Maximum intensity projections of the organ of Corti from the right ear of a P17 mouse injected at P6 with 1-2 µl of AAV9.hSyn.iGluSnFR suspension, immunolabeled for iGluSnFR (GFP), IHCs, OHCs and SGNs (parvalbumin) and synaptic ribbons and nucleus (CtBP2/RIBEYE). Close-up (right panel) highlights IHCs innervated by several iGluSnFR-expressing SGN boutons. Step sizes are 0.6 (left) and 0.5 µm (right), respectively. (Scale bars: 10 µm) Simultaneous perforated patch-clamp and imaging of IHCs (1.3 mM [Ca2+]e). IHCs were patch-clamped from the pillar side, with a patch pipette containing TAMRA-conjugated CtBP2-binding peptide. Towards the end of the recording, the membrane patch was ruptured to fill the IHC with the peptide, which stains synaptic ribbons and nucleus. (Left) Two exemplary confocal sections of IHCs showing baseline fluorescence of iGluSnFR-expressing afferent boutons from both modiolar (IHC1) and pillar (IHC2) side of the cell. Glutamate release from IHCs was evoked upon step depolarizations and detected as fluorescence change (ΔF) of iGluSnFR signal located on the SGN membrane (See B'). (Right) Overlaid images of the IHC1 and IHC2, displaying the boutons (iGluSnFR) and the synaptic ribbons (CtBP2), after the recording. (*: transduced afferent boutons, >: synaptic ribbons) (Scale bar: 2 µm) (see Appendix Fig S2 for the iGluSnFR ROI detection routine) The stimulus protocol of the example IHCs from B, displaying the voltage stimulation (Vstim), whole-cell Ca2+ influx (ICa) and single iGluSnFR responses. IHCs were stimulated by 10-ms-long step depolarizations to -22 mV from a holding potential of -87 mV (1.3 mM [Ca2+]e), and iGluSnFR fluorescence was recorded at 50 Hz. 