FIG. 4.
NKAα3 is expressed throughout all calyx microdomains. High magnification micrographs of the central region of the horizontal crista (A–C) and striolar region of the utricle (D–F) double immunolabeled with a mouse monoclonal anti-NKAα3 (green, A, D, and G and H) and rabbit polyclonal anti- KV7.4 (red, B, E, G, and H) to identify the molecular microdomains (Lysakowski et al. 2011) expressing the NKAα3. These micrographs reveal NKAα3 immunoreactivity colocalized with the KV7.4-positive deep calyx inner face termed Domain 1 (C, F). G, H Higher magnification micrographs of the boxes in C and F. Vertically oriented calyces visible in the preparation of the crista (G) show that NKAα3 immunoreactivity additionally extends into Domain 2, the apical part of the calyx, as well as Domain 3, the basolateral calyx and axon segment. Similar patterns of immunoreactivity were observed in high magnification views of the anterior crista (data not shown). Higher magnification micrographs of the horizontally oriented calyces visible in the preparation of the utricle (H) show that NKAα3 immunoreactivity colocalizes with the KV7.4-positive calyx inner face but is additionally expressed in the outer basolateral calyx membrane (H). Micrographs represent projections through the minimal number of optical sections required to completely visualize the hair cells and their afferent terminals (A–C and G) or single optical sections (D–F and H). Where clearly resolved, the inner-face (open arrowhead) and outer-face (closed arrowhead) calyx membranes are indicated. Scale bars measure 10 μm.