Laser confocal micrographs of double immunofluorescence using antibodies against the GluR2, NR1 and NR2A/B glutamate receptors subunits as indicated (red labelling) and neurofilaments (green labelling). Immunoreactivities to the glutamate receptor subunits appear as bright spots at the contact between the type I hair cell and its calyx terminal. Calyx terminals of afferent fibres innervating type I sensory cells are identified by neurofilament staining. Scale bar = 10 μm and applies to all panels. Western blot analysis of SDS gels of turtle (T) and mouse (M) whole brain using indicated antibodies. Apparent molecular masses are given on the left.

Hair bundles were displaced using a velocity controlled perfusion system represented by a small line below each trace. A and B, representative traces of mechanically evoked discharges at calyx terminals displaying background activity (A) or at a silent calyx terminal (B). C and D, blocking effects of TTX (C) and gentamicin (D) on the background and the mechanically evoked activities. Representative traces in control conditions (above) and after exposure for 30 s to the indicated compounds. Scale voltage value in bottom trace apply to all traces.

A, micrograph illustrating the approach of the recording pipette from the left through the surface of the posterior crista to directly access the calyx terminals for loose patch recordings. B, stack of 20 optical sections of another calyx terminal loaded with Lucifer Yellow after recording, taken using a two-photon microscope. Inset, single optical section of the same calyx terminal. The pipette is seen on the right of the calyx being recorded (arrow). Scale bars = 10 μm in A and B. C–E, blocking effect of indicated compounds on the background activity recorded at calyx terminals. Drugs were applied for 30 s and traces demonstrate their effects on the background activity. (gap in C: 30 s; gap in E: 25 min). Scale values in the bottom trace apply to all traces.

A, representative traces of background activity in one calyx terminal under control condition (upper trace), after 3 min exposure to the non-competitive NMDA receptor antagonist MK-801 (20 μm; middle trace), and after 2 min subsequent exposure to the AMPA receptor blocker NBQX (5 μm; lower trace). Corresponding effect on the discharge rate is illustrated on the right. (Bins 2 s; mean background frequency before drug application: 3.1 spikes s⁻¹.) B, representative traces of mechanically evoked firing activities at a calyx terminal under control condition (left trace), after 5 min exposure to MK-801 (20 μm; middle trace) and after 2 min subsequent exposure to NBQX (5 μm; right trace). (Bins 200 ms; mean background frequency: 3.5 spikes s⁻¹; mean mechanically evoked frequency: 34 spikes s⁻¹.) C, effect of GYKI 53784 (15 μm) on the firing activities at calyx terminal (left traces), 2 min after drug application (upper trace), and 50 and 60 min after rinsing with APS (lower traces). Corresponding discharge rate histogram is illustrated on the right. (Bins 5 s; mean frequency before drug application: 5.2 spikes s⁻¹; gaps: 40 min; 15 min.) D, functionality of NMDA receptors at calyx synapse. Representative traces of background activity in one calyx terminal under control condition (upper trace), 20 min after the start of the Mg²⁺ free APS + 10 μm glycine (left lower trace), and after 8 min subsequent exposure to d-AP5 (50 μm; right lower trace) (gaps: 40 min; 8 min). Corresponding discharge rate histogram is illustrated on the right. (Bins 5 s; control mean frequency: 0.7 spikes s⁻¹; mean frequency after Mg²⁺ free APS: 4.1 spikes s⁻¹; mean frequency after d-AP5 block: 0.5 spikes s⁻¹.) Traces in A, B, C and D are from different calyx terminals. M.S. mechanical stimulation.