Transfer characteristics of the hair cell's afferent synapse

Abstract

The sense of hearing depends on fast, finely graded neurotransmission at the ribbon synapses connecting hair cells to afferent nerve fibers. The processing that occurs at this first chemical synapse in the auditory pathway determines the quality and extent of the information conveyed to the central nervous system. Knowledge of the synapse's input-output function is therefore essential for understanding how auditory stimuli are encoded. To investigate the transfer function at the hair cell's synapse, we developed a preparation of the bullfrog's amphibian papilla. In the portion of this receptor organ representing stimuli of 400-800 Hz, each afferent nerve fiber forms several synaptic terminals onto one to three hair cells. By performing simultaneous voltage-clamp recordings from presynaptic hair cells and postsynaptic afferent fibers, we established that the rate of evoked vesicle release, as determined from the average postsynaptic current, depends linearly on the amplitude of the presynaptic Ca(2+) current. This result implies that, for receptor potentials in the physiological range, the hair cell's synapse transmits information with high fidelity.

Fig. 1.

In vitro preparation of the amphibian papilla. (A) A differential-interference-contrast micrograph depicts a portion of the split sensory epithelium in which the unmyelinated eighth-nerve fibers contact the basolateral surfaces of two hair cells (HC). Arrowheads, nerve terminals; SC, supporting cell; N, nucleus of dead supporting cell. (B) A stack of confocal images of the intact epithelium depicts hair cells immunolabeled for parvalbumin 3 (red) and afferent nerve terminals labeled for neurofilament-associated proteins (green). (C) A higher-magnification view of a split preparation shows the fine branches of afferent terminals juxtaposed to the basolateral surfaces of hair cells. (D) The terminal branches of a fiber labeled with a fluorescent lipophilic tracer surround a single hair cell. (E) An afferent terminal is labeled with a fluorescent tracer after whole-cell recording. (F) As visualized by transmission electron microscopy, an afferent synapse from the amphibian papilla displays prominent pre- and postsynaptic densities and an osmiophilic presynaptic dense body, or synaptic ribbon, to which a halo of synaptic vesicles is tethered.

Fig. 2.

Spontaneous EPSCs in afferent fibers. (A) The spontaneous EPSCs recorded from two terminals vary in amplitude and timing but display predominantly monophasic waveforms. (B) A subset of EPSCs display complex waveforms suggestive of multivesicular release slightly dispersed in time. (C) The amplitude distribution of spontaneous EPSCs (black) recorded in a particular afferent fiber extends over more than one order of magnitude. Analysis of these 1,216 events showed a mean amplitude of 100 ± 39 pA and an occurrence rate of 24 s⁻¹. The scaled distribution of noise amplitudes is shown in gray. (D) The 10–90% rise times (Left) and time constants of decay (Right) for EPSCs from the same fiber display tight distributions, suggesting that multiple release events are highly synchronized.

Fig. 3.

Spontaneous EPSCs mediated by AMPA receptors. (A) Superimposed records from one fiber reveal the rapid onset and slower, exponential decay of monophasic EPSCs, as well as the variation in rise time. (B) As the postsynaptic membrane potential was varied, averaged EPSCs reversed sign near 0 mV and slowed at more positive potentials. From top to bottom, the holding potentials were +50 mV, +30 mV, 0 mV, −20 mV, −70 mV, and −100 mV. (C) Addition of 100 μM cyclothiazide halved the average decay rate of spontaneous EPSCs, increasing the time constant from 0.65 ± 0.13 ms (n ] 91) to 1.25 ± 0.27 ms (n ] 38). (D) An antagonist of AMPA and kainate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), reversibly blocked EPSCs at a concentration of 10 μM. (E) Nifedipine, a specific antagonist of L-type Ca²⁺ channels, reversibly blocked EPSCs at a concentration of 50 μM.

Fig. 4.

Relationship between presynaptic voltage and evoked EPSCs. In a simultaneous voltage-clamp experiment, the hair cell was confronted with a series of 100-ms depolarizations whose timing is displayed at the bottom of the figure. For depolarizations that evoked near-maximal Ca²⁺ currents, the latency between stimulus onset and the first response decreased, and evoked EPSCs persisted after the termination of the pulse.

Fig. 5.

Voltage and Ca²⁺ sensitivity of transmitter release. (A) Recordings during a simultaneous voltage-clamp experiment depict presynaptic Ca²⁺ currents (middle traces) and EPSCs (bottom traces) evoked by 100-ms hair-cell depolarizations (top traces). (B) Both the presynaptic Ca²⁺ current (circles) and the postsynaptic current (triangles) display a sigmoidal dependence on the test potential. Data are presented as normalized means ± standard errors of the means for four experiments. (C) In each of four paired recordings, the dependence of the postsynaptic current on the presynaptic Ca²⁺ current is approximately linear over a physiologically relevant range of hair-cell membrane potentials, here −56 mV to −31 mV. Clockwise from the upper left, the linear regression coefficients (r²) are 0.88, 0.97, 0.97, and 0.95.