Figure 6. Supporting cell expansion in gentamicin-damaged BP. A–C, Examples of recordings in slice preparations of BP explants cultured for 24 h in the presence of 1 mm gentamicin under GJIC-permissive conditions. Ai, IR-DIC videomicrograph taken during a whole-cell recording from supporting cells at the neural edge of the proximal sensory epithelium. Aii, Confocal projection showing neurobiotin (red) and Lucifer yellow (green) distributions in the same slice after formaldehyde fixation. The injected cell is denoted by an asterisk. Nuclei are stained using DAPI (blue). Neurobiotin/Lucifer yellow fluorescence highlights the expanded cell bodies of supporting cells after hair cell ejection and also fine cytoplasmic extensions connecting supporting cells to the basement membrane (arrows). B, Neurobiotin and Lucifer yellow transfer in supporting cells at the neural edge of the distal region. C, Neurobiotin and Lucifer yellow transfer in supporting cells at the abneural edge of the distal region. Pyknotic nuclei of ejected hair cells (HC) are evident above the luminal surface of the slice. D, Single confocal image after a separate recording showing the distribution of neurobiotin in neural region supporting cells. Hair cells are no longer present and supporting cells have expanded to fill the spaces left behind. E–G, Dye injections in slices from gentamicin-treated BPs during transient bath application of 200 μm MFA. Distinct expanded cyto-architecture is observed in cells toward the neural edge (E, F) and toward the abneural edge (G) of the sensory epithelium. The neural cells measured 7.2 μm (E) and 5.4 μm (F) across the apical region. Pyknotic nuclei of ejected hair cells are evident in the DAPI channel. Ei, An apoptotic body is apparent within an intracellular vacuole of a neural edge supporting cell (arrow), delineated by Lucifer yellow fluorescence in Eii. In all regions, dye-filled cells have a foot structure extending toward the basilar membrane and nuclei are located apically. H–I, After some experiments using MFA, dyes localize to adjacent cell pairs. Hi, In the neural region, neurobiotin and Lucifer yellow have apparently transferred from one supporting cell (SC1) to another (SC2). Lucifer yellow fluorescence alone is shown in Hii. Ii, Both dyes are detected in an overlying supporting cell pair after an injection in the midregion (Iii shows Lucifer yellow alone). Iiii, The spatial relationship between the two cells is demonstrated by an orthogonal projection of neurobiotin and Lucifer yellow fluorescence (Lucifer yellow alone is shown in Iiv). Scale bars, 20 μm.