Synaptic transfer from outer hair cells to type II afferent fibers in the rat cochlea

Abstract

Type II cochlear afferents receive glutamatergic synaptic excitation from outer hair cells (OHCs) in the rat cochlea. However, it remains uncertain whether this connection is capable of providing auditory information to the brain. The functional efficacy of this connection depends in part on the number of presynaptic OHCs, their probability of transmitter release, and the effective electrical distance for spatial summation in the type II fiber. The present work addresses these questions using whole-cell recordings from the spiral process of type II afferents that run below OHCs in the apical turn of young (5-9 d postnatal) rat cochlea. A "high potassium puffer" was used to elicit calcium action potentials from individual OHCs and thereby show that the average probability of transmitter release was 0.26 (range 0.02-0.73). Electron microscopy showed relatively few vesicles tethered to ribbons in equivalent OHCs. A "receptive field" map for individual type II fibers was constructed by successively puffing onto OHCs along the cochlear spiral, up to 180 μm from the recording pipette. These revealed a conservative estimate of 7 presynaptic OHCs per type II fiber (range 1-11). EPSCs evoked from presynaptic OHCs separated by >100 μm did not differ in amplitude or waveform, implying that the type II fiber's length constant exceeded the length of the synaptic input zone. Together these data suggest that type II fibers could communicate centrally by maximal activation of their entire pool of presynaptic OHCs.

Figure 1.

Procedure. A, Differential interference contrast image of the experimental preparation. Stereociliary bundles of the OHCs are visible. Modiolus is to the left, out of view. Electrode (bottom, tip below plane of focus) patched onto a type II afferent dendrite spiraling under OHCs. Puff pipette (top) positioned at the apical pole of a targeted OHC. Scale bar, 10 μm. B, Exemplar map of OHCs targeted by puffer pipette while recording from a postsynaptic type II dendrite (different scale than A). Three rows of circles indicate three rows of OHCs. Black circles: OHCs removed to expose dendrites at recording site. White circles: unstimulated OHCs. Blue circles: stimulated OHCs that did not evoke postsynaptic EPSCs. Red circles: stimulated OHCs that evoked EPSCs in the postsynaptic type II afferent.

Figure 2.

Stimulation of individual OHCs by puffs of high potassium saline. A, Current-clamp recording from a row 3 OHC; 10 ms duration puff of 40 mm KCl extracellular solution at vertical red line elicited an action potential. B, Overlay of responses from a single OHC when high potassium solution was puffed directly onto that cell, or onto the adjacent OHC (green) or two hair cells away (blue) in the same row. C, Overlay of responses from a single OHC when high potassium solution was puffed directly onto that cell (black trace, row 3) or onto the adjacent row 2 (green) or row 1 (blue) OHC. D, A single OHC spiked without fail in response to repeated 10 ms puffs of high potassium solution (at red lines). With an interpuff interval of ∼1.2 s (puffs 1–6) there was no change in baseline membrane potential. At interpuff intervals of ∼400 ms the baseline membrane potential depolarized by ∼5 mV.

Figure 3.

In separate experiments, high potassium puffs evoked action potentials in OHCs and EPSCs in type II afferents. A, Overlay of current-clamp recordings of responses from 4 OHCs; 10 ms duration puff of 40 mm KCl solution at t = 0 in A–C. B, Voltage-clamp traces from two type II afferents (different experiments from A) showing waveform of evoked EPSCs. EPSCs were in the interval between 7 and 40 ms in which OHC spikes occurred, and during which EPSCs were classified as “evoked.” Red trace from a different experiment than remaining traces. C, Scatterplot showing timing of all EPSCs evoked by puff stimulation of a single OHC, 109 μm from the recording site. A different experiment than in B.

Figure 4.

EPSCs evoked in type II afferents by OHC stimulation. A, Voltage-clamp recording from a type II afferent dendrite, holding potential −90 mV. Vertical red lines indicate the timing of a puff of high potassium solution onto a nearby OHC, here 30 μm away. EPSCs visible as downward deflections followed a fraction of puffs onto the OHC. B, Zoom of evoked EPSC from A indicated by star. Ci, Amplitude histogram of EPSCs evoked in a type II afferent by repeated stimulation of a single OHC located 30 μm from the recording site. Red curve represents Gaussian fit of data. Cii, Amplitude by 10–90% rise time scatterplot of EPSCs from Ci. Ciii, Amplitude by time constant of decay (single exponential fit) scatterplot of EPSCs from Ci. Di, Amplitude histogram of EPSCs evoked in the same type II afferent as in Ci, during repeated stimulation of a different OHC 109 μm from the recording site. Red curve represents Gaussian fit of data. Dii, Amplitude by 10–90% rise time scatterplot of EPSCs from Di. Diii, Amplitude by time constant of decay (single exponential fit) scatterplot of EPSCs from Di.

Figure 5.

Electron micrographs of synaptic ribbons in IHCs and OHCs of P7–P9 rat cochleae (apical turn). A, Synaptic ribbon in an IHC (P7 rat). B, Synaptic ribbon in an IHC (P9 rat). C, Serial reconstruction (4 sections) of IHC ribbon from A. D, Synaptic ribbon in an OHC (P9 rat). E, Synaptic ribbon in a different OHC (P9 rat). F, Serial reconstruction (4 sections) of OHC ribbon from D. Reconstructions (C, F) show ribbon in turquoise, hair cell membrane in green, vesicles in yellow and afferent in burnt orange. Scale bars, 200 nm.

Figure 6.

Mapping of functional synaptic inputs to type II afferent dendrites. A, Tracing of AlexaFluor 488 hydrazide fill from a type II afferent from which recordings were made while stimulating OHCs. Overlay: Diagram illustrating position of stimulated OHCs, same scale as fiber tracing. Three rows of circles indicate three rows of OHCs. Black circles: OHCs removed to expose dendrites at recording site. White circles: unstimulated OHCs. Blue circles: stimulated OHCs that did not evoke postsynaptic EPSCs. Red circles: stimulated OHCs that evoked EPSCs in the postsynaptic type II afferent. B, Amplitude of EPSCs evoked from single OHCs by distance from the recording electrode, same experiment as shown in A. EPSC data aligned with map in A. Red line indicates linear fit of data. C, Scatterplot of EPSC amplitude plotted by distance from the recording site in 8 type II fibers. Each color represents EPSCs recorded from a different type II dendrite with linear regression in same color for each dataset.

Figure 7.

Receptive field maps for eight type II afferent dendrites. Three rows of circles indicate three rows of OHCs. Black circles: OHCs removed to expose dendrites at recording site. White circles: unstimulated OHCs. Blue circles: stimulated OHCs that did not evoke postsynaptic EPSCs. Red circles: stimulated OHCs that evoked EPSCs in the postsynaptic type II afferent. A–D, 1 s duration puffs evoked multiple EPSCs from OHCs indicated by red circles. Due to tissue orientation, OHC row number could not be determined in experiment in A. E, 100 or 10 ms duration puffs of high potassium solution evoked EPSCs from some OHCs. F–H, 10 ms puffs of high potassium solution were used to evoke synaptic release from OHCs.

Figure 8.

OHC depolarization by “whole-field” application of high potassium saline. Representative OHC recording shown. A large bore perfusion pipette was used to apply 40 mm potassium extracellular solution to the tissue while performing whole-cell current-clamp recordings from an OHC. The OHC responded with a slow depolarization, followed by an average of 8 spikes (n = 4 OHCs). The OHC membrane potential then reached a plateau depolarization for the remainder of the high potassium application.