Probing AMPA Receptors Activity at Ribbon Synapses in the Mammalian Cochlea by Glutamate Uncaging

JUAN D. GOUTMAN; MONA JAWAD; JAMES HUETTNER; MARK RUTHERFORD

Congreso; 47th Annual Mid Winter Meeting of the Association for Research in Otolaryngology; 2024

BackgroundAMPA receptors in the mammalian brain mediate fast neurotransmission. These glutamate receptors are concentrated in specific regions of the synapse called postsynaptic densities (PSDs), in close opposition to presynaptic active zones. Across synapses a great variability of PSDs sizes and number of AMPA receptors have been described. This morphological and molecular heterogeneity is a determinant of functional responses in the postsynaptic neuron. In the mammalian inner ear, glutamatergic synapses are also formed between inner hair cells (IHCs) and spiral ganglion neurons (SGNs). Postsynaptic terminals of SGNs are characterized by large PSDs, 5-10 times bigger than those found in the brain. Presynaptically, the IHC ribbon synapse supports high rates of vesicular exocytosis onto a single SGN PSD. However, the sizes and locations of events of glutamate release relative to the PSD are not well understood. We speculate that PSDs are not saturated by glutamate during low/moderate rates of release and are able to accommodate large amounts of glutamate released by IHCs. MethodsExplants of the organ of Corti from C57/BL6 mice of 15-17 days of age were used. A glutamate photolysis method was implemented on an upright microscope within an electrophysiology set up. The expanded beam of a 405 nm diode-laser was projected onto the back aperture of a 60x objective producing a diffraction-limited illumination spot at the objective focal plane. The laser light was flashed upon the tissue previously bathed with a caged-glutamate compound (MNI-glutamate), producing fast transients in glutamate concentration. The lateral width of the laser spot was approximately 0.2 m. ResultsResponses to glutamate uncaging were recorded by patch-clamp directly on SGNs terminals. Both intensity and duration of laser pulses could be modulated to generate glutamate transients of different sizes. The relationship was drawn between amplitudes of responses to glutamate photolysis (Iphotol) and laser intensities, obtaining complete curves with maximal responses for n = 4 SGNs. The average maximal Iphotol was 1,483 ± 261 pA with a holding potential (Vh) of -50 mV, which corresponds to 29.6 ± 5.2 nS (or 2,076 ± 366 pA with Vh = -70 mV). In parallel, spontaneous EPSCs were recorded in n = 5 SGNs (>1000 events each) evoked by the superfusion of a high K+ extracellular solution. In agreement with previous results, the EPSC average amplitude was 381 ± 57 pA (Vh = -70 mV), whereas the largest EPSC in each recording averaged 887 ± 129 pA. ConclusionsIn summary, Iphotol were recorded at SGNs terminals in response to brief (0.05 – 0.5 ms) laser pulses of different intensities. The maximal Iphotol was 2.3X greater than the largest spontaneous EPSC recorded in high-K+ solution and 3.9X larger than the average EPSC, suggesting that AMPA receptors on PSDs at SGNs are not saturated during low/moderate neurotransmission.