Figure 1.
Immunostaining of Slc26a7 in the cochlea.
Slc26a7+/+ in panels A–D: A) Slc26a7 was immunolocalized to Reissner’s membrane epithelial cells in a mid-modiolar section of the cochlea of a representative adult Slc26a7+/+ mouse. The four cross-sections of the cochlear duct are labeled in scala media from base to apex. B) Magnified image of cross-section #2. C) Slc26a7 was observed in Reissner’s membrane (RM) adjacent to the stria vascularis, but expression did not extend into any cells of the lateral wall. The antibody was detected only on the basolateral membrane of the epithelial cells and was not observed in the mesothelial cells. D) Slc26a7 was observed in Reissner’s membrane near the insertion at the spiral limbus. The antibody was detected only on the basolateral membrane of the epithelial cells and was not observed in the mesothelial cells. E), F), G) No staining was observed in the cochlea of Slc26a7Δ/Δ mice. Slc26a7, red; F-actin (phalloidin), green; Nuclei (DAPI), dark blue.
Figure 2.
Onset of expression of Slc26a7.
A–D) immunolocalization of Slc26a7 (red) in sections of Reissner’s membrane at the ages P1, P4, P5 and P16. The first expression was detected at P5. E–G) surface preparation of Reissner’s membrane of a P8 mouse stained for Slc26a7 and F-actin. The actin ring is known to associate with the lateral junctions.
Figure 3.
Density of Reissner’s membrane epithelial cells.
A) Representative image of mid-modiolar cochlear section of Slc26a7+/+ mouse stained with the nuclear dye, DAPI. RM, Reissner’s membrane. B) Summary of counts of nuclei per 100 µm length of Reissner’s membrane. No significant difference (ns) was found in the number of cells per 100 µm between Slc26a7+/+ and Slc26a7Δ/Δ mice at P15 through 8 months old.
Figure 4.
Organ of Corti in Slc26a7Δ/Δ and Slc26a7Δ/Δ Mice.
The morphology of mid-modiolar sections of the organ of Corti was compared between heterozygous (Slc26a7Δ/+ top panel; A, B, C, D) and knockout mice (Slc26a7Δ/Δ, bottom panel; E, F, G, H) of P15 littermates. No differences were observed between corresponding cochlear half-turns (labeled I, II, III, IV from the base to the apex). An open tunnel of Corti in each turn is consistent with no delay in development at this age.
Figure 5.
Stria vascularis in Slc26a7+/+ and Slc26a7Δ/Δ Mice.
Expression of key functional transport proteins that are known to be in the stria vascularis and that mediate generation of the endocochlear potential and the high potassium concentration of the luminal fluid, endolymph. There were no differences in the expression between Slc26a7+/+ (top panel; A, B, C) and Slc26a7Δ/Δ (lower panel; D, E, F) mice for the potassium channels Kcnq1 and Kcnj10 and for the Nkcc1 transporter Slc12a2.
Figure 6.
Nitrate currents in Reissner’s membrane epithelial cells from wild-type mice.
A) Whole-cell patch clamp currents from Reissner’s membrane epithelial cells from Slc26a7+/+ mice were recorded with Cl–-rich pipette solution and either Cl–-rich or NO3–-rich bath solution. B) The command voltage was held at 0 mV and stepped for 300 ms to +80 mV through −80 mV in 20 mV increments. Representative recordings of step responses before (left) and at the end (right) of the NO3− bath perfusion are shown. C) Summary of the mean currents at the end of each step are plotted with SEM error bars. Light green panel shows values used for statistical tests of differences in currents and conductances. Green triangles point to the reversal voltages. D) A representative continuous recording during perfusion of Cl–-rich and NO3–-rich bath solutions at the times marked.
Figure 7.
Nitrate currents in Reissner’s membrane epithelial cells from knockout mice.
A) Whole-cell patch clamp currents from Reissner’s membrane epithelial cells from Slc26a7Δ/Δ mice were recorded with Cl–-rich pipette solution and either Cl–-rich or NO3–-rich bath solution. B) The command voltage was held at 0 mV and stepped for 300 ms to +80 mV through −80 mV in 20 mV increments. Representative recordings of step responses before (left) and at the end (right) of the NO3− bath perfusion are shown. C) Summary of the mean currents at the end of each step is plotted with SEM error bars. Light green panel shows values used for statistical tests of differences in currents and conductances. Green triangles point to the reversal voltages. D) A representative continuous recording during perfusion of Cl–-rich and NO3–-rich bath solutions at the times marked.
Figure 8.
Nitrate currents exceed chloride currents in wild-type but not knockout mice.
Currents in NO3–-rich bath minus currents in Cl–-rich bath are plotted for wild-type and for knockout mice. Excess NO3− currents (at +80 mV) are significantly greater in wild-type animals, consistent with the contribution of anion currents mediated by Slc26a7 channels.
Figure 9.
Iodide currents in Reissner’s membrane epithelial cells from wild-type mice.
A) Whole-cell patch clamp currents from Reissner’s membrane epithelial cells from Slc26a7+/+ mice were recorded with Cl–-rich pipette solution and either Cl–-rich or I–-rich bath solution. B) The command voltage was held at 0 mV and stepped for 300 ms to +80 mV through −80 mV in 20 mV increments. C) Summary of the mean currents at the end of each step are plotted with SEM error bars. Light green panels show values used for statistical tests of differences in currents and conductances. Green triangles point to the reversal voltages. I− in the bath reduced Cl− efflux current and conductance (at −80 mV) and the I− slope conductance (at +80 mV) was also reduced compared to the Cl− bath, consistent with reduced permeability to I− and partial inhibition of the Slc26a7 channel.
Figure 10.
Inhibition of chloride currents by NPPB.
A) Whole-cell patch clamp currents from Reissner’s membrane epithelial cells from Slc26a7+/+ mice were recorded with Cl–-rich pipette and bath solutions in the absence or presence of NPPB. B) The command voltage was held at 0 mV and stepped for 300 ms to +120 mV through −140 mV in 20 mV increments. Representative recordings of step responses before and at the end of the 100 µM NPPB bath perfusion are shown. C) Currents at the end of each step between +80 and −80 mV are summarized. The currents were inhibited throughout the voltage range and conductances at the light green panels were also reduced.
Figure 11.
Auditory brainstem recordings.
A–C) Auditory brainstem responses (ABR) at 8 kHz, 16 kHz and 32 kHz were made on Slc26a7+/+, Slc26a7Δ/+ and Slc26a7Δ/Δ mice at ages P22 through P100. There were no differences in auditory threshold between either Slc26a7Δ/Δ or Slc26a7Δ/+ and Slc26a7+/+ mice during their early adult life. The auditory thresholds were unchanged among the genotypes. The numbers of animals for each mean threshold at P22, P56 and P100 are +/+: 11, 4, 7; Δ/+: 16, 13, 14; Δ/Δ: 10, 13, 12. D) The ABR of Slc26a7+/+, Slc26a7Δ/+ and Slc26a7Δ/Δ mice was measured before and after exposure to noise that produced a temporary auditory threshold shift. E) The ABR of Slc26a7+/+, Slc26a7Δ/+ and Slc26a7Δ/Δ mice was measured before and after exposure to noise that produced a permanent auditory threshold shift. The magnitude of the threshold shifts was not affected by the level of expression of Slc26a7. Numbers of animals are given by each bar.
Figure 12.
Slc26a7+/+, Slc26a7Δ/Δ mice and Slc26a7Δ/Δ mice were tested for balance on a Rotarod apparatus. There were no significant differences in vestibular function among any of the genotypes on any test day (ANOVA).; +/+: n = 3, 2 male, 1 female, 32 g, 10 mo; Δ/+: n = 5, 1 male, 4 female, 28 g, 10 mo; Δ/Δ: n = 7, 4 male, 3 female, 29 g, 11 mo.