Fig. 3.

Gene expression, immunolocalization, and in vitro ligand binding of the muscarinic (M3) acetylcholine receptor (M3R) in outer sulcus cells (OSCs) of the mouse (p14) cochlea. a, b Quantitative real-time PCR (qRT-PCR) data for the M3R expression in tissue samples from the mouse (p14) cochlea and positive control tissue (SL spiral ligament, SV stria vascularis, OC organ of Corti, AN auditory nerve with spiral ganglia, SG salivary (parotid) gland, - negative control). Error bars indicate the SD, Student’s t test: **p ≤ 0.01, *p ≤ 0.05. c–c”’ Immunolabeling of M3R and the inward rectifier type potassium channel Kir4.1 in OSCs in the apical cochlear turn. M3R fluorescence was detected in the soma region (white arrowheads) and root processes (hollow arrowheads) of OSCs. d–f”’ Spiral ligament whole-mount specimens that were directly d–d”’ incubated with the M3R fluorescent ligand M3-633-AN, incubated with M3-633-AN after e–e”’ pre-incubation with the M3R antagonist 4-DAMP, or f–f”’ incubated with 0.1 % DMSO. Vertical white lines in d”’, e”’, and f”’ indicate the radial extent of the stria vascularis (SV) and the outer sulcus cells (OSC); white arrowheads d”’, e”’ and f”’ point to the M3-633-AN fluorescence in the area of the d”’ OSCs, which is absent in e”’ and f”’. Scale bars: c–c”’ 10 μm; d–f”’ 20 μm