Amplification of cDNA derived

from the papilla using primers designed against chicken prestin produced a band of the correct size (382 nt) identical to a band produced by amplification of plasmid containing chicken prestin (Figure 8A). These results confirm the original cloning of prestin from chicken inner ear (Schaechinger and Oliver, 2007; Tan et al., Protein Tyrosine Kinase inhibitor 2011). Localization to the hair cells was demonstrated by immunolabeling with an antibody against an N-terminal peptide sequence of mammalian prestin. Immunoblots of protein extracts of chicken basilar papilla and mouse cochlea labeled with the antibody displayed a principal band at ∼80 kDa, appropriate for prestin in both animals (mouse, 81.3 kDa; chicken, 81.1 kDa). Similar results were seen in three other blots. When applied to the papilla, the antibody labeled the lateral membrane of SHCs (Figure 8C) and THCs (Figure 8D). Z projections of the stack (Figure 8C) showed the label as a ring around the basolateral Lenvatinib molecular weight aspect of the cell; in some SHCs (Figure 8E, middle) the label was denser

on the side where the top of the cell has a lip projecting toward the neural limb. Hair cell bounds were defined by immunolabeling also for otoferlin which is present in the cell membrane and cytoplasm (Goodyear et al., 2010); comparison with the prestin demonstrated that the prestin label was in the hair cell (Figures 8C and 8E), and none was present in the supporting cells. SHC labeling at other positions confirmed that prestin occurred along the entire epithelium, the label being consistently weaker at the

apex (d = 0.2) and stronger at the base (d = 0.8). We did not quantify the change in labeling intensity with location, but the results suggest a tonotopic gradient in prestin as for other hair cell proteins ( Tan et al., 2013). We investigated likely electromechanical processes in chicken auditory hair cells by measuring “active” hair bundle motion because this might underlie acoustic amplification and extension of the auditory frequency range Rolziracetam in birds. Our main findings were as follows: (1) depolarizing a hair cell elicited biphasic bundle displacements consisting of two processes, one inhibited by MT channel blockers and the other by Na+ salicylate; (2) salicylate had no effect on the maximum current, gating, or adaptation of the MT channels but did block a nonlinear capacitance sensitive to the intracellular chloride concentration; (3) hair bundle deflection with a flexible glass fiber could produce a fast bundle “recoil” or “mechanical twitch” that also appeared to possess two components: one observed in voltage clamp probably reflecting MT channel adaptation (Benser et al., 1996; Ricci et al.