Figure 6. EPSC inward rectification was due to endogenous polyamine block and Ca2+-permeable AMPARs.
(A) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents in medial olivocochlear (MOC) neurons evoked by 1 mM pressure-puffed glutamate near the cell soma. The soma of MOC neurons were dialyzed with an internal pipette solution containing 100 µM or no spermine. In the presence of spermine, glutamate-evoked currents resulted in an inwardly rectifying I-V relation. In the absence of spermine, the rectification was relieved though dialysis, which resulted in a linear I-V relation. Voltage steps ranged from −62.8 mV to +57.2 mV in 20 mV steps; average of 3–10 sweeps per trace. Each sweep was baselined to 0 pA, Bessel filtered at 3000 Hz, and normalized to glutamate-current decays and maximum amplitudes at −62.8 mV. (B) An I-V curve showing the average amplitudes (normalized to -62.8 mV) of glutamate-evoked currents in spermine-free (N = 3) and 100-µM spermine (N = 4) conditions. Error bars are ± SEM. Conditions were significantly different at +37.2 and +57.2 mV (p = 0.019 and 1.2 × 10-6, respectively; two-way analysis of variance (ANOVA) with post-hoc Tukey test). (C) At -82.8 mV, AMPAR-mediated currents were reduced by 55.29 ± 1.60% with bath application of Ca2+-permeable AMPAR antagonist, N-(1-phenylcyclohexyl)-1,5-pentanediamine dihydrobromide (IEM 1925; 25 µM). Wash-out of IEM 1925 resulted in inward currents that recovered to 81.20 ± 2.97% of control. (D) Average of 582 mEPSCs from one neuron. The fast component (τfast) was responsible for 93.2% of the decay amplitude. Fast decay kinetics are indicative of GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs). (E) Inter-event-interval (IEI) distribution of mEPSC activity, 0.02 s bins, 1873 events from three neurons. (F) Amplitude distribution of mEPSCs, 1.5 pA bins.