Fig. 5. Exposure to BDNF increased KCaantibody staining, whereas NT-3 decreased it. a–l, Representative images of spiral ganglion neurons from the apex and base in control, BDNF, and NT-3 conditions. The top panels(a, c, e, g, i, k) show neurons labeled with FITC-conjugated KCa antibody, and the bottom panels (b, d, f, h, j,l) show the overlay of the green FITC-conjugated KCa antibody and the red TRITC-conjugated NF200 antibody. Because a pure count of the number of stained cells would not accurately represent the obvious difference in label intensity that we consistently observed (compare Fig. 6a,b), these results also illustrate the need for a weighted ranking method of analysis. a–d, Spiral ganglion neurons in control cultures label significantly more with anti-KCa in the base compared with the apex.a, The faint profiles of KCa-labeled neurons are discernable; however, they were considerably lighter than those in the base (compare arrow in a witharrow in c). b, The double exposure shows prominent NF200-positive labeling, indicating that neurons were present in the culture, and demonstrating that the faintly stained cells in a were neurons. Thearrows in a and b indicate an NF200-positive neuron that minimally expressed the protein for KCa, therefore in b it retains most of the red (TRITC) color in the overlay.c, Anti-KCa stained a population of neurons removed from the base of the cochlea. The arrowindicates the round profile of a neuron strongly labeled with the anti-KCa. d, A double exposure of KCa (green) and NF200 (red) staining revealed that the majority of neurons were labeled with both antibodies, as indicated by theyellow color of each of the neurons.e–h, Neurons isolated from the cochlea and grown in culture supplemented with 5 ng/ml BDNF showed an increase in KCa antibody labeling. e, Neurons from the apical cochlea stained similarly to those from the base.f, The double exposure shows yellowneuron cell bodies, verifying that the basal neurons exposed to 5 ng/ml BDNF were labeled with both NF200 and KCa antibodies.g, Neurons from the basal, high-frequency region of the cochlea stained similarly to those from the base control.h, The double exposure shows yellowneuron cell bodies, verifying that the basal neurons exposed to 5 ng/ml BDNF are labeled with both NF200 and KCa antibodies.i–l, Cultures of spiral ganglion neurons supplemented with 5 ng/ml NT-3 demonstrated a decrease in KCa antibody labeling in neurons isolated from the base, while preserving the apex–base differences observed in controls. i, Neurons isolated from the apex of the cochlea lightly labeled with the KCa antibody. j, The double exposure shows that every neuron identified with anti-NF200 weakly labeled with anti-KCa, resulting in a predominance of thered NF200 color. k, Neurons isolated from the base of the cochlea stained significantly darker than the apex; there was, however, significantly less staining than that observed in the base control neurons. l, Double exposure of KCa-positive neurons (green) and NF200-positive neurons (red). For a–l, spiral ganglia were isolated from postnatal day 5 CBA/CaJ mice and maintained for 7 d in vitro. Tissues were incubated in a 1:800 dilution of anti-KCa overnight at 4°C.m, Histogram of the weighted percentage of KCa antibody staining of apical and basal spiral ganglion neurons in each condition (Control, BDNF, andNT-3) for three experiments. The error bar is not shown for the base BDNF condition because all three values were 100%. **p < 0.01 for comparisons between the experimental conditions denoted by the solid lines.