Maturation of Spontaneous Firing Properties after Hearing Onset in Rat Auditory Nerve Fibers: Spontaneous Rates, Refractoriness, and Interfiber Correlations

Abstract

Auditory nerve fibers (ANFs) exhibit a range of spontaneous firing rates (SRs) that are inversely correlated with threshold for sounds. To probe the underlying mechanisms and time course of SR differentiation during cochlear maturation, loose-patch extracellular recordings were made from ANF dendrites using acutely excised rat cochlear preparations of different ages after hearing onset. Diversification of SRs occurred mostly between the second and the third postnatal week. Statistical properties of ANF spike trains showed developmental changes that approach adult-like features in older preparations. Comparison with intracellularly recorded EPSCs revealed that most properties of ANF spike trains derive from the characteristics of presynaptic transmitter release. Pharmacological tests and waveform analysis showed that endogenous firing produces some fraction of ANF spikes, accounting for their unusual properties; the endogenous firing diminishes gradually during maturation. Paired recordings showed that ANFs contacting the same inner hair cell could have different SRs, with no correlation in their spike timing.

Significance statement: The inner hair cell (IHC)/auditory nerve fiber (ANF) synapse is the first synapse of the auditory pathway. Remarkably, each IHC is the sole partner of 10-30 ANFs with a range of spontaneous firing rates (SRs). Low and high SR ANFs respond to sound differently, and both are important for encoding sound information across varying acoustical environments. Here we demonstrate SR diversification after hearing onset by afferent recordings in acutely excised rat cochlear preparations. We describe developmental changes in spike train statistics and endogenous firing in immature ANFs. Dual afferent recordings provide the first direct evidence that fibers with different SRs contact the same IHCs and do not show correlated spike timing at rest. These results lay the groundwork for understanding the differential sensitivity of ANFs to acoustic trauma.

Keywords: auditory nerve fiber; development; hair cell; intrinsic firing; refractoriness; spontaneous rate.

Figure 1.

APs can be unambiguously identified in loose-patch extracellular recordings. A, B, Representative traces of a loose-patch extracellular recording from an ANF ending. Spikes in the recording were blocked by TTX (A). Remaining smaller events were blocked by NBQX in the same fiber (applied previously and washed out), and thereby identified as EPSPs (B). C, Average waveform of spikes in A shows inflection in the rising phase (arrowhead). D, Amplitude histogram with normalized numbers of APs (blue), EPSPs (red), and noise (yellow) showing a clear separation of these three groups. E, Superimposed extracellularly recorded spikes from 10 ANFs (left in light blue), intracellularly recorded spikes from 6 ANFs (middle in gray), and their average waveforms (left in dark blue and middle in black). At right, the average waveforms are compared, along with the scaled first derivative of the intracellular waveform. Note the similarity of the first derivative with the extracellular waveform. F, Whole-cell voltage-clamp recording from an ANF bouton showing typical spontaneous EPSCs. Two EPSCs are “multiphasic” with several peaks and one is “monophasic,” with a single peak. Detected event times are marked with red X's. Holding potential was −99 mV.

Figure 2.

Extracellular spike trains and intracellular EPSCs recorded in vitro show fractal behavior. A1, A2, Rate plots from one ANF showing fluctuations of SR at different bin sizes (30 and 3 s). A1, Black bar represents the region covered by the abscissa in A2. A3, Rate plot of the same recording after shuffling the order of IEIs; bin size is the same as A1. Note the reduced fluctuation of rate. A4, Fano factor time curve for the same recording, showing a power-law increase in Fano factor at longer bin widths (navy blue) and the loss of this increase when the order of the IEIs was shuffled (green). FD, Fractal dimension (slope of the cyan line on log-log coordinates). A4, The bin width for A1, A2, and A3 are marked (red circles). B, Fano factor time curve of an in vivo cat ANF recording, for comparison (T. Ropp and E.D.Y., unpublished data). C, A second example of an in vitro recording from an ANF terminal with lower SR. D1, E1, Rate plots of EPSC counts from two representative whole-cell voltage-clamp recordings, showing rate fluctuations. Bin width is 10 s. D2, E2, Fano factor time curves of the same voltage-clamp recordings showing the same fractal features in presynaptic release as in spike trains.

Figure 3.

Diversification of SRs after hearing onset in ANFs recorded in vitro. A, Plot of SRs of ANFs recorded at different ages. Navy blue dots represent the mean SR for an individual ANF. Cyan error bars indicate SEs with bin size of 1 s. Red symbols represent the median rates. Red error bars indicate the first and third quartiles of each age group. Median SR for P15-P17 (n = 23), P19-P21 (n = 50), and P29-P32 (n = 51) were 3.87, 6.80, and 12.85 spikes/s, respectively. Three-sample permutation test was used to compare medians due to the skewed distribution of values. The median SR was significantly higher at P29-P32 compared with P19-P21 (p = 0.04), also at P19-P21 compared with P15-P17 (p = 0.03), and P29-P32 compared with P15-P17 (p = 0.003). B, Normalized histograms of SRs for ANFs recorded at P15-P17, P19-P21, and P29-P32. Bin size is 3 spikes/s. C, SR (blue) during one experiment in which bath temperature (red) was varied. SR increased when the bath temperature was increased and stayed at the elevated rate when the bath temperature stabilized at 29°C–30°C (pink bar). SR reversed to control values with restoration of the 23°C bath in this case. D, SR versus temperature for 6 such experiments during the temperature increase only. The navy blue fiber is the same unit as in C. Note the wide range of slopes of the linear fits for the data points (1.93–17.15 spikes/s °C). *p < 0.05. **p < 0.01.

Figure 4.

Characteristics of in vitro ANF recordings. A–D, Example traces (A1, B1, C1, D1), IEI histogram (A2, B2, C2, D2), and hazard function plots (A3, B3, C3, D3) of in vitro ANF recordings in rat. A, C, D, Spikes in loose-patch extracellular recordings. B, EPSCs in an intracellular recording. Short intervals with a few events in the hazard function represent the ARP (e.g., C3, D3, green bars). Some recordings exhibited a peak at IEIs slightly larger than the ARP (green arrows), demonstrating the preferred occurrence of these IEIs. Bin size for IEI histogram and hazard function plots are shown on the IEI histogram. D1, Inset, One of the AP bursts in D1 at an expanded time scale, demonstrating the regular intervals between APs; these form the large peak at the green arrows in D2, D3. E, IEI histogram and hazard function plot of an in vivo ANF recording in cat. Red lines indicate least-squares fits to the IEI histogram; they show exponential decay of IEI probability for reference. The hazard rate is the absolute value of the slope of these lines; the hazard rate is shown as a horizontal red line on the hazard functions. Note slightly faster than exponential decay in most examples (A2, B2, E1).

Figure 5.

Changes in the properties of ANF spike trains with age. A–D, Histogram and box plots represent properties of ANF spike trains in three age groups. A, The percentage of fibers with peak in their hazard function plot decreased from 79% at P15-P17 to 42% at P19-P21 and then to 33% at P29-P32. B, The relative peak size decreased significantly over age (H₍₂₎ = 16.91, p = 0.0002): from P15-P17 (median = 16.75) to P19-P21 (median = 1.35, p = 0.003) and to P29-P32 (median = 1.23, p = 0.78) (between P15-P17 and P29-P32, p = 0.0003). C, The peak time for fibers with peak shifted to significantly smaller values over age (H₍₂₎ = 8.77, p = 0.01): from P15-P17 (median = 15.5 ms) to P19-P21 (median = 13.5 ms, p = 0.35), and to P29-P32 (median = 12.5 ms, p = 0.28) (between P15-P17 and P29-P32, p = 0.01). D, The ARP was significantly shortened over age (H₍₂₎ = 7.78, p = 0.02): from P15-P17 (median = 10.05 ms) to P19-P21 (median = 3 ms, p = 0.03) and to P29-P32 (median = 5.85 ms, p = 0.88) (between P15-P17 and P29-P32, p = 0.06). B–D, Kruskal–Wallis rank sum test was used with post hoc pairwise comparisons using Nemenyi test with χ² approximation for independent samples. χ² values were corrected for ties present in the data. E, Among recordings obtained at P19-P21 and P29-P32, the ARP of fibers with peak (blue triangle, median = 7.95 ms) was significantly longer than those without peak (navy blue dot, median = 2.5 ms, W = 900.5, p = 2.40 × 10⁻⁵). F, Among all fibers, the duration of the ARP was significantly correlated with the logarithm of the relative peak size (τ = 0.37, p = 7.60 × 10⁻⁷). G, Among all fibers with a peak, the duration of the ARP was significantly correlated with peak position (τ = 0.45, p = 0.0002). H, Fibers with peak (blue triangle, median = 23.39 spikes/s) did not differ significantly in their median SRs from fibers without peak (navy blue dot, median = 14.82 spikes/s, W = 696, p = 0.09). E, H, The median of group data with first and third quartiles (error bars) is shown left to the individual data. Wilcoxon rank sum test was used due to non-normal data distributions or the small sample size. I, J, Among all fibers, SR did not have a significant correlation with either the logarithm of the relative peak size (τ = −0.10, p = 0.17) or the duration of the ARP (τ = 0.02, p = 0.74). Only fibers with spike counts ≥2000 were included in this analysis. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 6.

Presynaptic and postsynaptic origin of spontaneous activity in ANFs. A, Percentage of spontaneous activity blocked by addition of 0.5 mm CdCl₂ to block calcium channels. The significant higher percentage of block that was achieved in fibers without peak (without preferred IEIs, navy blue circles, median = 100%) compared with fibers with peak (with preferred IEIs, blue triangles, median = 70%, W = 17, p = 0.003) suggests the presence of nonsynaptically activated APs in fibers with preferred IEIs. B, Percentage of spontaneous activity blocked by NBQX (filled symbols; 10–50 μm) or GluR blocker mixture (open symbols; 20 μm NBQX, 20 μm (RS)-CPP for NMDA receptors, 1 mm (RS)-α-methyl-4-carboxyphenylglycine for mGluRs, 50 μm CNQX for kainate/AMPA receptors). Percentage of block was not significantly different between fibers with (open and filled blue triangles, median = 30%) and without preferred IEIs (open and filled navy blue circles, median = 90%, W = 74, p = 0.18). A, B, The median of group data with first and third quartiles (error bars) is shown left to the individual data. A, B, The percentage of block was calculated by comparing the average rates of 1 min recording segments before and after 2 min of drug application. Wilcoxon rank sum test was used due to non-normal data distributions. C, D, APs were color-coded according to the waveform. Blue represents APs with inflections (arrowhead) in the rising phase. Red represents APs without inflections in the rising phase. C, Left, Representative traces of one fiber before or during NBQX application. Middle, Events indicated by arrows in traces on the left are enlarged for better visualization of the detailed waveform. Right, Average waveform and its first derivative for events in this recording before or during NBQX application. Arrowheads point to inflections in the rising phase. Events with inflections in the rising phase were lost during NBQX application, suggesting that they were initiated by synaptic activity. D, Left, Representative trace of one fiber with preferred IEIs, in which two bursts are identified as “a” and “b.” The first AP in burst “a” shows an inflection in the rising phase but not the following APs. None of the APs in burst “b” shows an inflection in the rising phase. Middle, Right, Average waveform and its first derivative for the first spikes and the following spikes, respectively, within 50 identified bursts in this recording. **p < 0.01.

Figure 7.

Double recordings of two ANFs contacting the same IHC. A, For 16 pairs, two ANFs (fiber 1 and 2) that terminate on the same IHC were recorded simultaneously and showed different SRs. B1, C1, Representative traces from two paired recordings of ANFs contacting the same IHC. B2, C2, Cross-correlograms of spike timing for two ANF pairs in B1 and C1, respectively. Bin size is 1 ms.