L-type calcium channels refine the neural population code of sound level

Abstract

The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (Ca_L: Ca_V1.1-1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of Ca_L to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. Ca_L is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, Ca_L activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, Ca_L boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, Ca_L either suppresses or enhances firing at sound levels that evoke maximum firing. Ca_L multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level.

Keywords: auditory midbrain; dynamic range; inferior colliculus; level tuning; local circuits; rate-level functions.

Fig. 1.

Calcium currents in ICC neurons. A: calcium currents (I_Ca) are illustrated for a series of voltage steps to +50 mV. Holding potential is −90 mV. B: the current remaining after addition of NNC-HCl (20 μM) and ωCTx-GVIA (100 nM) is abolished by nimodipine (10 μM). C: I_Ca current-voltage (I-V) curves illustrate differences in activation thresholds as well as maximum currents of the different components. Currents plotted are the maximum inward current at each membrane potential. All I-V curves are from currents evoked from a −90-mV holding potential (16 neurons). D: nimodipine dose-response curve shows an IC₅₀ of 4.68 μM and saturation at ∼10 μM. To obtain dose-response curves, different concentrations of nimodipine were tested on the I_Ca that remained in NNC-HCl and ωCTx-GVIA (5 cells).

Fig. 2.

Establishment of an effective nimodipine concentration for in vivo recordings. A: i, nimodipine dose-response curve shows responses saturating at 50 μM. Firing rates were measured at sound levels that evoked maximum firing in each neuron (11 cells: 5 monotonic, 6 nonmonotonic). ii, Nimodipine (50 μM) does not change spike heights or widths. Spikes were evoked by a 100-ms CF tone at 40 dB SPL. Center insets: expanded traces of spikes before (top) and after (bottom) nimodipine was added. Population average: 100 spikes, 8 cells. B: slice recordings show effects of 50 μM nimodipine on K⁺ currents evoked in an external calcium concentration of 0.1 mM. i, K⁺ currents evoked by a step to +30 mV from a holding potential of −60 mV show no change in peak current or inactivation in nimodipine. ii, I-V curves shown before and after nimodipine overlap and are not significantly different.

Fig. 3.

Consistency of nimodipine effects in vivo. A: absence of artifacts from pressure application of nimodipine. i, Spike rasters show uninterrupted firing during nimodipine puffs (arrow; 3 puffs at sweep 66). 120 sweeps were recorded at 60 dB SPL. ii, First spike latencies (ΔFSL) are unchanged in nimodipine (8 cells). B: repeated applications of 50 μM nimodipine yield consistent effects on firing rates. i and ii, monotonic cells; iii, nonmonotonic cells. Firing rates were allowed to recover completely between nimodipine applications. Error bars are SE. iv, Population average of responses to the second and third nimodipine applications compared with the first application (second application, 9 cells; third application, 6 cells). Values in A, Bii, Cii, and Div are means ± SD.

Fig. 4.

Nimodipine effects on monotonic RLFs vary proportionally with dynamic range. A: measurement of dynamic range (DR). RLFs were fit with sigmoidal functions, and DR was measured between 10 and 90% of the maximum firing rate. B: nimodipine effects on 5 monotonic RLFs with decreasing DR (top to bottom). Firing rates decreased in cells with DRs of 65, 50, and 40 dB and increased in cells with DRs of 14 and 10 dB. CFs (top to bottom) are 32, 21, 26, 14, and 9 kHz. Error bars are SE. C: bimodal distribution of control DRs (39 cells; 5-dB bin width). D: cluster analysis. i, Change in maximum firing rate after nimodipine application as a function of the DR of the control RLF. Top, 2-cluster (C1, C2) grouping. Gray lines are linear fits. Bottom, 3-cluster (c1, c2, c3) grouping. Cent, centroid of each cluster. ii, Silhouette values for the 2- and 3-cluster groups (n = 39 cells). E: i, Ca_L gain as a function of sound level for clusters C1 and C2. Ca_L gain was obtained by dividing the control RLF by the RLF in nimodipine [C1, sigmoidal fits are plotted (r² ≥ 0.99874 for all cells); C2, unfitted curves; 6 cells in each panel]. ii, Population average of Ca_L gain (C1, n = 10 cells; C2, n = 29 cells). Averaged curves were fit with a sigmoid function. Error bars (SD) are shown at 20, 40, 60, and 80 dB SPL. F: maximum Ca_L gain as a function of DR. Best fits (red lines) were linear for C1 and exponential for C2 (n = 39 cells). G: DRs themselves did not change in nimodipine. Gray bars represent individual cells. Black bar represents population mean (n = 39 cells).

Fig. 5.

Nimodipine effects on nonmonotonic RLFs vary inversely with their width. A: nonmonotonic RLFs before and after nimodipine application in 6 cells with increasing RLF width (top to bottom). CFs (top to bottom) are 12, 11, 41, 22, 27, and 31 kHz. Error bars are SE. B: unimodal distribution of control RLF widths. RLF widths were measured at half-maximal firing (37 cells; 5-dB bin width). C: relationship between changes in peak firing in nimodipine and RLF width. Red line indicates sigmoidal fit. D: Ca_L gain as a function of sound level for cells with RLF widths >30 dB (i) and <30 dB (ii). Ca_L gain was obtained by dividing the control RLF by the RLF in nimodipine. Individual functions were fitted with a Gaussian function (r² ≥ 0.96612 for all cells). Gains are plotted with reference from a gain of 1, irrespective of whether nimodipine increased or decreased firing. E: Ca_L gain as a function of RLF width (37 cells). A divisive gain (<1) increases as RLF width narrows. Red line indicates double-exponential fit. F: RLF widths do not change in nimodipine (37 cells). Gray lines represent individual cells. Black line represents population mean. G: asymmetric effects of nimodipine on RLFs. i, Difference integrals of the rising and falling phases of the RLF for 1 cell. Integrals are the product spikes/s × dB. ii, Difference integrals in the nonmonotonic population. Ca_L gain is greater during the falling phase compared with the rising phase in all cells (31 cells).

Fig. 6.

Temporal integration increases Ca_L contribution to monotonic firing. A–C: raster plots, RLFs, and changes in gain for 1 monotonic neuron at different times during a 100-ms tone. A: single sweeps recorded at different sound levels in control (top) and nimodipine (middle). In nimodipine, the decrease in firing occurs during the sustained portion of the tone (e.g., at >50 ms after tone onset). Bottom, sweeps at high sound levels (76, 81, and 86 dB SPL) clearly illustrate the loss of spikes at later times during the tone (e.g., boxed area). B: RLFs measured over the whole tone (cumulative; 5–105 ms) and in successive 20-ms windows. C: Ca_L gain in different time windows. Among the windowed gains, Ca_L gain is the highest between 47 and 67 ms, and stays at that level at later times. Ca_L gain integrated over the whole tone (Cum) is similar to the gain in the 47- to 67-ms window. Ca_L gain was obtained by dividing the control RLF by the RLF after nimodipine application. Sigmoidal fits of the resultant RLF ratio are plotted. D: population average of Ca_L gains. The maximum increase in Ca_L gain is reached after 40–60 ms of integration time. The cumulative Ca_L gain is similar to the gain in the 40- to 60-ms window. Each point on the abscissa represents the firing rate averaged over the preceding 20 ms (20 ms = 0–20 ms, 40 ms = 20–40 ms, etc.). All time values are referenced to the first-spike latency for each neuron. Data are from monotonic RLFs with DRs ≥45 dB (n = 27). Values are means ± SD.

Fig. 7.

Temporal integration does not change Ca_L contribution to nonmonotonic firing. A–C: raster plots, RLFs, and changes in gain for 1 nonmonotonic neuron at different times during a 100-ms tone. A: single sweeps recorded at different sound levels in control (top) and nimodipine (middle). Increased firing in nimodipine occurs throughout the tone. Bottom, single sweeps at the preferred level of 51 dB SPL and two flanking levels, 46 and 56 dB SPL, illustrate increased spiking in nimodipine throughout the tone. B: RLFs measured over the whole tone (cumulative; 5–109 ms) and in successive 20-ms windows. RLFs remain nonmonotonic in the different time windows. C: Ca_L gain in different time windows. The maximum gain is similar in different time windows and when integrated over the whole tone (Cum). Ca_L gain was obtained by dividing control RLFs by RLFs in nimodipine and fitting the resultant RLF with a Gaussian function (r² ≥ 0.953 for all time windows). D: population averages of Ca_L gains in different time windows during the tone. Each point on the x-axis represents the firing rate averaged over the preceding 20 ms (20 ms = 0–20 ms, 40 ms = 20–40 ms, etc.). All time values are referenced to the first-spike latency for each neuron. Data are from nonmonotonic RLFs that exhibited changes in firing rate in nimodipine (n = 31). Values are means ± SD.

Fig. 8.

Summary of multiplicative and divisive effects of Ca_L on sound level codes. Exponential fits of Ca_L gain vs. the dynamic range of monotonic RLFs and the width of nonmonotonic RLFs. Fits are extracted from Figs. 4F and 5E, respectively. In level-variant RLFs with wide dynamic range, Ca_L raises firing rates proportionally with dynamic range. In level-tuned RLFs, Ca_L decreases peak firing rates inversely with the width of level tuning. In sum, Ca_L refines the representation of sound level in the ICC by increasing the sensitivity of level-variant responses to high sound levels and lowering the sensitivity of level-tuned responses at preferred sound levels.