A "synaptoplasmic cistern" mediates rapid inhibition of cochlear hair cells

Abstract

Cochlear hair cells are inhibited by cholinergic efferent neurons. The acetylcholine (ACh) receptor of the hair cell is a ligand-gated cation channel through which calcium enters to activate potassium channels and hyperpolarize the cell. It has been proposed that calcium-induced calcium release (CICR) from a near-membrane postsynaptic store supplements this process. Here, we demonstrate expression of type I ryanodine receptors in outer hair cells in the apical turn of the rat cochlea. Consistent with this finding, ryanodine and other store-active compounds alter the amplitude of transient currents produced by synaptic release of ACh, as well as the response of the hair cell to exogenous ACh. Like the sarcoplasmic reticulum of muscle, the "synaptoplasmic" cistern of the hair cell efficiently couples synaptic input to CICR.

Figure 1.

Ryanodine receptors in hair cells. a, Schematic of the efferent synapse and synaptoplasmic cistern. b, RT-PCR for the three RyRs and IP₃R reveal that all but RyR2 was found in microdissected samples of OHCs and Dieters' cells. Lanes 1 and 9, 100 bp ladder (arrow at 500 bp). Samples 2-7 (run concurrently as a single gel), Combined OHCs and Deiters' cells, IP₃R1, IP₃R2, IP₃R3, RyR1, RyR2, and RyR3 primers, respectively, plus and minus RT each lane. Sample 8, α9 positive control for OHCs. When OHCs alone were used as template, only RyR1 was found (sample 10), along with the positive α9 control (sample 11). c, A cross section of the adult rat organ of Corti is highlighted to show the position of OHCs and IHCs. The section outline, tunnel of Corti, four OHCs, and one IHC are drawn in d, the same cross section as in c. Black dots are precipitated silver grains attributable to emission from radioactive in situ probe to RyR1. OHCs, but not IHCs, reveal RyR1 in apical turns of the adult rat cochlea.

Figure 2.

Intracellular calcium store modulators on ACh-evoked potassium currents in OHCs. All effects were observed at -30 mV. a, The SERCA antagonist CPA (10 μm) reduced the ACh-evoked current by 43 ± 6% (5 of 5 OHCs). b, Caffeine (5 mm) produced an initial augmentation (14 ± 4%; 3 of 4 OHCs) followed by a gradual reduction of the ACh-evoked current (65 ± 6%; SE, 4 of 4 OHCs). c, At 0.5-1 μm, ryanodine facilitated the ACh response by 60 ± 29% (6 of 6 OHCs). d, The amplitude of the ACh-evoked response was reduced by 39 ± 4% (6 of 6 OHCs) in the presence of 100 μm ryanodine. e, Ryanodine (100 μm) did not reduce the isolated nAChR current in three BAPTA-buffered OHCs.

Figure 3.

Synaptic currents (IPSCs) in rat OHCs. a, Potassium saline (40 mm) caused a burst of IPSCs.b, Averaged IPSC waveform obtained before (n = 700), during (n = 440), and after (n = 406) exposure to 100 μm ryanodine. c, Cumulative fraction plot of IPSC amplitude. IPSC amplitudes shifted to smaller values in 100 μm ryanodine.d, IPSC amplitude, but not time course, was changed by 100 μm ryanodine. Average peak amplitude was -95 ± 2.0 pA (n = 700; 5 cells) in control, -64 ± 1.6 pA (n = 440; 5 cells) in ryanodine (6 min), and -79.4 ± 2.3 pA (n = 406; 5 cells) after washing. Rise and decay times of single, well segregated IPSCs showed no effect of ryanodine. The mean 10-90% rise time was 17.4 ± 0.5, 17.1 ± 0.5, and 16.9 ± 0.5 msec respectively in control, ryanodine, and wash conditions. Single exponential decay time constants (in milliseconds) were 45.5 ± 1.8 in control, 43.4 ± 1.4 in ryanodine, and 46.8 ± 2.3 after washing. IPSC frequency (f) dropped from 4.5 to 2.9 Hz in the presence of 100 μm ryanodine but did not recover. e, IPSC amplitude distributions shifted to larger values with 1 μm ryanodine or 3 μm cADPR intracellular. f, IPSCs were -145 ± 2.3 pA in control (n = 849; 17 haircells), -207 ± 2.5 pA in 3 μm cADPR (n = 1446; 11 cells), -210 ± 2.5 in 1 μm ryanodine (n = 883; 4 cells). Rise times (in milliseconds; 10-90%) were 18.8 ± 0.2, 18.4 ± 0.1, and 17.0 ± 0.1. Decay times (in milliseconds) were 51.2 ± 0.6, 54.9 ± 0.5, and 44.6 ± 0.4. IPSC frequency was 0.25 Hz in control cells, 0.41 Hz in cADPR, and 0.57 Hz in ryanodine. Error bars represent SE.