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Additional Supporting Information may be found in the online version of this article.

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CNE_22576_sm_suppinfofig1.tif1227KSupplementary Figure 1. Type I and type II spiral ganglion neurons in vitro were immunolabeled similarly with anti-GluR2/3 antibody. a. Two different fields from the same culture dish show that basal type I and type II spiral ganglion neurons in vitro were comparably stained with anti-GluR2/3 antibody (green). b. Both types of spiral ganglion neurons were also stained similarly with anti-β-tubulin antibody (blue). c. Anti-peripherin antibody labeling of presumptive type II neurons (magenta). d. Merged image of neurons shown in panels a and b show that although clusters of neurons contained both putative type I and type II neurons, there were no significant differences in their staining patterns with anti-GluR2/3 and anti-β-tubulin antibodies. e & f. Frequency histograms of anti-GluR2/3 antibody irradiance measurements from a single experiment; apical type I and type II neurons and basal type I and type II neurons were all well fitted with single Gaussians. g. Normalized Gaussian fits for histograms shown in e and f show that average irradiance levels of both basal type I and type II neurons are greater than those obtained from both classes of apical neuron. h & i. Frequency histograms composed of anti-GluR2/3 antibody irradiance measurements from 6 experiments; apical type I and type II neurons and basal type I and type II neurons were well fitted with single Gaussians. j. Normalized Gaussian fits for histograms shown in h and i. Type I and type II distributions were essentially overlapping from each area. As in the single experiment, irradiance levels were consistently highest for both classes of basal neuron. Scale bar in panel d applies to a-d. Corresponds to text Figure 1.
CNE_22576_sm_suppinfofig2.tif1256KSupplementary Figure 2. Type I and type II spiral ganglion neurons in vitro isolated from apical and basal regions each showed heterogeneous immunolabeling with anti-synaptophysin antibody. a-d. Apical type I spiral ganglion neurons displayed uniform anti-β-tubulin antibody labeling (b, blue) without anti-peripherin antibody labeling (c, magenta), and showed heterogeneous anti-synaptophysin labeling when viewed alone (a, green) or when panels a and b were merged (d). e-h. Apical type II spiral ganglion neurons displayed uniform anti β-tubulin labeling (f, blue) with robust antiperipherin antibody labeling (g, magenta), and showed heterogeneous antisynaptophysin labeling when viewed alone (e, green) or when panels e and f were merged (h). i & j. Frequency histograms of anti-synaptophysin antibody irradiance measurements from a single experiment; apical type I and type II neurons and basal type I and type II neurons were all fitted with single Gaussians. k. Normalized Gaussian fits for histograms shown in i and j show that average irradiance levels of both apical type I and type II neurons were greater than those obtained from both classes of basal neurons. l & m. Frequency histograms composed of anti-synaptophysin antibody irradiance measurements from 6 experiments; apical type I and type II neurons were well fitted with double Gaussians and basal type I and type II neurons were well fitted with single Gaussians. n. Normalized Gaussian fits for histograms shown in l and m. Within each region, type I and type II distributions were similar, yet irradiance levels of apical neurons were consistently higher than both classes of basal neuron. Scale bar in panel h applies to a-h. Corresponds to text Figure 3.
CNE_22576_sm_suppinfofig3.tif671KSupplementary Figure 3. Inner and outer hair cells located in the basal cochlea had a higher level of anti-BDNF antibody labeling than those located in the apical cochlea. a. Low magnification view of a postnatal tissue section labeled with anti-BDNF (green) and anti-β-tubulin (magenta) antibodies. b-c. High magnification merged images of sensory hair cells outlined in panel a (white boxes). Regions of measurement are demarcated with a dashed line for inner hair cells and dotted lines for outer hair cells. d. Averages of 15 and 20 measurements (apex and base, respectively) taken from 10 separate sections from 3 different preparations with two anti-BDNF antibodies showed that irradiance was significantly different between the hair cells from the apical and basal cochlear. Significance of the paired Student's t-test, **p<0.01. Scale bar in panel c applies to b and c. Corresponds to text Figure 5.
CNE_22576_sm_suppinfofig4.tif662KSupplementary Figure 4. Inner and outer hair cells located in the apical cochlea had a higher level of anti-NT-3 antibody labeling than those located in the basal cochlea. a. Low magnification view of a postnatal tissue section labeled with anti-NT-3 (green) and anti-β-tubulin (magenta) antibodies. b-c. High magnification merged images of sensory hair cells outlined in panel a (white boxes). Regions of measurement are demarcated with a dashed line for inner hair cells and dotted lines for outer hair cells. d. Averages of 7 and 10 measurements (apex and base, respectively) taken from 5 separate sections from 2 different preparations showed that irradiance measurements were significantly different between the hair cells from apical and basal cochlear regions. Significance of the paired Students t-test, **p<0.01. Scale bar in panel c applies to b and c. Asterisks indicate areas between inner and outer hair cells that were not measured. Corresponds to text Figure 6.
CNE_22576_sm_suppinfofig5.tif1925KSupplementary Figure 5. Anti-GluR2/3 antibody labeling of spiral ganglion neurons was systematically graded along the tonotopic axis. a. Low magnification view of a postnatal cochlear section labeled with anti-GluR2/3 (green) and anti-β-tubulin (magenta) antibodies indicating the location of spiral ganglion neurons in different cochlear turns. b-e. Anti-GluR2/3 (green) antibody labeling of spiral ganglion neurons was lowest in apical neurons (b), intermediate in mid-cochlear regions (c & d), and highest in the most basal region (e). f-i. Spiral ganglion neurons shown in b-e were uniformly labeled with anti-β-tubulin (magenta). j-m. High magnification view of neurons outlined with white boxes shown in panels b-e. n. Average values from 5 experiments confirmed that anti-GluR2/3 irradiance levels were highest in basal neurons and decreased systematically in neurons innervating more apical cochlear regions. Significance of the paired Students t-test, **p<0.01. Scale bar in i applies to b-i; scale in m applies to j-m. Corresponds to text Figure 7.
CNE_22576_sm_suppinfofig6.tif1811KSupplementary Figure 6. Anti-synaptophysin antibody labeling of spiral ganglion neurons was systematically graded along the tonotopic axis. a. Low magnification view of a postnatal cochlear section labeled with antisynaptophysin (green) and anti-β-tubulin (magenta) indicating the location of spiral ganglion neurons in different cochlear turns. b-d. Anti-synaptophysin (green) antibody labeling of spiral ganglion neurons was highest in apical neurons (b), intermediate in a mid-cochlear region (c), and lowest in the base (d). e-g. Spiral ganglion neurons shown from b-d, respectively, were uniformly labeled with anti-β-tubulin (magenta). h-j. High magnification view of neurons outlined with white boxes shown in panels b-d. k. Average values from 7 experiments confirmed that anti-synaptophysin irradiance levels were highest in apical neurons and decreased systematically in neurons innervating more basal cochlear regions. Significance of the paired Students t-test, *p<0.05; **p<0.01. Scale bar in g applies to b-g; scale in j applies to h-j. Corresponds to text Figure 8.
CNE_22576_sm_suppinfofig7.tif1098KSupplementary Figure 7. Local gradients of synaptophysin in postnatal sections are systematically distributed from the scala vestibuli to the scala tympani. a. Low magnification view of a postnatal cochlear section labeled with anti-synaptophysin (green) and anti-β-tubulin (magenta) antibodies indicating the location of spiral ganglion neurons in an apical cochlear turn. b. Antisynaptophysin (green) antibody labeling of spiral ganglion neurons showed two gradients. The first extends from base to apex (solid arrow) the second extends from scala vestibuli to scala tympani (dashed arrow). Therefore, antisynaptophysin irradiance was highest in the apical neurons closest to the scala tympani (between the two arrowheads). c. Anti-β-tubulin antibody labeling was relatively uniform throughout the region. d. Average anti-synaptophysin antibody irradiance measured from neurons within the upper (SV), middle, and lower (ST) regions shown in panel b, Row 1 (white boxes contain 8, 9, and 9 neurons, respectively). e. Average anti-synaptophysin antibody irradiance measured from neurons within the upper (SV), middle, and lower (ST) regions shown in panel b, Row 2 (white boxes contain 10, 8, and 7 neurons, respectively). f. Average anti-synaptophysin antibody irradiance measured from neurons within the upper (SV), middle, and lower (ST) regions shown in panel b, Row 3 (white boxes contain 7, 8, and 8 neurons, respectively). g-i, Average antisynaptophysin antibody irradiance from 3 separate experiments measured from neurons within the upper (SV), middle, and lower (ST) regions g, Row 1 measurements were made from the most apical region within each section. h, Row 2 measurements were made from the mid-frequency region within each section. i, Row 3 measurements were made from the most basal region within each section. Scale bar in c applies to b and c. SV: scala vestibuli; ST: scala tympani. Corresponds to text Figure 9.

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