the responses of these two pulses, the artifact can be reduced . In addiRecording parameters like number of steps, number of averages, minimum and maximum stimulation level, and pulse duration were chosen differently depending on the aim of the measurement. To investigate the electrode–nerve interface, a few measurement points are sufficient. To calculate an ECAP threshold, more points are necessary to establish a regression line through the linear region of the AGF. Each recording was reviewed with respect to ECAP responses. The linear regression line, automatically generated by the clinical software MAESTRO, was corrected to fit data points within the linear region, as shown in the examples of In MED-EL CI implant devices, electrode array lengths are 16, 20, 24, 28, and 31.5 mm. All arrays were inserted to their full length. Current level and charge level are approximately equal to SI units: 1 cu ≈ 1 µA, 1 qu ≈ 1 nC.Whole array: electrodes 1–12Apical region: electrodes 1–3Medial region: electrodes 4–9Basal region: electrodes 10–12Single apical electrode no. 1Single apical electrode no. 2Single apical electrode no. 3Since the electrode arrays consisted of 12 electrode contacts, the postoperative ECAP measurement dataset can have a maximum of 12 measurements. There are patients where all 12 electrodes were stimulated successfully. However, there are also patients in whom the ECAP AGF slope of some electrodes is documented as not measurable  or not available (which means no measurement was made on this electrode for unknown reasons). Those missing values were either very conservatively replaced by single imputation of the worst possible value 0 , or excluded from analysis  . ECAP slThis retrospective study investigated whether the ECAP AGF slope, and therefore presumably the function of SGN, correlates with data that are available before cochlear implantation: the duration of deafness, the age at implantation, the etiology, or the residual hearing. For all variables, the data from 139 patients were analyzed.All necessary data were selected from the patient medical files and the CI database at Hannover Medical School and supplemented with the available measurements. The influencing factors were determined as follows:The duration of deafness is expressed in years and measured from the time of deafness to the date of surgery. Patients deaf for less than one year were set to 0.5 years. All remaining time periods were rounded down to half year steps. There were patients for whom no exact date of deafness was given, because they could not give or did not know the exact date of deafness, or because they had some residual hearing before surgery and were therefore not considered deaf. In these cases, the date when the patient was considered hard of hearing was used for calculation.Age at implantation is indicated in years. The period from birth to implantation is given. Rounding up and down was the same as for the duration of deafness.In some cases, the age at implantation can be determined more exactly than the duration of deafness, since the latter is often a subjective parameter and should therefore be considered less strongly when judging on anamnestic parameters.unknown (N = 39)not documented (N = 39)acute (N = 50)progressive (N = 11)and regarding the cause of the hearing loss:unknown (N = 7)not documented (N = 40)sudden idiopathic hearing loss, acute hearing loss (N = 42)infection (N = 12)syndrome, hereditary (N = 15)trauma, ototoxins (N = 23)presbyacusis (N = 0)The influencing factor etiology was classified regarding the occurrence:The etiology and mean ECAP AFG slope of all electrodes were plotted  to decidTo determine the residual hearing, all available pure tone audiograms of each individual patient were examined, and the audiogram, which was created as close as possible to the date of the first fitting, was taken. The frequencies 125, 250, 500, and 1000 Hz were evaluated for the respective hearing loss (dB SPL), whereas the higher frequencies  were not taken into account, since hearing was lost in these frequency regions; 750 Hz was not included into the analysis, since not every audiogram included this measurement. To detect the frequency-specific acoustic hearing loss, air conduction hearing loss was measured using a calibrated audiometer according to DIN EN 60318 as previously described in detail .Analysis: SPSS  and GraphPad Prism  were used for analysis. Data were checked for normal distribution using D’Agostino–Pearson Test, and ECAP AGF slopes of grouped and single electrodes were compared using Mann–Whitney test. Pearson correlation was subsequently applied. A model was created in which univariate linear regressions were performed followed by examination of all significant variables in a common model by multivariate analysis. The analysis was performed with the data set of missing values being set to zero  as well as with a data set where missing values were excluded .Boxplots show the median, quartiles, and extreme values. The box represents the interquartile (IQ) range, which contains the middle 50% of the records. The whiskers are lines that extend from the upper and lower edge of the box to the highest and lowest values, which are not greater than 1.5 times the IQ range. The line across the box indicates the median. Outliers are cases with values between 1.5 and 3 times the IQ range, i.e., beyond the whiskers and marked with a small circle. Extremes are cases with values more than three times the IQ range and marked with a star.2r was calculated. The factors duration of deafness, age at implantation, etiology grouped by occurrence, and etiology grouped by cause were correlated with the mean ECAP AGF slope values of electrodes covering different regions of the cochlea.In the univariate analysis, the ECAP AGF slope was defined as the dependent variable and the respective influencing factor as an independent variable. Each influencing factor was examined individually in a regression model to the significance level of 5%. Pearsons squared correlation coefficient To describe the differences between patients with residual hearing in the low-frequency  region and patients without any residual hearing, ECAP AGF slopes of the apical electrodes 1–3 were included into the analysis. The mean ECAP AGF slope of electrodes 1–3 was correlated with the hearing loss values of the frequencies 125, 250, 500, and 1000 Hz. Additionally, the frequency specific hearing loss was correlated with each single electrodes  ECAP AGF slope.All linear regressions of the residual hearing were made separately for each electrode array length. Since there are only two patients for the 16 mm implant model, it was omitted. Finally, a multivariate regression was used to determine whether all significant analyses correlate.p < 0.001 for both pairings). Additionally, the slopes measured on the basal electrodes were significantly reduced compared to the medial electrodes (p < 0.001).The mean ECAP AGF slope of all patients electrodes for analysis A = missing values set to 0 and analysis B = missing values excluded are 31.10 µV/nC and 37.26 µV/nC, respectively. Grouped for electrodes 1–3, 4–9, and 10–12, the mean ECAP AGF slopes are 46.52 µV/nC, 29.77 µV/nC, and 18.32 µV/nC , and electrode 10, 11, and 12 below 25 µV/nC . The hearing loss at 125, 250, 500, and 1000 Hz was correlated (Pearson correlation) to the mean slope values of the separate electrodes 1, 2, and 3, and to the mean of electrodes 1, 2, and 3. Only few of these 128 performed analyses have shown significant correlations, which are listed in In a multivariate analysis of all significant factors in a common model, the factors duration of deafness and age at implantation have a strong negative correlation to the mean ECAP AGF slope of all electrodes, but after multivariate analysis, the age at implantation is the only significant factor affecting the ECAP AGF slope .p = 0.036) but not for the duration of deafness (p = 0.077).The multivariate analysis of the mean ECAP AGF slopes of electrodes 1–3 was performed for dataset B but not for data set A, because for the latter, only age at implantation but not duration of deafness is significantly correlated to the respective slopes . The mulFor electrodes 4–9, the duration of deafness, and the age at implantation, significant negative correlations to the mean ECAP AGF slopes measured were detected  and therThe mean ECAP AGF slopes of electrodes 10–12 correlated with the duration of deafness and age at implantation , and theThe mean ECAP AGF slopes of all electrodes and of the grouped electrodes of the different cochlear implant array lengths used are reported in The purpose of cochlear implantation is to restore functional hearing via an implanted device that electrically stimulates the auditory nerve. Over the last decades, technological advances helped to significantly improve speech perception in implanted patients. Nevertheless there is a large inter-patient variability in the benefit patients have by using their implant. This uncertainty and parameter variability have been shown to affect CI outcomes ,26,27,28From animal models, we know that SGN degenerate over time due to lack of neurotrophic support from the Organ of Corti ,33. The We report results of two different approaches on how to deal with missing values. For analysis A, missing slope values were replaced by zero, while for analysis B, missing slope values were excluded. By the conservative statistical approach of setting missing values to zero, averages may be shifted to lower AGF slopes as they are in reality, leading to misinterpretation in terms of judging the slope and therefore the SGN function to be lower than it is. The rationale for this conservative approach is that a missing slope value could indeed refer to a dead region or poor SGN function. On the other hand, spread of excitation usually spans several electrode contacts. If an ECAP response is measurable on one electrode contact, there should be a response on an adjacent contact as well. An exception would represent extracochlear contacts, caused by a migrated electrode array or partial insertion. However, such cases were not within our study group. All patients had auditory perceptions on each of the electrode contacts. ECAP measurements may become unpleasantly loud at higher levels, and this is in clinical routine a reason to abort the measurement. Therefore, analysis B seems to be a more appropriate approach. We report that excluding values results in a small increase of mean ECAP AGF slopes , medium values at electrodes positioned in the middle part of the cochlear , and the highest slopes in the more apical region  lead to poorer speech perception outcomes in patients with single sided deafness . The cat + years The ECAP AGF slope is steeper in the apical region in comparison to the basal region of the cochlea. This finding suggests that in the more apical region, there are more and/or healthier neurons than in the basal part. Whether this has an impact on the CI outcome has to be investigated in future studies.The etiology categories used in this study do not correlate with the AGF slopes measured and can therefore not be used for prediction. The age at implantation and duration of deafness are negatively related to the AGF slope and are therefore perfectly suitable to be used for prediction of the benefit the patient may have by cochlear implantation, since a prediction about the neuronal health status is possible. By this, our study supports the conviction of the CI community that an early implantation is crucial for its success. The consequence should be that patients should be cochlear implanted as early in life and as soon as possible after hearing loss.


Since it is known that many natural siderophores are also involved in the uptake and metabolism of other essential elements than iron, this study reports some results on the investigation of 8-HQA interactions with molybdate , in order to understand the possible role of this ligand as molybdophore. A multi-technique approach has been adopted, in order to derive a comprehensive set of information necessary to assess the chemical speciation of the 8-HQA/MoO42− system, as well as the coordination behavior and the sequestering ability of 8-HQA towards molybdate. Chemical speciation studies have been performed in KCl(aq) at I = 0.2 mol·dm−3 and T = 298.15 K by ISE-H+ (glass electrode) potentiometric and UV/Vis spectrophotometric titrations. CV (Cyclic Voltammetry), DP-ASV , ESI-MS experiments and quantum mechanical calculations have been also performed to derive information about the nature and possible structure of species formed. These results are also compared with those reported for the 8-HQA/Fe3+ system in terms of chemical speciation and sequestering ability of 8-HQA.8-hydroxyquinoline-2-carboxylic acid ( By this procedure, the pH scale used is the free scale, pH ≡ −log [H+], where [H+] is the free proton concentration (not activity). The reliability of electrode calibration in the alkaline pH range was checked by determining appropriate pKw values. A total of 80–100 data points were collected for each titration, and the equilibrium state during titrations was checked by adopting the usual precautions, such as the check of the time required to reach equilibrium and the execution of back titrations.ISE-HA) vs. wavelength (λ/nm) was made using Varian Cary WinUV (version 3.00) software. Measurements were performed in thermostatted cells at T = 298.15 ± 0.1 K in the wavelength range 200 < λ/nm ≤ 800. During the measurements, a 602 Metrohm Biotrode combined metrosensor glass electrode was also introduced in the cell, and was connected to a Model 713 Metrohm potentiometer. In this way, it was possible to simultaneously record both the A vs. λ and e.m.f. vs. titrant volumes for each alkalimetric titration point. The combined glass electrode was calibrated before each experiment in terms of free proton concentration, as reported in previous section. The titrant was delivered in the measurement vessel by means of a 665 Metrohm motorized burette. Solutions were maintained under magnetic stirring and N2(g) was bubbled in order to exclude the presence of CO2(g) and O2(g), paying attention to avoid interference with both the electrode and the optical fiber probe. Spectrophotometric measurements were carried out by titrating 25 cm3 of the titrand solution with standard NaOH solutions, up to pH ≈ 10.5–11. The titrand solutions consisted of different amounts of 8-HQA and molybdate , hydrochloric acid (1.0 ≤ cH/mmol·dm−3 ≤ 10.0), and KCl in order to obtain the pre-established ionic strength value (I = 0.2 mol·dm−3). As for potentiometric measurements, titrand solutions were prepared considering different concentration ratios, i.e., 1:1 ≤ cMoO4:c8-HQA ≤ 1:3.UV/Vis spectrophotometric titrations were carried out using a Varian Cary 50 UV/Vis spectrophotometer equipped with an optical fiber probe with a fixed 1 cm path length. The spectrophotometer was connected to a PC and the data acquisition of the couples absorbance ) by a Metrohm 663 VA Stand (Series 05) workstation, equipped with a three electrode system supplied by Metrohm, consisting of: a Multimode Mercury Electrode  working in SMDE mode (Static Mercury Drop Electrode), a double junction Ag/AgCl/KCl (3 mol·dm−3) reference electrode (RE) (model 6.0728.000 + 6.1245.000), and a glassy carbon (GC) auxiliary electrode (AE) (model 6.1247.000). The MME was filled with 99.9999% Mercury . The workstation was connected to an Autolab potentiostat/galvanostat (Eco Chemie) with an IME663 interface (Eco Chemie). The whole system was controlled using NOVA v. 1.10 software (Metrohm). The free hydrogen ion concentration in the DP-ASV and CV experiments was measured before and after each voltammetric run by using the same apparatus and procedure already described in the previous sections. Purified N2(g) was bubbled into the solutions for 300 s prior to any experiment.Cyclic Voltammetry (CV) and Differential Pulse-Anodic Stripping Voltammetry (DP-ASV) experiments were carried out (at 3 of solutions containing known amounts of molybdate (10−4 ≤ cMoO4 ≤ 5 × 10−4 mol·dm−3), 8-HQA in suitable concentrations to reach desired 8-HQA:MoO42− concentration ratios (1:1 ≤ cMoO4:c8-HQA ≤ 1:3), KCl in order to obtain the pre-established ionic strength value (I = 0.2 mol·dm−3), and adding KOH or HCl to adjust pH at the desired values (3.5 < pH < 10.5). Two series of CV measurements were performed for various 8-HQA:MoO42− ratios: in one, different scan rates (20–500 mV/s) were applied to solutions at fixed pH (pH = 7.3); in the other, scan rate was fixed at 100 mV/s and pH was varied (3.5 < pH < 10.5). Scan conditions for the first CV series were: starting potential = ending potential = 0.000 V, upper vertex potential = 0.050 V, lower vertex potential = −0.400 V, number of stop crossings 10, step potential = −0.008 V. For the second CV series they were: starting potential = ending potential = −0.020 V, upper vertex potential = 0.000 V, lower vertex potential = −1.000 V, number of stop crossings 10, step potential = −0.008 V.CV measurements were performed on 25 cm3 of solutions containing known amounts of molybdate (10−4 ≤ cMoO4 ≤ 5 × 10−4 mol·dm−3), KCl in order to obtain the pre-established ionic strength value (I = 0.2 mol·dm−3), KOH or HCl to adjust pH at the desired values (3.5 < pH < 10.5), and adding 8-HQA into the solutions up to 8-HQA:MoO42− ~3:1. Voltammograms were recorded after each 8-HQA addition, while pH was checked both before and after 8-HQA additions. Working conditions were as follows: deposition potential = −1.000 V, deposition time = 2 s, equilibration time 10 s, start potential = −1.000 V, end potential = 0.000 V, step potential = 0.005 V, modulation amplitude = 0.05 V, interval time = 0.5 s, scan rate 10 mV/s. Further experimental details on voltammetric measurements can be found in refs. ), m/z = 188.035 , m/z = 225.991 , m/z = 228.006 , m/z = 242.284 ([MoO4H3K2]), m/z = 265.961 . Even at m/z > 350 it is still possible to detect “free” 8-HQA adducts, like the signal at m/z = 377.078, corresponding to [C20H13N2O6] , and m/z = 431.997 . The presence of these signals is worth noting because, despite these ligand dimeric species being observed with high intensities, any signal was detected in all the spectra for eventual dimers containing molybdate in all the investigated m/z range. According to m/z range of interest, the following signals were identified, corresponding to molecular adducts containing one at least one molecule of molybdate, one of 8-HQA and one of water. In particular, isotopic patterns of peaks at m/z = 367.927 correspond to [C10H8NO8Mo], i.e., (MoO4)(8-HQA)H(H2O); m/z = 386.634 is [C10H11NO9Mo], i.e., (MoO4)(8-HQA)H2(H2O)2; m/z = 406.630 is [C10H8NO8KMo], i.e., (MoO4)(8-HQA)H(H2O)K; m/z = 446.992 is [C10H9NO8K2Mo], i.e., (MoO4)(8-HQA)H2(H2O)2K2. Other than the fact that these findings are in perfect agreement with the speciation scheme proposed, the most important aspect to consider is that no adducts compatible with the presence of MoO3 and/or MoO22+ moieties were observed in any of the spectra recorded under all the investigated conditions, as well as for eventual presence of poly-8-HQA molybdate complexes. Again, though this is not an absolute proof of the presence/absence of these species, this finding represents a further evidence in support of the correctness of the proposed speciation model.4)p(8-HQA)qHr species, quantum mechanical calculations have been performed. Based on previous findings on the coordination of 8-HQA on Fe  adduct was detected an confirmed by HRMS. By means of quantomechanical calculations, it was proven how the presence of this water molecule stabilizes the configuration of the complex through hydrogen bonds, leading to a structure that is compatible with all results obtained in this work. No stable configurations were obtained for poly-8-HQA complexes, as well as structures with MoO3 and/or MoO22+ moieties.The main results obtained in the present work can be summarized as follows. A series of potentiometric (ISE-H17O- and 95Mo-NMR) was hampered due to the relatively low 8-HQA solubility in water , though some tests were initially performed with unsatisfactory results.Worth mentioning is also the fact that, for the system investigated in this work, the use of NMR techniques (including 42− by 8-HQA was assessed by means of pL0.5 calculations at different pH, and was compared with that of Fe3+. 8-HQA proved to be a good sequestering agent towards molybdate in a wide pH range, suggesting its possible role as molybdophore, especially in relation to the pH-mediated selectivity towards MoO42− and Fe3+.Finally, the sequestration of MoO


Deafness in both ears is highly disruptive to communication in everyday listening situations. Many individuals with profound deafness receive bilateral cochlear implants (CIs) to gain access to spatial cues used in localization and speech understanding in noise. However, the benefit of bilateral CIs, in particular sensitivity to interaural time and level differences (ITD and ILDs), varies among patients. We measured binaural sensitivity in 46 adult bilateral CI patients to explore the relationship between binaural sensitivity and three classes of patient-related factors: age, acoustic exposure, and electric hearing experience. Results show that ILD sensitivity increased with shorter years of acoustic exposure, younger age at testing, or an interaction between these factors, moderated by the duration of bilateral hearing impairment. ITD sensitivity was impacted by a moderating effect between years of bilateral hearing impairment and CI experience. When age at onset of deafness was treated as two categories (<18 vs. >18 years of age), there was no clear effect for ILD sensitivity, but some differences were observed for ITD sensitivity. Our findings imply that maximal binaural sensitivity is obtained by listeners with a shorter bilateral hearing impairment, a longer duration of CI experience, and potentially a younger age at testing. 198/200. Normal hearing (NH) listeners develop sensitivity to acoustic differences between the ears at a very young age. This allows listeners to unmask speech from noise and localize sound sources . In ordeStudies to date have shown that patients with bilateral CIs primarily use ILDs for sound localization because clinical processors can provide usable ILDs to some extent ,7,8. In The present study investigated the relationship between several etiological and experiential factors and sensitivity to ILDs and ITDs in adult bilateral CI patients. These factors were categorized as (1) age-related factors ; (2) acoustic experience/exposure factors ; (3) experience with electric hearing . The goal of the present study was to uncover how these factors concurrently contribute to ILD and ITD sensitivity.Two age-related factors have demonstrated reliable relationships with binaural sensitivity and were investigated in the present study: the age at onset of deafness and the age at experimental testing. An earlier age of onset of deafness has been associated with poorer binaural hearing performance, specifically ITD sensitivity. Prior studies that examined the relationship between age at onset of deafness and binaural sensitivity in bilateral CI users have found an association between ITD sensitivity and age at onset of deafness between adult, late-onset (>18 years), mid-late childhood onset (between 18- and 5-years-old), and early onset (<5-years-old) groups ,15. PatiThe relationship between binaural sensitivity and age at onset of deafness is also supported by animal studies. Recent work in rabbits has found that the response of neurons in the inferior colliculus (IC) to ITDs presented through electrical pulse trains were more likely to show sustained, excitatory responses and less likely to show suppression in early-deafened rabbits than adult-deafened rabbits. In addition, early-deafened rabbits had reduced overall firing in ITD-sensitive neurons compared to adult-deafened animals. This work implies that sensitivity to ITDs of neurons involved in binaural processing in adult animals is significantly reduced following an early onset of deafness, thus leading to a sub-optimal encoding of ITDs .Aging during adulthood can also play a role in binaural sensitivity, as seen in human studies conducted with older adults. Both behavioral and electrophysiological measures have shown that aging leads to declines in the ability to process binaural cues including ITDs and interaural phase differences (IPDs) in individuals with normal audiometric thresholds ,18. BehaElectrophysiological measures have been used in prior studies to understand the physiological correlates of sensitivity to interaural differences in older and younger listeners, specifically for ITDs. For example, cortical responses to ITDs differ in older (mean = 70.0 years) versus younger mean = 24.9 years) listeners . In olde.9 years The degree of acoustic exposure and bilateral impairment can also contribute to binaural sensitivity. Thus, we investigated the total years of acoustic exposure and years of bilateral hearing impairment in this study. The best outcome attainable by patients might be limited if they experienced long periods of auditory deprivation. Prior work has shown that patients who are fitted with CIs and tested on speech understanding tasks between two post-operative visits have demonstrated that, with longer durations of severe-to-profound hearing loss before implantation, they show less improvement in behavioral tasks than in patients with a shorter duration .Studies with single-neuron recordings in non-human species provide insight into the role of acoustic exposure on binaural sensitivity. In acutely deafened animals, the encoding of neurally evoked ITDs presented through electrical stimulation of a CI can provide just as accurate encoding of ITDs as those presented with acoustic stimulation . This fiAnother factor for consideration is the degree of relative impairment in each ear, either neurally or behaviorally, that can be affected by prolonged periods of monaural input when one ear receives significantly more stimulation than the other ear ,28,29. AThe impact of bilateral hearing impairment on binaural sensitivity has also been investigated in children that use hearing aids. One study investigated whether bilateral hearing impairment in children with hearing aids impacted binaural processing. They found that ITD perception is abnormal in children that used bilateral hearing aids, but that ILD perception was similar to the NH control group . This stIn summary, these studies indicate that prolonged periods with no auditory stimulation or bilateral hearing impairment prior to CI stimulation can lead to poorer processing of ITDs. Because it is difficult to determine the degree of asymmetry in patients, in this study, we will characterize the effect of patients’ impairment on binaural functionality using the total years of bilateral hearing impairment.It is still unclear how the length of experience with electric hearing directly influences binaural sensitivity in patients with bilateral CIs. As there are limited data on the relationship between experience with electric hearing and binaural sensitivity, research conducted in children may provide insight into the influence of electric hearing.Gordon and colleagues have conducted much of the work on this topic in children. Overall, the perception of ILDs develops early in life in children with bilateral CIs ,34,35. WIn contrast to ILDs, longer experience with bilateral CI use is needed for ITD sensitivity to develop ,35. UsinWhile typical candidates for cochlear implantation have severe-to-profound sensorineural hearing loss , the hisForty-six adult bilateral CI listeners with at least one year of experience with bilateral CI hearing participated in this study. All 46 listeners participated in an ITD discrimination experiment, while 31 of the listeners participated in an ILD discrimination experiment. Listeners traveled to the University of Wisconsin–Madison for three days of testing and were paid a stipend for their participation. Listener etiology is displayed in Stimuli were delivered via specialized bilateral research processors. Thus, the listeners’ external processors were removed and replaced with bilaterally-synchronized L34 research processors or RF Generator XS . The research processors were controlled using MATLAB  via the Nucleus Implant Communicator . This setup has been used extensively by the authors for psychophysical experiments with bilateral CI patients  for all even-numbered electrodes.  The loudness map defines the range of current units that generate an audible percept between threshold (T) and most comfortable (M) loudness levels at each electrode. During the mapping session, listeners reported the perceived loudness of the electrical stimulus. The T-level is the level (in CUs) that the listener can detect before the sound becomes inaudible. The comfortable level, or C-level, is the level that the listener reported to be comfortably loud and was willing to listen to all day. Finally, the M-level is defined as the highest current level that the listener can tolerate without being uncomfortably loud. Levels for odd-numbered electrodes were interpolated from the levels found for the even-numbered electrodes. C-levels were adjusted to ensure that the loudness perceived at each electrode, both within and across ears, was equal. C-levels were compared across electrodes within an ear to ensure the same loudness. This was achieved by stimulating the electrodes one at a time with an inter-stimulus interval of 100 ms. Listeners indicated the electrodes that were noticeably louder than others and those C-levels were adjusted. The experimenter adjusted levels to attain equal loudness across all electrodes. Across-ear loudness balancing was completed after the pitch matching procedure (described in the next paragraph). A single pitch-matched pair was played simultaneously, varying the C-level across ears, until the listener perceived the sound as equally loud and coming from the center of the head.Three bilateral, pitch-matched electrode pairs along the length of the electrode array were determined for each listener. Across-ear pitch-matching of electrodes serves as a means of matching place-of-stimulation in the cochlea in each ear. Other methods exist for place matching, including finding the interaural electrode pair that yields the “best ITD” sensitivity using psychophysical testing and measurement of the binaural interaction component in the brainstem ,44. AlthTwo tasks were used to determine pitch-matched pairs: a pitch magnitude estimation (PME) and a direct pitch comparison (DPC) task. For both tasks, the stimulus was presented at C-level. In PME, listeners were asked to rate the pitch of the stimulus on a scale from 1 (low pitch) to 100 (high pitch), while stimulation was presented to a single electrode in either the left or right ear. Listeners were first familiarized with the range of pitch that they are likely to perceive by stimulating each electrode individually. The results of PME were used to determine the range of electrodes that should be used for the DPC task. In the DPC task, a reference electrode in one ear was compared against a subset of electrodes in the other ear using a two-interval, five-alternative forced-choice task. On each trial, the reference electrode was played first, followed by one of the electrodes in the other ear, chosen from the small subset identified from the PME task. The listener’s task was to judge whether the stimulus in the second interval was: “much higher,” “higher,” “same,” “lower,” or “much lower” in pitch compared to the reference electrode. The three pitch-matched electrode pairs were usually tested within the same block, where each of the three reference electrodes was chosen randomly for each trial. From the DPC task, the pair that yielded the highest number of “same” results was chosen as the pitch-matched pair for further testing (see [ILD just-noticeable-difference (JND) thresholds were estimated using a two-interval, two-alternative forced-choice paradigm with a 300-ms inter-stimulus interval. In each trial, a bilateral stimulus was presented to one pitched-matched electrode pair. The first interval of a trial had an ILD imposed either in the left- or the right-ward direction. In the second interval, an ILD of the same magnitude was applied in the opposing direction to that of the first interval. Subjects responded by indicating the direction of the second interval relative to that of the first interval . No training or feedback was provided to the listeners. A method of constant stimuli procedure was used, where ILDs of ±1, ±2, ±5, and ±10 CUs were tested in all listeners. In some instances, additional ILDs were included (such as ±20 CUs) to complete a psychometric function. Each ILD was tested 40 times with 20 left-ward and 20 right-ward ILD trials. The experiment was conducted in blocks, where each block consisted of 10 trials at each ILD (five left-ward and five right-ward). ILDs within each block were tested in random order. The percent correct data were fitted using the “psignift” MATLAB toolbox version 2.5.6 . The ILDITD JND thresholds were estimated using a similar task to the ILD task. ITDs of 100, 200, 400, and 800 μs were tested in all listeners. In some listeners, additional ITDs were included  in an attempt to completely cover the range of ITD values where the listener could achieve up to 100% correctness. Each ITD was tested 40 times with 20 left-ward and 20 right-ward ITD trials. The experiment was conducted in blocks, where each block consisted of 10 trials at each ITD (five left-leading and five right-leading). Psychometric functions were fit using the same method in the ILD task. Again, the JNDs reported in this paper are approximately 50% smaller than that typically reported using threshold estimation tasks that have a 0-μs ITD in the first interval ,52,53,54To investigate the factors listed in (1) To understand the effect of place of stimulation, the “lme4” package (version 1.1-13) was used to fit a generalized linear mixed-effects model (GLMM) with a binomial link function for the ILD and ITD JNDs. In this model, “electrode location”  served as the fixed effect and “subject” served as the random effect.(2) To explore the impact of etiological factors on JNDs, the “stats” package (version 3.4.4) was used for the linear regression (function: “lm”) and the “MASS” package (version 7.3-49) was used to perform a stepwise model selection (function: “stepAIC”) using the Akaike’s information criterion (AIC). These functions were used concurrently to perform a stepwise multiple regression to determine the effect of the factors mentioned in All predictors were mean-centered prior to including them in the model. This simply indicates that the mean of each predictor was subtracted from every value of that predictor. Thus, the zero-point for each predictor is redefined as the mean, but units and their interpretation remain unchanged. Mean centering was completed to combat multicollinearity (linear relationships between combinations of factors) between predictors since many predictors were related to one another .(3) Categorical analysis was also conducted to further understand the effects of age at onset of deafness because a number of listeners had “unmeasurable,” or “could not determined” JNDs. This analysis allowed us to include both "measurable" and "unmeasurable" JNDs by comparing the number of listeners in two groups with different ages of onset of deafness. (4) A linear regression analysis was conducted to explore the relationship between ILD and ITD sensitivity using only “measurable” JNDs.p = 0.65). Therefore, all measurable ILD JND thresholds were averaged across electrode locations for the remainder of analyses.p = 0.27). Therefore, all measurable ITD JNDs were also averaged across electrode pair locations in the remaining analyses because of the lack of effect of electrode location.p < 0.05; p < 0.05, VIF = 1.89) but not for years of bilateral hearing impairment  or years with at least one CI . The positive coefficient for years of acoustic exposure indicates that ILD JNDs increased  with more years of acoustic exposure. Additionally, the interaction between years of bilateral hearing impairment and years of acoustic exposure was significant , implying that bilateral hearing impairment moderates the effects of years of acoustic exposure . In order to predict ILD JNDs from the factors listed in p < 0.05; p < 0.05, VIF = 1.87) but not years of bilateral hearing impairment  or years with at least one CI . Additionally, there was a significant interaction between years of bilateral hearing impairment and age at testing . Thus, the positive coefficient for age at testing suggests that sensitivity to ILDs decreased as age increased  = 3.49, ncreased B. This ip < 0.05; p = 0.25; VIF = 1.01) or years with one CI  were found for the final model. Only the interaction term between years of CI experience and years of bilateral hearing impairment reached significance . In order to predict ITD JNDs from the factors listed in Prior work suggests that if the developmental time window for the emergence of binaural hearing is interrupted at a young age, the ability to use binaural cues, in particular ITDs, deteriorates in humans ,34,38. AILD JNDs were binned into five categories: <2.0, 2.01–5.0, 5.01–10.0, and >10.0 CUs, where <2.0 CUs was considered “good” and >10.0 CUs was considered very poor. “Unmeasurable” JNDs were identified as those that were >19.0 CUs. ITD JNDs were binned into five categories that also ranged from “good” to “very poor”: <200, 201–400, 401–800, 801–1600, and >1600 μs. p = 0.36; mid: p = 0.19; base: p = 0.18). There was only one instance in which a JND was considered “unmeasurable” for the mid electrode location. For the ITD data set, a significant difference in observed frequencies between the two groups was found for the mid electrode pair location, but not the other two locations . The histograms in A Chi-square test of independence was conducted on the distributions of JNDs to determine whether the observed frequencies, according to the defined bins, differed between the childhood and adulthood groups. Distributions based on the aforementioned bins are shown using a histogram for each electrode pair location  in Finally, we found that there was only one case (1.1%) of unmeasurable ILD JNDs, while, 18.6% of ITD JNDs were unmeasurable. This result suggests that all bilateral CI users can access ILDs, regardless of whether they lost their hearing before or after the age of 18. Our results also suggest that listeners are more prone to losing access to ITDs than to ILDs.p < 0.05, Given that both ILD and ITD tasks probe the efficacy of the binaural system, we were also interested in determining if both JND measures could be best explained by matched performance across the two cues. We ran a simple linear regression using the “lm” function only on the listeners that participated in both the ILD and ITD discrimination task, using their mean JNDs. ILD JNDs were highly predictive of ITD JNDs. Thus, increased ILD JNDs corresponded to increased ITD JND  = 6.34, Binaural hearing has become highly relevant in the clinical domain, due to the appreciation of patients’ needs to function in realistic, complex auditory environments. The research to date in patients with bilateral CIs shows a gap in performance relative to NH listeners on tasks such as sound localization, speech-in-noise understanding, and binaural sensitivity ,13,14,56Prior studies have shown that early access to binaural acoustic information promotes the emergence of spatial and binaural hearing ,16,57,58For age at onset of deafness, all listeners that participated in the ILD task exhibited some ILD sensitivity for at least one electrode location. In addition, we found no effect of electrode location on all measurable ILD JNDs. This is not surprising as ILDs are generally robust and are delivered reliably well through clinical processors ,69. WhenWhen comparing the distributions of ITD JNDs between childhood- and adulthood-onset of deafness groups, differences were found for the mid electrode pair location but not the other locations. A critical finding from  was thatSome key work in animals also supports the notion that age at onset of deafness matters for binaural sensitivity, particularly ITD sensitivity. Prior work from recordings in the inferior colliculus of rabbits has shown that early onset of deafness has a permanent effect on ITD coding because it can reduce the number of ITD-sensitive neurons and reduce their temporal coding . A later onset of deafness, with a prolonged period of deafness, could simply “blur” neural ITD tuning, suggesting ITD sensitivity is potentially recoverable with appropriate binaural experience ,70. AddiTaken together, both the behavioral and animal data reveal that an early onset of deafness has a greater detrimental impact on ITD than ILD sensitivity. We observed a weak effect of age at onset of deafness on ITD sensitivity. In the following sections, we discuss how heterogeneity and complex interactions with multiple etiological factors may have contributed to this result. One reason we may have observed this outcome is because of the distribution of ages in our cohort: only 14 of our listeners had an age at onset less than five years of age. Hence, recruiting a greater number of listeners in the “early-onset” age group might reveal the impact of age at onset of deafness on ITD sensitivity.Another reason we did not observe an effect could be because age at onset of deafness in humans is often difficult to determine accurately and usually relies on self-reporting from study participants. Inaccurate reporting of age at onset of deafness may introduce additional variability that led to the difference in results between studies. Further, binaural hearing in this population is impacted by the combined effects of onset of deafness with other patient-related factors. For example, age at testing had a very high correlation with years of acoustic exposure, which was included in the ILD model but was not used as a predictor in our regression to achieve the most parsimonious model determined according to Akaike’s information criterion. Other work has suggested a relationship of binaural sensitivity with age, particularly for ITDs ,22,72,73Our investigation on the impact of age at onset of deafness revealed that most listeners have binaural sensitivity and that the influence of acoustic factors on binaural sensitivity may play a larger role. Factors relating to acoustic exposure were total years of acoustic exposure and years of bilateral hearing impairment. A rather surprising finding for ILD JNDs was that the multiple regression revealed a significant and negative contribution of total years of acoustic exposure, as well as a significant interaction of years of bilateral hearing impairment with total years of acoustic exposure, for ILD JNDs . This reWe discovered a strong correlation between years of acoustic exposure and age at testing see A. When rThe interaction of age at testing with years of bilateral hearing impairment  explainsFor ITD sensitivity, we found a significant interaction between years of bilateral hearing impairment and years with at least one CI . This reThe third group of factors investigated was the experience with electric hearing, including years with at least one CI and years with bilateral CIs. Prior work has demonstrated the importance of experience with electric hearing for communication and everyday functioning in patients with CIs ,75. In tWhile we found that CI experience appears to play a role in ITD sensitivity, it is unclear how the ITD JNDs collected with carefully-controlled stimuli presented through synchronized research processors relate to ITD performance with unsynchronized clinical processors. It is likely that the data presented here represent the “best case” scenario for binaural acuity with CIs. Hence, in order to maximize ITD/ILD sensitivity in bilateral CI users, synchronized processing combined with a new sound coding strategy, such as those proposed in other work in our laboratory ,79, willA striking finding in our data set was that sensitivity to ILDs and ITDs was correlated. Thus far we have discussed why ITD and ILD coding might differ. In this final analysis, we found that behavioral performance on ILD and ITD sensitivity measures are related. This finding falls in line with other studies where both ILD and ITD JNDs were predictive of one another ,80. SpenIn the present study, sensitivity to ITDs and ILDs were measured behaviorally in adult CI patients using bilaterally synchronized research processors. This study aimed to uncover how age-related, acoustic, and electric experience impact binaural sensitivity in adults with bilateral CIs. We determined that between the childhood- and adulthood-onset of deafness groups, and across a spectrum of performance from “good” to “unmeasurable” thresholds, differences in the distributions of ITD JNDs were found for only one electrode pair location. While this might suggest that the two groups differ, it is important to keep in mind that we found no effect of electrode location in the mixed-effects model, suggesting that factors other than the age at onset of deafness play a more important role in binaural sensitivity. We found that as years of acoustic exposure increased, ILD JND thresholds increased , contrar


RDX. Radixin is important for hearing but remains functionally obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to visualize stereocilia motion while measuring electrical potential amplitudes during acoustic stimulation. Radixin inhibition decreased sound-evoked electrical potentials. Other functional measures, including electrically induced sensory cell motility and sound-evoked stereocilia deflections, showed a minor amplitude increase. These unique functional alterations demonstrate radixin as necessary for conversion of sound into electrical signals at acoustic rates. We identified patients with RDX variants with normal hearing at birth who showed rapidly deteriorating hearing during the first months of life. This may be overlooked by newborn hearing screening and explained by multiple disturbances in postnatal sensory cells. We conclude radixin is necessary for ensuring normal conversion of sound to electrical signals in the inner ear.The stereocilia of the inner ear sensory cells contain the actin-binding protein radixin, encoded by  Sonal Prasad et al. identify several mutations in the radixin (RDX) gene that are associated with early-life hearing loss. Using a guinea pig model, they propose that radixin helps convert sound into electrical signals in the mature inner ear. While the functional relevance of these interactions has not been clarified, it is evident that phosphorylated radixin links the actin cytoskeleton with various transmembrane adhesion proteins, such as CD448.Radixin is enriched within stereocilia10, acting in concert with forces generated within the soma of outer hair cells12 to establish normal hearing sensitivity and frequency selectivity.Given radixin’s important role in the network of proteins within stereocilia, we hypothesized that radixin may contribute to the regulation of stereocilia function in the mature inner ear. Little is known about this regulation, but we note that stereocilia may be capable of active force generationTo determine the influence of radixin on cochlear amplification and sensory cell function, we used a custom rapid confocal imaging technique to examine stereocilia motion while recording the electrical potentials produced by the sensory cells during acoustic stimulation. These measurements revealed an unusual pattern of functional changes when radixin was disabled. The sound-evoked electrical potentials were substantially reduced despite other important functional measures, such as stereocilia deflections and electrically induced motility being intact. This shows that radixin is necessary for the normal function of mechanically sensitive ion channels, allowing them to work at acoustic rates.RDX variants. Their hearing was normal early in life, presumably because ezrin partially substitutes for radixin, but hearing was lost during the first months of life. This causes a delay in diagnosis, and also means that a brief therapeutic window exists in the event that specific therapies aimed at DFNB24 become available.We also provide a clinical characterization of patients with Toxoplasma gondii, and syphilis, ruling out these agents as contributors to hearing loss. There was no risk for chromosomal abnormalities or metabolopathies, as revealed by standard screening. The only risk factor was consanguinity, as her parents were cousins. Importantly, hearing screening before the third day of life revealed that otoacoustic emissions, faint sounds produced by the inner ear in response to low-level acoustic clicks, were present. Since the sensory outer hair cells must be intact for otoacoustic emissions to be generated, this ruled out peripheral hearing loss13.The first patient was a 2-year old girl of Moroccan origin born to term after a normal pregnancy and delivery. Maternal serology was positive for rubella and negative for hepatitis B, human immunodeficiency virus, However, at the age of 16 months, the patient was referred to the ENT department because of suspected hearing loss. Otoacoustic emissions could not be detected, suggesting that peripheral hearing loss had developed. Auditory evoked potentials were absent and steady-state evoked potential testing revealed a bilateral threshold of 90 dB hearing level at 0.5 and 1 kHz , which was confirmed with Sanger sequencing. The result is a truncation of the protein in exon 3 (of 14), leaving only a part of the membrane-binding domain but stripping all of the actin-binding C-terminus, a change that completely disables radixin since most of its length is lost.Genetic testing with the OTOgenics panelRDX’s second exon, where the initiation codon is located (NM_002906.3: c.-64-1215_12 + 348del). Notably, an in-frame start codon present in exon 3 may mean that a protein 11 amino acids shorter is present in this patient. This shortened protein should be capable of attaching to the actin cytoskeleton, but membrane binding will be affected.The second patient was female and adopted at 6 months of age. Early hearing screening was performed with brainstem auditory-evoked potentials and the patient passed. However, she was referred to the ENT department at 8 months of age with a suspicion of hearing loss. Testing with steady-state evoked potentials showed moderate hearing loss Fig. . GeneticRDX . This removes the highly conserved actin-binding motif (exons 13 and 14)2, preventing radixin from interacting with actin filaments. Otoacoustic emissions were absent, and a younger sister was similarly affected with symmetrical profound sensorineural hearing loss without otoacoustic emissions. Both siblings had the same homozygous RDX variant. Neonatal hearing screening was not performed in either case.Our third case was diagnosed with hearing loss in infancy and underwent pure-tone audiometry at the age of 8 years, revealing a bilaterally symmetrical profound sensorineural hearing loss Fig. . Exome sRDX alterations that result in a non-functional protein can pass newborn hearing screening on the first days of life, but hearing sensitivity deteriorates thereafter. It is not clear why this hearing loss develops, so we performed experiments to determine the functional role of radixin.These clinical data show that patients with 15, direct visualization of sound-evoked stereocilia motion is possible in a nearly native environment , and in vivo measurements of hearing sensitivity in animals whose inner ears were treated with radixin inhibitors.The radixin labeling pattern is consistent with the one found in chick20. The other electrode barrel was used for delivering the radixin blocker DX-52-1, which disrupts radixin’s ability to link the actin cytoskeleton with the cell membrane22 . The change induced by DX-52-1 was apparent 10 min after its application, deflections remained elevated for at least 10 min, and a gradual recovery took place thereafter .We also evaluated the effect of DX-52-1 on inner hair cell stereocilia. However, inner hair cells are less numerous than outer hair cells and their stereocilia often do not label as well with di-3-ANEPPDHQ and are slightly damage-prone. In a more limited sample of inner hair cells, there was no consistent effect of DX-52-1 on sound-evoked stereocilia motion , but this was not the case in preparations injected with the vehicle alone . Control injections in 14 preparations showed no significant change in the fluorescence recovery time , on the cell bodies of the outer hair cells  and inner hair cells , and on the dendrites of the auditory nerve . In neither case did we find a significant change in the mobility of membrane lipids after DX-52-1 injection, suggesting that this compound specifically affects outer hair cell stereocilia when it is injected in the scala media.We also performed FRAP experiments on inner hair cell stereocilia . A significant difference in amplitude was also evident between preparations injected with DX-52-1 and the controls .Upon injection of DX-52-1, a decrease in the cochlear microphonic (CM) amplitude Fig.  was evidThe decrease in the CM amplitude means that the ability to convert sound into rapidly alternating electrical potentials is impaired. This, however, is not due to a change in the stimulation of stereocilia, because stereocilia deflections were slightly increased Fig. . Also, tTo assess the influence of radixin on hearing sensitivity in vivo, we applied 1 µl of a 1 mM DX-52-1 solution directly to the round window membrane of anesthetized guinea pigs while measuring the amplitude of the auditory nerve compound action potential (CAP). The CAP represents the summed response of auditory nerve fibers to acoustic stimulation, and is most effectively elicited by high-frequency acoustic stimuli with rapid rise time. Ten to forty minutes after the application of DX-52-1, the CAP amplitude decreased significantly compared to control preparations where only the vehicle, perilymph with 1.8% DMSO, was applied Fig. .Fig. 5DXn = 18 for DX-52-1 vs. 10 controls; P = 0.00001; two-way ANOVA). While the overall shape of the CAP waveform remained similar after DX-52-1, there was a slight increase in the response latency 6. Although the rates of both fast and slow adaptation are affected by PAO6, its indirect inhibitory effect on radixin can be used to confirm some of the DX-52-1 effects described above.Radixin mediates interactions between the cytoskeleton and the cell membrane, but membrane attachment also requires the presence of PIPP = 0.00001, two-tailed paired t-test; Fig. P = 0.004, 2-tailed paired t-test; Fig. P = 0.00001, two-tailed paired t-test; Fig. P = 0.004, two-tailed unpaired t-test with Welch’s correction). PAO caused no morphological changes to stereocilia during the time frame of the present experiments . We did not find a significant change in the mobility of membrane lipids on the cell bodies of the outer hair cells after PAO injection .Next, we used FRAP to look for changes in the membrane lipid diffusion kinetics after PAO injection. Diffusion of di-3-ANEPPDHQ molecules within a defined region of interest Fig.  on the sion Fig. , n = 9.P = 0.00001, two-tailed paired t-test; n = 33). A significant difference in the amplitude was seen between preparations injected with PAO and the controls . Additional data showed a minor but no significant effect on the CAP amplitudes after PAO application .PAO injection also led to a decrease in the CM amplitude Fig. . The droion Fig. , n = 6.FAlthough neither DMSO, DX-52-1, or PAO resulted in any detectable morphological change in stereocilia . The subsequent development of hearing loss could be due to a combination of reduced transduction currents and an inability to maintain stereocilia structure, including their stiffness, in the long term in the absence of membrane-cytoskeletal links. However, hearing loss was profound in three of our patients and moderate in one. At first sight, the removal of the start codon in exon 2 in this patient should lead to complete absence of radixin expression. Due to an in-frame start codon present in exon 3, it is however possible that a protein 11 amino acids shorter could be produced. We speculate that such a shorter protein could retain some functionality, explaining the less severe hearing loss in this patient and suggesting a clinically relevant genotype–phenotype correlation for pathogenic 38. The resulting delays in speech and language development may contribute to impairment of social skills and cognition39. No previous study has examined the effect of radixin on stereocilia function. Therefore, understanding the physiopathology of genes such as RDX and increasing our awareness of its contribution to this burden of delayed diagnosis could improve the care of children with hearing impairment. This is important, especially for siblings of already diagnosed patients. Therefore, in these families, if a genetic diagnosis has not been obtained, close monitoring of the siblings who have passed initial newborn hearing screening is mandatory. Importantly, the fact that hearing appeared normal early in life could mean that a time window exists in the event that therapies for restoring radixin functionality become available. Nevertheless, any potential, gene-specific, therapeutic opportunity will always be enhanced by an early and comprehensive genetic diagnosis.The development of early onset hearing loss in children passing newborn hearing screening can confound both patients and their physicians, causing diagnosis and intervention to be postponedThe clinical data collection was approved by the Institutional Review board at the University of Miami (USA) and by the Comité de Ética de Investigación del Principado de Asturias (research project #75/14), Spain. A signed informed-consent form was obtained from the parents of each participant. The Regional Ethics Board in Linköping approved all animal experiments (DNR 16-14) and animal care was under the supervision of the Unit for Laboratory Animal Science at Linköping University.40. Briefly, the OTOgenetics library preparation followed the SureSelectXT protocol (Agilent). Sequencing was performed using a NextSeq500 sequencer (Illumina) using manufacturer’s specifications. An optimized diagnostic pipeline allowed the identification of single nucleotide variants/indels and copy number variations. Exome enrichment followed the SureSelect Human All Exon 50 Mb kit (Agilent) protocols. Sequencing was carried out using the HiSeq 2000 instrument (Illumina). An in house bioinformatics pipeline was used for variant and copy number variation calling. Validation and segregation testing of the variants was performed.Patients I and II Fig.  were eva2, 5% CO2) cell culture medium . The bone of the bulla was gently removed to expose the middle ear and the basal turn of the cochlea, including the round window niche. Thereafter, a small triangular or trapezoidal opening was made at the apex using a #11 scalpel blade and a hole of 0.6 mm diameter was drilled at the base of the cochlea using a straight point shaped pin. These openings allowed continuous perfusion of oxygenated tissue culture medium through an external syringe tube connected to the basal hole with a plastic microtube. Sound stimulation occurred through a calibrated loudspeaker connected to the chamber with a plastic tube. Because of the immersion of the middle ear and the opening at the apex, the effective sound pressure level was reduced by ~20 dB. The values given throughout the text are corrected for this attenuation. The whole preparation was maintained at room temperature (22–24 °C). The apical opening allowed confocal imaging of the hearing organ and permitted insertion of a double-barrel glass microelectrode filled with artificial endolymph-like solution  into the scala media through the Reissner’s membrane. This special electrode with septum is used for CM recordings, electrical stimulation, endocochlear potential recordings, bundle membrane staining, and delivery of pharmacological substances, as specified.Young mature Dunkin-Hartley guinea pigs of both sexes  were used for all experiments. Prior to decapitation all animals were tested for the Preyer reflex and then anesthetized with 18–24 mg of sodium pentobarbital intraperitoneally, according to their body weight. The left temporal bone was excised and attached to a custom-built holder. The holder allowed immersion of the cochlea and the middle ear in oxygenated (95% O16. Phenylarsine Oxide (P3075-1G Sigma Aldrich): 45 mM in pure DMSO diluted to 1 mM for use.The following stock solutions were prepared and further diluted in artificial endolymph to the desired concentration. Di-3-ANEPPDHQ (D36801 ThermoFisher Scientific): 4 mM in pure DMSO diluted 100 times for use. Quinocarmycin analog DX-52-1 : 22 mM in 50% DMSO and phosphate-buffered saline (PBS) diluted to 1 mM for use. Note that the effective concentration in the endolymph is lower than 1 mM because the agent is diluted in the scala media fluids upon injection. Previous estimates suggest a 10× dilution factorSamples were imaged with an upright laser scanning confocal microscope (Zeiss LSM 780 Axio Imager) controlled with the ZEN 2012 software. Outer hair cell bundle displacement movements were acquired with a 40×, 0.80 numerical aperture water immersion objective lens (Zeiss Achroplan or Nikon CFI Apo lens); immunofluorescence imaging was made with a 100× oil immersion, 1.40 NA objective (Zeiss Plan-Apochromat). Images were processed in ImageJ 1.50i software, Imaris 9.2, ZEN 2012 and Matlab  and schemes drawn in Inkscape 0.92.3.Hair bundles were labeled with the membrane dye di-3-ANEPPDHQ, which was dissolved in endolymph solution and delivered by electrophoresis. This protocol ensured minimal dye release into the scala media and produced strong labeling of stereocilia while preserving the barrier function of Reissner’s membrane. Double-barrel microelectrodes with an outer diameter of 1.5 mm were pulled with a standard electrode puller and beveled at 20° to a final resistance of ~4–6 MΩ. The electrodes were mounted in a manual micromanipulator at an angle of 30° and positioned in the apical opening. Reissner’s membrane was penetrated using a hydraulic stepping motor. Current injections were performed with a linear stimulus isolator  sending positive steady-state currents of up to +14 µA. These currents restored the normal potential around the hair bundles, leading to an increase in the currents through the MET channel, and in the force produced by the hair cells. The endocochlear potential upon penetration of Reissner’s membrane was 25–30 mV. CM potentials were measured with an IX1 amplifier (Dagan Instruments) and digitized with a 24-bit A/D board  at 10 kHz, using custom Labview software. Tuning curves were recorded in response to a series of tone bursts at 60 dB SPL ranging from 60 to 820 Hz. The rise and fall time was 1 ms, using a Hanning window. The sampled signals were Fourier-transformed and the peak amplitude plotted as a function of stimulus frequency. Before applying drugs, tuning curve measurements were repeated every 5 min for 15–20 min to verify that the response was stable. We thereafter proceeded with the measurements described below.20, to generate a sequence of 12 images at equally spaced phases of the sine wave. Images for positive and negative current stimulation were also reconstructed at 12 equally spaced phases. These image sequences were low-pass filtered and subjected to optical flow analysis20. To improve the signal-to-noise ratio, trajectories for all pixels in 3 × 3 or 5 × 5 region were averaged. For combined sound and electrical stimuli, current injection is switched directly from positive to negative at 5 Hz to avoid charge build-up in the scala media. When imaging bundle movements, each experiment began by acquiring a baseline of 3–5 sets of images during a 20–30 min time period. Once a stable response was verified, DX-52-1, PAO, or vehicle were applied.To measure sound-evoked bundle motion, the hearing organ was stained with 1 µl of dye di-3-ANEPPDHQ added in the perfusion tube. Subsequently, the sensory hair cell bundles were stained with di-3-ANEPPDHQ dissolved in the electrode solution and delivered to the hair bundles iontophoretically with a current stimulus of 3–5 µA. The preparation was stimulated acoustically near the bundles’ best frequency (180–220 Hz). The best frequency was selected from the highest peak of the tuning curve of the CM recordings. Image acquisition triggered both the acoustical and electrical stimulus. A series of 37 images was acquired; each series requiring ~40 s for combined sound and current stimulus. Custom Labview software ensured that every pixel in the image series had a known phase both of the acoustic and electrical stimuli. To obtain images free from motion artifacts, the software tracked the temporal relation between the pixels and the sound stimulus. Image sequences free from motion artifacts were then reconstructed using a Fourier series approachFor experiments in which blockers (DX-52-1 or PAO) were injected into the endolymphatic space through the double-barrel microelectrode, one barrel of the electrode was filled with the dye di-3-ANEPPDHQ dissolved in endolymph and the other contained the blocker dissolved in endolymph. Pipettes had 1.5–3 µm tip diameter and were positioned 50–70 µm from the hair bundles The blocker was pressure-injected by a 2 pound-per-square inch pressure pulse lasting for 10 s. To verify the injection, a time series of confocal images, 60–100 s in length was acquired during each injection. CM potentials were recorded before and at 5-min intervals after the injection. Sets of confocal images of hair bundle displacement were recorded before (2 sets) and after injection (3–4 sets), and continued every 5 min for the next 30–40 min of the experiment time at a stimulus level of 80 dB SPL, 10 µA at 220 Hz best frequency. The argon laser line at 488 nm and matching beamsplitter was used. To avoid bleaching, the laser power was set to the minimum value consistent with an acceptable signal-to-noise ratio.41. The recovery of fluorescence was tracked by acquiring a series of 30 images at 1- or 3-s intervals over a time of 100–140 s. The images were 256 × 512 pixels, 12-bit pixel depth, with an integration time of 6.30 µs per pixel, and a pinhole of 1.50 Airy units. Confocal images were obtained before and 10 and 20 min apart after the blocker injection. Statistical analysis was performed by fitting the experimental data to a one-phase decay model.FRAP was performed using Zeiss software, by outlining a region of interest on the stained stereocilia membrane. Following an acquisition of a series of 10 baseline images, a 2-μm spot on the stereocilia membrane was photobleached by focusing the laser at a maximum power into the region of interest2, 1 mM MgCl2, 5 mM D-glucose, 5 mM HEPES, pH 7.4, 300 mOsmol/kg) and introduced on the round window membrane. All recording softwares were written in LabVIEW.To record CAPs, animals were anaesthetized with an initial dose of an intra-muscular injection of Xylazine (10 mg/kg) and Ketamine (40 mg/kg). Three to four minutes after the animal was adequately anesthesized, the surgical site of the left bulla was shaved and the animal placed on a thermostatically controlled heating blanket to maintain a core body temperature of 38 °C. Bupivacain (0.2 mg/kg), a long-acting local anaesthetic, was administered near the surgical site before skin incision. A retroauricular incision was made in order to reach the temporal bone. Muscle and other soft tissues were dissected, and the posterio-lateral part of the auditory bulla was opened to access the round window niche. A thin Teflon-insulated Ag/AgCl silver ball recording electrode was placed in close contact with the round window membrane. The electrode wire was fixed to the temporal bone with dental cement to ensure that the position of the recording electrode remained stable throughout the experiment. The animal was then placed inside a sound-proof recording booth where an Ag/AgCl electrode was inserted subcutaneously at the vertex of the skull. Cochlear CAPs were recorded sequentially from the left ear of the animal. Standardized input–output functions were generated by varying the intensity of stimulus . The recorded evoked-CAP signal was then filtered , amplified at a gain of 10,000 and stored for offline analysis. The responses to 200 repetitions of each stimulus were averaged with a sampling rate of 100 kHz. Three sets of recordings were obtained before the blocker application, and recordings were repeated at 5-min intervals for the next 40 min after application of the blocker. Blockers were dissolved in artificial perilymph .The sensory epithelium was exposed by carefully removing the cochlear bone, the spiral ligament, and the tectorial membrane. After washing the samples in PBS, they were permeabilized using 0.3% Triton X-100 soaked in 3% bovine serum albumin , dissolved in PBS for 10 min at room temperature followed with one-time wash with PBS for 5 min. Permeabilization was followed with blocking step by incubating the samples for 2 h in PBS containing 3% normal goat serum  and 3% BSA and then stained overnight at 4 °C with the primary monoclonal antibody  at a dilution of 1:500. This antibody was shown to be specific for radixin by Shin et al.® 488–conjugated IgG, ab150113 Abcam) and Alexa Fluor 568 conjugated Phalloidin  at a dilution of 1:500. The antibody solutions were prepared in blocking solution. After three washes with PBS for 20 min each, sections were readied for surface preparations. Sections of organ of Corti starting from apex to base were carefully dissected and mounted on glass slides with mounting media Fluorosave reagent . The slides were sealed and allowed to rest for ~2 h before proceeding with imaging. Confocal images of the mounted sections were obtained in two track channel mode with excitation at 488 nm for Alexa Fluor 488 fluorescence and at 561 nm for Alexa Fluor 568 fluorescence. Z-stacks were acquired at 12-bit pixel depth, 512 × 512 pixels, with an integration time of 6.30 µs per pixel, pinhole of 1.0 Airy units and a spacing 1.0 or 3.0 µm per slice with 20 slices up to 10 µm in total depth.Samples were then washed three times with PBS for 10 min each, followed by a 2 h incubation with a mixture of the secondary antibody . Plots were generated in the Matlab and Prism softwares. A Kolmogorov-Smirnov test verified that the data were normally distributed. Differences were analyzed with Student’s paired or unpaired Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2