Spike encoding of neurotransmitter release timing by spiral ganglion neurons of the cochlea

Abstract

Mammalian cochlear spiral ganglion neurons (SGNs) encode sound with microsecond precision. Spike triggering relies upon input from a single ribbon-type active zone of a presynaptic inner hair cell (IHC). Using patch-clamp recordings of rat SGN postsynaptic boutons innervating the modiolar face of IHCs from the cochlear apex, at room temperature, we studied how spike generation contributes to spike timing relative to synaptic input. SGNs were phasic, firing a single short-latency spike for sustained currents of sufficient onset slope. Almost every EPSP elicited a spike, but latency (300-1500 μs) varied with EPSP size and kinetics. When current-clamp stimuli approximated the mean physiological EPSC (≈300 pA), several times larger than threshold current (rheobase, ≈50 pA), spikes were triggered rapidly (latency, ≈500 μs) and precisely (SD, <50 μs). This demonstrated the significance of strong synaptic input. However, increasing EPSC size beyond the physiological mean resulted in less-potent reduction of latency and jitter. Differences in EPSC charge and SGN baseline potential influenced spike timing less as EPSC onset slope and peak amplitude increased. Moreover, the effect of baseline potential on relative threshold was small due to compensatory shift of absolute threshold potential. Experimental first-spike latencies in response to a broad range of stimuli were predicted by a two-compartment exponential integrate-and-fire model, with latency prediction error of <100 μs. In conclusion, the close anatomical coupling between a strong synapse and spike generator along with the phasic firing property lock SGN spikes to IHC exocytosis timing to generate the auditory temporal code with high fidelity.

Figure 1.

Type I spiral ganglion neuron physiology in the acutely explanted organ of Corti with cochlear ganglion. A, Schematic cross-section of the cochlear explant, showing the organ of Corti (right) and the peripheral neurites of type I SGNs (one highlighted in black) connected to somata in the cochlear ganglion (left). Each type I SGN is nonbranched and receives input from one IHC presynapse via a single, postsynaptic bouton-type connection. The synaptic region at the base of the IHC is enlarged in B. B, Intracellular patch-clamp recordings were made from the postsynaptic boutons, connected to their nonmyelinated neurites ≈30 μm in length, which exit the organ of Corti at the foramina nervosa. Beyond this small perforation in the basilar membrane (not on the scheme) are the Na_V1.6-positive heminodes and the beginnings of the myelinated segments, and then several nodes of Ranvier before the cochlear ganglion. Due to the approach vector of the electrode, recorded boutons most likely innervated the hemisphere of the IHC that faced the cochlear ganglion (i.e., the 4 SGNs highlighted in black). Drawings are approximately to scale. C, In current-clamp mode, excitatory stimuli (e.g., arrow) were injected into boutons to measure the time course of depolarization and spike generation (top trace, left). Ca²⁺-dependent synaptic transmission (e.g., arrowheads) from IHCs evoked EPSPs and so-called spontaneously occurring action potentials. D, In the same recording in voltage-clamp mode, neurotransmitter release events from the IHC were measured as EPSCs of variable size and shape.

Figure 2.

Nearly every neurotransmitter release event triggered a spike. A, Synaptic transmission from an IHC evoked spikes in a SGN under current clamp. The arrowhead marks an EPSP that failed to evoke a spike. Age, P19; 356 spikes and 7 failures in 60 s; holding current, −10 pA. B, Left, Distribution of spike heights, measured from a mean baseline potential of −83 mV. Right, Effect of baseline potential on spike height and peak voltage. C, Voltage-clamp recording from the same SGN. Ongoing EPSCs (enlarged in inset) had maximum amplitudes of ∼500 pA. EPSCs measured in voltage-clamp mode did not evoke action currents, which could be differentiated by their shape and larger amplitude. In contrast, a step to −65 mV from the holding potential of −80 mV evoked a stimulus artifact and an action current (arrowheads). D, Interval distributions for action potentials (black) and EPSCs (gray) measured in interleaved current-clamp and voltage-clamp recordings (3 × 20 s in each mode) demonstrate irregular timing; the distributions are similar and suggestive of a Poisson process. Bin size, 50 ms. Overlaid, cumulative probability density functions for spikes and EPSCs were well fit by single exponentials with τ = 151 ms and τ = 182 ms, respectively.

Figure 3.

Ribbon synapse exocytosis evoked spikes with variable latency. A, Histogram of EPSP maximum slopes for IHC-evoked spikes (mean, 148 ± 75 mV/ms; n = 333). Successful EPSPs are color coded from slowest (blue) to fastest (red). Four slow EPSPs failed to evoke a spike (black). B, Histogram of spike onset latencies measured from EPSP onset (mean, 0.59 ± 0.3 ms; n = 333). C, Scatter plot of spike onset latency versus EPSP maximum slope for IHC-evoked spikes (crosses), color-coded as in A. The larger crosses correspond to the short-latency (red) and long-latency (blue) IHC-evoked spikes in D. The large circles are the CC-evoked spikes in D. D, Comparison of IHC-evoked spikes (solid lines) and CC-evoked spikes (dashed lines). Left, Similar IHC-evoked and CC-evoked spikes with long latencies (1.09 and 1.16 ms) and small EPSP maximum slopes (34 and 46 mV/ms). Right, Similar IHC-evoked and CC-evoked spikes with short latencies (0.36 and 0.34 ms) and large EPSP maximum slopes (230 and 228 mV/ms). Short-latency IHC-evoked spikes were smaller in height and thinner at the peak when overshooting 0 mV, consistent with the presence of greater synaptic conductance compared with long-latency IHC-evoked spikes. Bottom, The stimuli for CC-evoked spikes. E, Membrane potential slope versus membrane potential for IHC-evoked spikes (n = 161). The asterisks mark the onset of the spike segments for one fast and one slow EPSP. Each trace is color-coded according to its EPSP maximum slope (as in A and C). The bold traces correspond to the IHC-evoked spikes (solid) and CC-evoked spikes (dashed) in D. Smaller EPSP maximum slopes were associated with larger spike slopes and vice versa. Data are from one SGN aged P19.

Figure 4.

Spiral ganglion neurons respond as phasic high-pass filters with low rheobase. A1, Depolarizing current steps evoked one spike (and rarely a second, smaller spike) near stimulus onset (A2). Hyperpolarizing current steps evoked inward rectification, and rebound spikes at stimulus offset (A3). B, Top, V–I relationships: steady-state voltage (V_SS) as a function of current amplitude for three SGNs (V_SS estimated as mean V_m over last 20 ms of 200 ms current-clamp steps, as in A1). Bottom, Steady-state slope resistance (R_slope) as a function of current amplitude. Zero-current potentials (denoted by perpendicular gray dotted lines) were between −80 and −70 mV, and corresponded roughly to the peak in R_slope. C, Strength–duration functions for eight SGNs stimulated from baseline potentials (V_base) of approximately −80 mV. Rheobase ranged from 35 to 75 pA. Current pulses were applied at 5–20 pA increments. The dotted lines in C and D show the 50 pA level, and insets show double-log scale. D, Strength–duration functions for one SGN from different V_base, demonstrating similar rheobase but increased chronaxie with hyperpolarized V_base. E, Averages of subthreshold responses to 20 pA current steps on absolute and relative scales exhibit a smaller membrane time constant with depolarization of V_base, due to activation of hyperpolarizing membrane conductance. The dashed lines are double-exponential fits up to the response peak: τ_fast and τ_slow were 0.09 and 0.74 ms from −72 mV; 0.09 and 0.81 ms from −82 mV; 0.16 and 3.0 ms from −95 mV. F, Bottom, Ramp stimuli from −35 to +50 pA, for durations of 1–19 ms in 2 ms increments. Top, Ramps briefer than 9 ms failed to generate spikes at these small current levels, demonstrating dependence of spike generation on a minimum charge. Ramps >15 ms also failed, demonstrating dependence of spike generation on a minimum rate of depolarization. G, Ramp from −100 to +100 pA in 100 ms (bottom) demonstrates the increase in membrane conductance during subthreshold depolarization, due to K⁺ current activation before spike onset (top). The arrow labels a spontaneous spike. The dotted line marks the spike onset potential of −68 mV.

Figure 5.

EPSCs of diverse size and waveform were used as stimulus templates. A, EPSC amplitude versus 10–90% rise time demonstrates heterogeneity in size and onset time course (n = 304 EPSCs in 60 s for A–C). B, EPSC amplitude versus charge shows high variability, reflecting heterogeneity in the shape of excitation. C, EPSC FWHM versus charge illustrates the variety of synaptic event durations. D, E, Two EPSCs with a large difference in amplitude, rise time, and FWHM. One is slower and smaller in amplitude than the other, but the two EPSCs have a similar charge (284 vs 245 fC). The small gray EPSC in D is the same as in E, displayed on a different scale. Such broad ranges of EPSC size and kinetics were displayed in individual SGNs from hearing rats (e.g., age P19) and referenced for construction of EPSC-like stimuli for Figures 6–8.

Figure 6.

Effect of stimulus shape and SGN baseline potential on spike latency and jitter depended upon stimulus size. A, Four stimulus shapes (bottom) were scaled to have the same charge. Shapes 1 and 2 (red and black) had linear rise times of 0.3 ms, plateaus of 0.1 ms, and differed only in decay (τ of 0.5 and 1 ms). Shapes 3 and 4 (gold and blue) had linear rise times of 0.8 ms, plateaus of 1 ms, and differed only in decay (τ of 1 and 2 ms). Shapes 1–3 evoked spikes with variable latency (top), but shape 4 failed. In this example, each stimulus delivered 125 fC. B, Each stimulus shape was scaled over a range of amplitudes, conserving charge between shapes. The smallest amplitudes for shapes 1–4 were 83.3, 50, 26, and 18.4 pA, respectively. Larger amplitudes were integer multiples of the smallest ones. Shown are the range of amplitudes for one series of shape 2 (50–700 pA). Selected stimulus–response pairs are in bold. Only the smallest (50 pA) stimulus failed to evoke a spike. C, Spike latency (+SD) versus charge for each stimulus shape (colored as in A) shows reduction of spike latency and jitter with increasing charge. Each data point is the mean of 5–10 repetitions. For small charges the relationship was similar to 1/x (bottom dashed line). For charges >400 fC, the relationship was closer to 1/√x (top dashed line), indicating reduced charge efficiency of spike generation. Note the double-log scale. Waveforms with faster kinetics evoked spikes with shorter latency and less jitter, especially when charge was small. D, Shape 2, Amplitude of 100 pA, delivered from three baseline potentials. Latency depended strongly on baseline potential for such small stimuli. E, Larger amplitude (shape 2, 300 pA) reduced the shift in spike latency associated with changing the baseline potential. F, Spike latency (±SD) versus stimulus amplitude for shape 2 delivered from three baseline potentials (−94 mV, ▿; −83 mV, ○; −72 mV, ▵). Note reduction of latency, jitter, and sensitivity to baseline potential as stimulus amplitude was increased. Inset, Jitter (SD) versus mean latency for 5–10 repetitions of shape 2 at each amplitude, from V_base = −83 mV. Similar trends were obtained with shapes 1, 3, and 4.

Figure 7.

Two-compartment LIF and EIF neuron models predicted spike latency with low error for a broad range of stimuli. A, Hundreds of EPSC-like stimuli (gray) were injected into SGNs (charge from 100 to 700 fC in steps of 100 fC; rise time from 0.1 to 0.8 ms in steps of 0.1 ms; plateau durations from 0 to 5 ms in steps of 0.5 ms; decay τ = 1 ms; amplitudes calculated). The bold colored traces show the largest- and smallest-amplitude waveforms for each of the seven charge sets. Inset, Characteristics used to define shapes. B, Schematic of the two-compartment circuit. Compartment 1 is connected to the pipette and, via an axial resistance (R_axial), to compartment 2. C, Two-compartment LIF model-predicted and experimentally measured voltage responses for one current stimulus. Shown are the data (black line) and the predicted voltages at both compartments (dashed magenta lines). After the voltage crossed threshold (V_Th) at compartment 2, a spike was predicted to occur at a fixed delay D (fixed for all stimuli). The gray dashed lines show the predicted voltage in both compartments for the case of purely passive membranes. Measured AP onset was defined at 0.15 ms before the voltage crossed 20 mV below AP peak. Prediction error (in milliseconds) = measured AP onset minus predicted AP onset. The stimulus, a 70 pA plateau with 0.4 ms linear rise time, started at 0 ms. D, Two-compartment EIF model. All same as in C, but here a spike is generated with a fixed delay D after the predicted voltage in compartment 2 (green) crosses V_T + 10 · Δ_T (see Materials and Methods). A long-latency spike is used for the example in C and D for clarity. E, Model-predicted spike onset latency versus measured spike onset latency for the LIF (magenta) and EIF model (green) demonstrates general accuracy of predictions for latencies from 0.3 to 5 ms. F, Top, Prediction errors versus measured spike onset latency for the LIF model in magenta and EIF model in green (501 responses). rms latency errors δL: LIF, 104 μs; EIF, 83 μs. Fraction of correctly predicted spike occurrences F: LIF, 98.7% (8 extra or missing spikes in a total of 600 stimuli with 506 spikes triggered); EIF, 98.3% (10 extra or missing spikes). Bottom, SD of the prediction error versus measured spike onset latency (calculated using groups of 20 successive points). Model parameters for baseline potential of −82 mV were as follows: double-exponential fit: τ_fast = 0.07 ms, R_fast = 40 MΩ, τ_slow = 2.3 ms, R_slow = 450 MΩ. Two-compartment circuit: R₁ = 1760 MΩ, C₁ = 1.3 pF, R₂ = 600 MΩ, C₂ = 3.8 pF, and R_axial = 75 MΩ. LIF: V_Th = −66.5 mV; fixed delay D = 0.23 ms. EIF: V_T = −68.6 mV; Δ_T = 1.3 mV; fixed delay D = 0.09 ms.

Figure 8.

Effect of current stimulus waveform kinetics on latency: data and model predictions. A, Latency contours in 200 fC parameter space. The black points on the graph represents 88 stimuli of variable amplitude (31–190 pA, y-axis), rise time (0.1–0.8 ms, x-axis), and plateau (0–5 ms, isoplateau bands labeled on right), each with a total charge of 200 fC. Stimuli evoked spikes for all but the smallest waveforms (black X, failure; n = 9). Measured spike onset latencies were plotted as solid black contour lines (1–4 ms, labeled in black). Spike onset latencies predicted by the EIF model are overlaid as green dashed contour lines (green X, predicted failure; n = 1). B, In the 300 fC parameter space, every stimulus evoked a spike. Latencies (black contour lines) were accurately predicted by the EIF model (green dashed contours). C, Spike latency contours for the 500 and 700 fC parameter spaces illustrate reduction of spike latency for larger stimuli; however, reduction in spike latency was reticent when stimuli were increased above 400 fC. D, Stimulus–response pairs for two subthreshold stimuli (100 fC). The stimulus (I_inj, bottom part of each panel) and the response of the cell (V_m) are shown in solid black. The passive response of the model circuit in compartment 1 is shown as a dashed gray line. The response of the EIF model in compartment 2 is shown as a solid green line, where the threshold of −67.5 mV is show by the dotted green line. D1, The data and the passive response in compartment 1 were virtually indistinguishable. D2, Some near-threshold behavior was not well predicted by the passive response. Note the voltage drop between the site of current injection (compartment 1) and compartment 2. E, Two stimulus–response pairs (as in D) from the 200 fC parameter space, labeled in A. The box shows area enlarged in inset. E1, A failure of spike generation where the model predicted a spike. E2, Similar near-threshold stimulus triggered a long-latency spike. F, Two stimulus–response pairs from the 300 fC parameter space, labeled in B. Each inset enlarges the area around spike threshold, where the response of the SGN and the EIF model deviated from the passive response. The EIF model predicted that spike onset occurred after a fixed delay D of 90 μs from when the membrane voltage diverged toward infinity (E, F, dotted green vertical lines). G, Comparing spike onset latency as a function of EPSP maximum slope for CC-evoked spikes (black) and IHC-evoked spikes (blue to red; replotted from Fig. 3C) revealed a very similar relationship.

Figure 9.

Absolute and relative spike threshold and onset potential covary with baseline potential. A, Individual responses to 50 pA current steps from three baseline potentials in one SGN. B, Phase plots for the action potentials in A. The open circles in A and B mark the spike onset potential, defined when slope reached 30 mV/ms. A and B reveal that the spike onset potential depolarized with the baseline potential. C, Absolute (V_m) and relative (V_m − V_base) spike onset potentials, measured using minimum stimulation (ovals). Absolute and relative threshold potentials, predicted at the spike generator, assuming possible solutions for the two-compartment LIF model (bars). All plotted versus baseline potential for four SGNs (black, cell from 3 baseline potentials; white, cell from 2 baseline potentials; 2 shades of gray, 2 cells from different baseline potentials). As a function of baseline potential, changes in onset potential and threshold potential were smaller in relative value than absolute value. Such an effect could reduce the influence of baseline potential on spike onset latency.