Enhanced Activation of HCN Channels Reduces Excitability and Spike-Timing Regularity in Maturing Vestibular Afferent Neurons

Step-evoked firing patterns

We classified neurons into two broad groups based on the firing patterns evoked by depolarizing current injections: transient-firing (83 of 146 neurons; Fig. 1A) and sustained-firing (63 of 146 neurons; Fig. 1B–D). These classifications are similar to those used by Kalluri et al. (2010) and Iwasaki et al. (2008). Transient-firing neurons displayed strong spike-train accommodation, firing a single spike at the onset of the current step. Beyond spike threshold, the firing pattern of most transient-spiking neurons was invariant to intensity. In a few rare cases, transient-firing neurons fired a second spike when injected with high amplitude currents (4/83 neurons; data not shown). Sustained-firing neurons displayed varying degrees of accommodation, ranging from cells that fired long trains of spikes (sustained-A firing patterns had spikes throughout the depolarizing step; Fig. 1D) to cells with intermediate degrees of accommodation (sustained-B/sustained-C firing patterns had shorter trains of spikes followed by voltage oscillations; Fig. 1C, B, respectively). Consistent with Kalluri et al. (2010), the degree of accommodation depended on intensity for the sustained-firing patterns. Figure 1E shows a sustained-firing pattern at a recording temperature between 35°C and 37°C.

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Figure 1.

Vestibular ganglion neurons produce different firing patterns in response to depolarizing current steps. A, Transient-firing neurons are characterized by a single action potential at the onset of suprathreshold depolarizing steps. B–D, Sustained-firing patterns are more variable. B, Sustained-C neurons have one or two spikes followed by prominent voltage oscillations. C, Sustained-B neurons show adaptation, producing several spikes at current onset and then cease firing (accommodate). D, Sustained-A neurons produce long spike trains throughout the current step. E, Sustained-A neuron at 35–37°C in 500 μm db-cAMP, which produces more spikes than in sustained-A neuron shown in D. The amplitude of the depolarizing current step that evokes spikes (current threshold) is shown in the label at the right of each trace and the dashed line represents −60 mV. Indicative of the presence of hyperpolarization-activated mixed cationic current (I_H), most VGNs produced a voltage sag (arrow labeled V_sag) in response to large hyperpolarizing currents (−200 pA shown). Scale bars (top right trace) apply to all traces. For this figure, recordings were made using whole-cell ruptured-patch with 100 μm cAMP, except where the recording pipette contained 500 μm db-cAMP.

When injected with hyperpolarizing currents, most neurons (140/146) responded with a deep voltage hyperpolarization followed by a slow depolarization that eventually reached steady state (Fig. 1A–D). Such hyperpolarization-induced voltage “sags” indicate the presence of the HCN channel-mediated current, I_H (Fig. 1C; Rennie and Streeter, 2006; Angelo and Margrie, 2011; Almanza et al., 2012). On average, voltage sags increased in amplitude with larger hyperpolarizing current injections and were first visible when the membrane potential fell below −80 mV.

Our goal was to understand how I_H current influences spike-timing regularity. One way to do this is to compare responses before and after selectively blocking the current with channel-specific antagonists such as ZD7288 and CsCl. This approach was not successful here because both antagonists may affect more than just HCN channels. For example, CsCl is known to block other kinds of potassium channels including inward rectifying potassium channels (Bond et al., 1994; Takumi et al., 1995). Previous studies have also shown that ZD7288 can partially block sodium, T-type calcium, and/or potassium channels (Felix et al., 2003; Sánchez-Alonso et al., 2008; Wu et al., 2012). We suspected similar nonspecific effects in VGNs because action potential amplitudes decreased, and net conductance increased when either ZD7288 or CsCl blocked I_H (5 of 8 cells; data not shown). This is a counter-intuitive result since blocking the channel should have decreased net conductance. Although not discussed, a similar effect in VGNs is evident in the recordings presented by Yoshimoto et al., 2015. We concluded that we could not rely on these channel antagonists to isolate the role of I_H on firing patterns. As shown next, we chose to amplify I_H by controlling the intracellular concentration of cAMP, which shifts the current's voltage activation range (Almanza et al., 2012).

Enhancing the activation of I_H by controlling cAMP concentration

In voltage-clamp, hyperpolarization produced inward currents with a near instantaneous component (I_inst) and a slowly activating component that eventually reached steady state (I_ss; Fig. 2A). Known I_H antagonists, CsCl (Fig. 2A2) and ZD7288 (data not shown), blocked the slow current, suggesting that it was HCN mediated. The current's reversal potential (−32.7 ± 2.8 mV, n = 8) and voltage activation range and kinetics (discussed further below; Figs. 2, 3) are consistent with reported values for I_H (Biel et al., 2009; Almanza et al., 2012; Yoshimoto et al., 2015). We measured the activation range of I_H under several experimental conditions. First, we varied the concentration of simple cAMP in the intracellular pipette solution (at 10, 100, 200, and 500 μm). Second, we used perforated-patch methods (where the intracellular milieu is left intact) to estimate the endogenous concentration of cAMP. Third, we used 100 and 500 μm db-cAMP, a non-hydrolysable version of cAMP that resists degradation by phosphodiesterases. Fourth, we tested if the activation of I_H is further enhanced by increasing recording temperature, as previously reported in the substantia nigra (Gambardella et al., 2012).

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Figure 2.

An electrophysiological and pharmacological method for characterizing the activation properties of I_H. Current excited by a family of long duration (1.7 s) conditioning voltage steps ranging from −135 to −35 mV in 5 mV increments followed by a 300 ms tail step to −100 mV. For clarity, not every voltage-step response is shown. Current under control conditions (A1) and with CsCl (A2) and the Cs⁺-sensitive current obtained by subtraction 1–2 (A3). Gray arrows, The amplitude of the I_H current was determined by taking the difference between the I_inst and the I_SS. B, The voltage-dependent activation of I_H is determined by plotting the magnitude of the tail current (I_tail) as a function of the conditioning voltage. Curves were fit by a Boltzmann function and normalized by the maximum current. The fits generated from the tail of the control condition (○) and from the current isolated from the Cs+ induced block (Δ) are similar. All voltage-dependent characterization of I_H in subsequent figures is derived from the control condition.

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Figure 3.

Effects of cAMP concentration and temperature on the activation of I_H currents. A, I_H currents activated by a family of voltage steps from −135 to −35 mV under several cAMP concentrations and temperature combinations. As in Figure 2, tail currents were used to construct current–voltage relationships. B, Normalized current–voltage relationships (means ± 95% confidence intervals) for I_H are compared for the 10 μm cAMP and 500 μm db-cAMP conditions. The difference between the two conditions illustrates the potential range of I_H activation. C, Box plots for each cAMP condition. V_1/2 depolarizes dependent on cAMP concentration, db-cAMP conditions produce the most depolarized V_1/2 values (shaded box plots). Increasing the concentration of db-cAMP from 100 to 500 μm did not further depolarize V_1/2 and neither did increasing the recording temperature to 35°C. The endogenous concentration was obtained in perforated patch configuration. Mean slope value (k) for all the conditions was 8.3 ± 0.2 mV and not affected by cAMP concentration. D, A single exponential was used to fit to the currents produced by voltage steps from −135 to −65 mV. The time constant of the fits, τ_single, is plotted as function of voltage for 100 μm cAMP (blue; n = 17), 500 μm db-cAMP (red; n = 10), perforated patch (black; n = 4), and 500 μm db-cAMP at 35°C (green; n = 8). Solid lines show fits were produced using Equation 5 to show the relationship of τ_single values and voltage. Parameters for the fits at 100 μm cAMP, 500 μm cAMP, and 500 μm db-cAMP at 35°C are summarized in Table 1. E, For all conditions except those with db-cAMP, mean V_1/2 hyperpolarized as a function of time. V_1/2 ± SEM is plotted as a function of recording time for 10 μm cAMP (diamonds), 100 μm cAMP (cyan circles), 100 μm db-cAMP (gray downward triangle), 500 μm cAMP (squares), and 500 μm db-cAMP (red open squares).

Estimating the voltage-dependent activation of I_H

To estimate the voltage-dependent activation properties of I_H, we used a voltage-clamp tail protocol, which consists of 1.7-s-long preconditioning steps from −135 to −35 mV in 5 mV increments followed by a tail step to −100 mV (Fig. 2A, bottom). The long preconditioning step allowed I_H to approach steady-state activation but was not so long as to kill cells because of prolonged hyperpolarization (see Materials and Methods). The tail step to −100 mV was within the typical activation range for HCN channels but hyperpolarized away from the activation range of most other channels known be expressed in VGNs (e.g., low-voltage-gated potassium channels). The currents measured during the tail step reflect the channels activated during the preconditioning steps but with a fixed driving force. The maximum current density (I_max/capacitance) was larger for sustained-firing neurons than for transient-firing neurons (11.8 ± 1.2 pA/pF vs 6.9 ± 0.6 pA/pF; t_(25.8) = −3.6, p = 0.0013, Welch's unpaired t test), consistent with results reported by Yoshimoto et al. (2015). The normalized tail current (I_tail/I_tail^max) is plotted as a function of the preconditioning step to show the voltage-dependent activation of I_H (Fig. 2B). The normalized activation curves generated from these whole-cell currents were similar to those extracted by pharmacologically isolating I_H (Fig. 2B). These results led us to conclude that the tail current measurements were adequate for characterizing I_H. All subsequent I_H activation parameters are derived from the unblocked whole-cell currents.

Modulating the voltage-dependent activation range of I_H

Examples of whole-cell currents recorded in response to the tail protocol from six cells, each at different intracellular cAMP concentrations and two obtained in 35°C are shown in Figure 3A. The activation curves shifted to more positive potentials as cAMP concentration increased; nearly 15 mV when two extreme concentrations were compared (10 and 500 μm db-cAMP; Fig. 3B). A similar shift has been reported for auditory afferents in response to cAMP analogues (Yi et al., 2010). We found a significant dependence of V_1/2 on cAMP condition (F_(6,44) = 11.67, p = 0.0001 based on one-way ANOVA; Fig. 3C). Based on similarity in V_1/2, we estimate that the endogenous concentration of cAMP (the perforated-patch condition) is between 10 and 100 μm. This estimate is consistent with reports by Almanza et al. (2012). At 100 μm cAMP, the mean V_1/2 and slope factor (k) are −94.32 ± 3.06 mV and 7.97 ± 1.31 mV (n = 14), respectively. This is also consistent with reported values for I_H at 100 μm cAMP (−93 ± 2 and 9 ± 0.9 mV; Almanza et al., 2012). Interestingly, mean V_1/2 values were not significantly different between the 100 and 500 μm db-cAMP conditions, suggesting that enzymatic degradation of cAMP is a critical parameter affecting cAMP concentration and I_H activation.

Temperature did not further depolarize the activation range of I_H. In the substantia nigra increasing recording temperature depolarizes the activation range of I_H by as much as 15–20 mV (Gambardella et al., 2012). Here we found that recording in 500 μm db-cAMP with an elevated temperature (35°C) did not further depolarize the activation range of I_H (Fig. 3C, compare gray box plots). These results suggest that the db-cAMP is at least as effective at shifting the activation range of I_H as temperature. We did not test the influence of temperature alone on the activation range of I_H, although such a temperature-dependent shift would offer a physiologically plausible mechanism for enhancing I_H.

Modulating the activation time course

Next, we show that the time course of I_H activation speeds up with increased concentrations of cAMP. We applied single exponential fits to the currents evoked by the family of voltage steps from −135 to −55 mV. A single exponential reliably fit our data between −135 and −55 mV. Double exponentials better fit the I_H current at −135 mV (Almanza et al., 2012; Liu et al., 2014) but failed for the smaller hyperpolarizations as the size of I_H current diminished. We relied on single exponential fits because they were robust in all cells and for wide range of voltage steps. In Figure 3D, the exponent τ is plotted as a function of voltage steps for recordings made under the four cAMP conditions (perforated-patch, black triangle; 100 μm cAMP, blue circle; 500 μm-dB cAMP, red square; 500 μm-dB cAMP @ 35°C, green triangles). The voltage-dependent trends for τ activation were fit with Equation 5 (see Materials and Methods) for each cAMP condition (Fig. 3D, solid lines). These same fits were used in the model described later. In each condition, the current activates fastest at extreme hyperpolarizations and slows as the voltage step approaches V_1/2. As the voltage steps become more positive the activation kinetics speed up. Like the cAMP-dependent shift in voltage activation, the activation kinetics shift such that at any particular voltage (e.g., −90 mV) the 500 μm db-cAMP was the fastest. Similar cAMP driven acceleration in activation kinetics have been reported in various types of neurons including in chick nucleus laminaris, substantia nigra pars compacta and spiral ganglion (Yamada et al., 2005; Yi et al., 2010; Gambardella et al., 2012). Although increasing temperature did not further depolarize half activation voltage beyond that produced by 500 μm dB-cAMP (Fig. 3C), it did speed up activation kinetics (Fig. 3D, green symbols).

Time-dependent stability of I_H modulation by cAMP

Although we found that the activation range of I_H could be shifted by increasing cAMP concentration, the shifts were most robust and prolonged in the dB-cAMP conditions. Since we were characterizing the activation properties of I_H and firing patterns in the same groups of cells, it was important for us to maintain stability for ∼30–40 min. To determine the time-stability of cAMP driven shift, we measured I_H activation curves at regular time intervals throughout the recording session. Figure 3E shows mean V_1/2 as a function of recording time and by cAMP condition. For all but the 500 μm dB-cAMP condition, the mean V_1/2 was most depolarized during early recordings (0–10 min) and became progressively more hyperpolarized as neurons were held for longer periods (30–40 min). In Figure 3C, activation parameters of I_H were taken from the earliest recording time possible. In db-cAMP, V_1/2 values remained time stable, showing no significant difference between early (0–10 min) and late recordings (30–40 min; t_(3.3) = −0.13, p = 0.907, paired t test). This is presumably because db-cAMP is a non-hydrolysable version of cAMP that can maintain elevated intracellular cAMP levels for hours (Campos-Toimil et al., 2008). We did not determine whether db-cAMP remains stable beyond 40 min. The ability to stably shift the activation range of I_H was ultimately important for characterizing the influence of I_H on firing patterns (presented in the next section). Most of our quantitative comparisons are between the 100 μm cAMP condition (which we estimate to be most like the endogenous condition) and the 500 μm db-cAMP condition (where we are confident that I_H activation was the most robust and stable).

Effects of cAMP on firing patterns

Step-evoked firing patterns with increased cAMP

To understand how cAMP driven enhancement of I_H influenced firing patterns, we compared spikes evoked by ∼100–120 pA step currents in 100 μm cAMP (n = 12) and 500 μm db-cAMP (n = 10). We also recorded at 35°C with 500 μm cAMP (n = 8) to test whether increasing temperature together with elevated cAMP concentrations had an additional influence on spike timing. Figure 4, A1 and A2, shows spike traces from example sustained-spiking neurons in the three recording conditions. For each neuron, we quantified the first spike latency (t_p) and the slope of the afterhyperpolarization trajectory (AHP slope; Fig. 4B1,B2, filled symbols are for the examples in A). We applied two-way ANOVAs to test the influence of the three cAMP conditions and firing-pattern category on each of the two spike features. Both, first spike latency and AHP slope were significantly dependent on cAMP condition (F_(2,29) = 4.47, p = 0.02; F_(2,30) = 5.10, p = 0.01, respectively) and AHP slope also depended on firing-pattern (F_(1,30) = 7.58, p = 0.01). Post hoc analysis by Tukey HSD indicated that first spike latency tended to be shorter and AHP slopes were steeper in the two 500 μm db-cAMP conditions than in the 100 μm cAMP condition (p = 0.030 and p = 0.086, respectively, for first spike latency; p = 0.026 and p = 0.031, respectively, for AHP slope). Neither feature was significantly different between the 500 μm db-cAMP and 500 μm db-cAMP at 35°C conditions (p = 0.96 for first spike latency; p = 0.99 for AHP slope). These results suggest that more I_H current was available in elevated cAMP to speed up afterhyperpolarization trajectories and spike timing, but that raising temperature had no additional influence on these two spike features (Fig. 4B,D).

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Figure 4.

The effects of a db-cAMP and temperature on step-evoked firing patterns. A1, Example responses to a depolarizing current step for sustained-firing VGNs; one recording in 100 μm cAMP (black) and the second in 500 μm db-cAMP at 25°C (red) conditions. A2, Example responses to a depolarizing current step for sustained-firing VGNs; one recording in 500 μm db-cAMP at 25°C (red) condition and the second in 500 μm db-cAMP at 35°C (cyan) condition. VGN firing patterns in A were obtained in response to similar current step (∼100–120 pA). First spike latency (t_p) and the slope of the afterhyperpolarization (AHP) trajectory were obtained as shown in A1. AHP slope was obtained by taking the slope of a line fit to the region illustrated in A. Dashed gray line shows −60 mV. B1, B2, Data for time to peak (ms) and AHP slope (mV/ms) for transient (□) and sustained (Δ) is shown for all three conditions 100 μm cAMP (black n = 11) and 500 μm db-cAMP at both 25°C (red; n = 10) and 35°C (cyan; n = 8). Solid symbols are data for the example VGN shown in A.

Noise (pseudo-EPSC) driven firing patterns with increased cAMP

Next, we examined whether the enhanced activation of I_H yielded regular spiking. Although spontaneous spiking in vivo is driven by quantal release of neurotransmitter from hair-cells, dissociated VGNs require current injection to induce spiking. To mimic the random timing of synaptic input, we injected “pseudo-synaptic” currents into VGN somata (n = 47; for additional detail see Materials and Methods; Kalluri et al., 2010). The size of the unitary EPSC was varied to drive neurons at different rates (Fig. 5A,B). Moreover, the average EPSC size needed to drive neurons to similar rates was larger in transient-firing neurons than sustained-firing neurons (Fig. 5C). Based on the steeper AHPs in step-evoked firing patterns, we hypothesized that firing rates should be higher in 500 μm db-cAMP where the enhanced activation of I_H contributed to the depolarization phase of spike generation. Recordings with 500 μm db-cAMP (n = 11) produced a mean firing rate of 30.4 ± 2.9 spikes/s, significantly higher than the 18.5 ± 1.0 spikes/s in the 10/100 μm condition (p < 0.001, Tukey HSD). However, when we looked at each condition by firing type we saw that this increase in mean firing rate was mainly driven by sustained-B-firing neurons (n = 5), which produced firing rates as high as 70 spikes/s in db-cAMP (Fig. 5C, black triangles). Again, increasing the temperature from 25°C to 35°C did not further increase mean firing rates beyond what was achieved with db-cAMP alone (32.6 ± 3.2 spikes/s, n = 8 vs 30.4 ± 2.9 spikes/s, n = 11).

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Figure 5.

Enhancing I_H activation by increasing cAMP concentration does not increase spike-timing regularity. A, Spike trains in response to pseudo-synaptic current injection. Spike rate increased as the amplitude of unitary pseudo EPSC increased. B, ISI histograms constructed from spike trains in response to frozen variations of pseudo-synaptic currents. Mean spike interval (ISI) decreased as unitary EPSC amplitude increased compare black and gray histograms. C, Firing rate (spikes/s) as a function of mean EPSC amplitude (pA) in different cAMP concentrations. Mean firing rate was impacted by cAMP condition and mean EPSC amplitude. D, CV as a function of mean ISI (ms). Symbols represent cAMP conditions: 10 μm cAMP (n = 4; X), 100 μm cAMP (n = 12; gray ●), 200 μm cAMP (n = 5; orange rectangle), 500 μm cAMP (n = 6; black ○), 500 μm db-cAMP (n = 12; blue ▿), and 500 μm db-cAMP at 35°C (n = 8; red ▶). Fourty-seven cells contributed to this plot, and an individual cell could have contributed multiple points.

Next, we quantified the regularity of spike timing with the CV of the ISI histograms (Fig. 5B). Smaller CV values correspond to narrower histograms and more regularly-timed spike intervals. Figure 5D plots CV as a function of mean ISI measured for all cAMP conditions for (n = 47), of which eight were in 500 μm db-cAMP between 35°C and 37°C. Recall (from Materials and Methods) that we drove each cell to spike at different rates to allow us to compare CV across neurons at similar spike rates. Consequently, there are >47 points on the plot because each cell contributes more than one point.

We conducted a slope regression analysis to test the influence of ISI and cAMP condition on CV. Spike-train regularity was significantly dependent on ISI (F_(1,130) = 7.015, p = 0.0092) but not on cAMP condition (F_(4,130) = 0.32, p = 0.8624). In other words, this analysis suggests that once we account for the effect that increasing cAMP has on ISI (recall from Fig. 5C that cAMP condition does affect spike rate or the inverse of ISI), cAMP has no further influence on regularity. For example, even in 500 μm db-cAMP, were the activation range of I_H is most depolarized and ISIs were as short as 10 ms, firing patterns were still very irregular (with CV values > 0.45; Fig. 5D, blue triangles). This is counter to the findings in neonatal calyx terminals (Horwitz et al., 2014), where increasing cAMP or blocking I_H reduced CV by an order of magnitude (ranging from CV = 0.8–0.08).

Increasing the temperature from 25°C to 35°C in 500 μm db-cAMP did not significantly change mean firing rates (30.4 ± 2.9 spikes/s, n = 11 vs 32.6 ± 3.2 spikes/s, n = 8; p = 0.56). There is significant overlap between the red triangles and blue triangles in Figure 5D, except for a few spike trains with CV < 0.5 (which we discuss in the next section). These results suggest that regular spike trains do not emerge even with faster channel kinetics at higher temperature.

Overall, firing patterns were more irregular here than observed by Kalluri et al. (2010), who recorded from neonatal VGNs without explicitly controlling the activation of I_H. Thus, although activation of I_H via cAMP was sufficient to increase firing rates, it did not produce highly regular firing.

Developmental changes in firing patterns indicate acquisition of I_KL

An explanation for why VGNs failed to produce highly regular firing can be seen by examining the firing pattern of each cell in response to step currents. In Figure 6A, we recolored the symbols in Figure 5D according to step-evoked firing pattern. Triangles indicate recordings made between 35°C and 37°C. Transient-firing neurons (Fig. 6A) had the highest CV values (blue symbols), sustained-B firing neurons had intermediate CV values (gray symbols), and sustained-A firing neurons had the lowest CV values (red symbols). One sustained-A spiking neuron encountered in 500 μm db-cAMP at 35°C produced the most regular spike train in this study and was similar to that seen for sustained-A neurons by Kalluri et al. (2010) (Fig. 6A, red triangle with CV ∼0.2). This suggests that the firing pattern is the dominant predictor for spike-train regularity.

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Figure 6.

Age-dependent changes in firing pattern and passive membrane properties. A, The influence of firing-type on spike-timing regularity. Same data as in Figure 5D but separated by step-evoked firing pattern; transient-firing (blue; n = 29), sustained-B (gray; n = 14), and sustained-A (red; n = 4). Data obtained in 35°C are distinguished by the symbol ▶ (n = 8). Shaded regions represent the CV range reported by Kalluri et al. (2010) before the first 2 postnatal weeks. B–D, Data from the present study from more mature neurons (open symbols; n = 130) are pooled with that from the younger cells reported by Kalluri et al. (2010) (solid symbols; n = 192). B, Examples of VGN firing types in response to a 750 ms long current step. Bar plots showing the proportion of cells that fall into each firing-pattern group from the first, second, and third postnatal weeks. C, Resting potential becomes more negative with age for both firing types. During early postnatal days, the resting potential for sustained-firing cells (red triangles) were more depolarized than for transient-firing cells (blue squares). As indicated by the convergence of the two trend lines, this difference diminished at older ages. Trend lines are exponential fits to the pooled dataset with 95% confidence intervals (shaded regions) for transient (blue) and sustained (red) firing neurons D, Input resistance (R_in) as a function of membrane capacitance (C_m), for 56 sustained and 71 transient neurons. On average, C_m is larger for older neurons, regardless of firing pattern. R_in becomes smaller for older sustained-spiking neurons. Together, A–D follow the idea that sustained-spiking cells are acquiring additional potassium currents as they mature.

The above described firing-pattern-dependent stratification in CV is qualitatively consistent with that previously reported in younger VGNs (Kalluri et al., 2010) but is quantitatively very different. The overall range of CV values is much more compressed within the irregular/high CV region at the older ages in this study than at younger ages (shaded regions indicate the range of CVs reported by Kalluri et al., 2010). One striking difference between the two studies is that here we saw fewer cells that responded to current injections with sustained-A spike patterns, which appears to be a developmentally driven change. Figure 6B illustrates that the proportion of VGNs that fire with sustained-firing patterns diminishes during the first 3 postnatal weeks. During the first postnatal week, equal numbers of cells had either transient-firing or sustained-firing patterns (Fig. 6B); among the sustained-firing group ∼12% had a fully sustained-A firing pattern. These proportions are very different by the third postnatal week when nearly 70% of cells had transient-firing patterns with very few cells having sustained-A firing patterns (∼5%).

A shift toward the more phasic sustained-B-spiking and transient-spiking patterns is consistent with an increase in the expression of low-voltage-gated potassium currents (Iwasaki et al., 2008; Kalluri et al., 2010; Yoshimoto et al., 2015). Also, consistent with this suggestion, we found that there was an age-dependent hyperpolarization in resting potential (V_rest) and decrease in input resistance (R_in) for all cell types. In Figure 6, C and D, we pooled data from the present study (n = 146) with those from Kalluri et al. (2010) (n = 192) to illustrate the age-dependent changes in these passive membrane properties. Resting potentials of sustained-spiking (red) and transient-spiking (blue) VGNs were different during early postnatal days but begin to converge with that of transient firing neurons by the third postnatal week (Fig. 6C). Membrane capacitance (C_m) was typically larger in the older dataset (Fig. 6D) with transient-firing neurons being larger (40.9 ± 1.4 pF, n = 77) than sustained-firing neurons (31.4 ± 1.7 pF, n = 60; t_(132.3) = 4.45 p = 0.0001, unpaired t test). When compared by cell size, R_in values were smaller in transient-firing neurons than sustained-firing neurons at younger ages (Fig. 6D, red triangles, blue squares,), but the differences are less striking at older ages. These data suggest that the ion channel properties of VGNs are becoming less heterogeneous with development. This developmental shift in firing patterns likely accounts for why we did not see highly regular firing, despite enhancing the activation of I_H.

Modeling the influence of I_H activation in the presence and absence of I_KL

Our initial prediction was that by recording from older ages and increasing the activation of I_H we would have seen increased sustained-A spiking in response to current steps and more regular spiking in response to pseudo-synaptic stimulation. However, on average, we saw the opposite result in that VGN responses shifted toward transient/irregular spiking. Next, we will show through model simulations that this result can be accounted for by considering the interaction between I_KL and I_H, both of which are present in nearly all VGNs at older ages.

We used a single-compartment conductance-based vestibular ganglion model in which we could control the conductance density of I_KL and the activation of I_H (see Materials and Methods; Hight and Kalluri, 2016). Sustained-A and sustained-B firing patterns were simulated (Fig. 7A1,B1) by setting g_KL to 0 and 0.65 (mS/cm²), respectively. The equations describing g_KL are the same as those described by Hight and Kalluri (2016) for currents carried through an equal combination of Kv1 and Kv7 type ion channels. Sodium channel density (g_Na) was set to 20 mS/cm² for all simulations. This value was large enough to avoid spike train adaptation because of sodium channel inactivation (Hight and Kalluri, 2016). A family of 1500 ms step depolarizing currents was used to elicit spike trains for each condition. Figure 7, A1 and B1, shows responses at current threshold. The conductance density for the HCN channel, g_H, was set from 0.07 through 0.91 mS/cm². This range is between 0.5 and 7 times the mean value we measured in VGNs (0.13 mS/cm²). Here we show the result for the largest value because conductance density for I_H is reported to be larger in calyx terminals than in somata (Horwitz et al., 2014). We repeated simulations with two I_H conditions that represented the current's most hyperpolarized and depolarized voltage-dependent activation (minimum and maximum activation, representative of the 100 μm cAMP and 500 μm db-cAMP conditions, respectively). The parameters describing the voltage activation range and kinetics for these conditions were based on the data shown in Figure 3 and are represented by Equations 3–5 (see Materials and Methods; parameters are summarized in Table 1).

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Figure 7.

The influence of shifting the activation range of I_H explored in a conductance-based model. Model results from sustained-A and -B type VGNs in response to a 1500 ms depolarizing current step (A3, B3, bottom). Two I_H conditions are shown in each panel; minimum activation (black) and maximum activation (dashed red). A, B, The sustained-A type model was produced by setting g_Na to 20 mS/cm² and g_KL to 0. The sustained-B type model was produced by setting g_Na to 20mS/cm² and g_KL to 0.65 mS/cm² composed equally of Kv1 and Kv7. A1, B1, Voltage-clamp responses for the sustained-A and -B models under model conditions for I_H current (minimum activation, where V_1/2 = −96 mV and maximum activation, where V_1/2 = −84 mV. Black triangle (▴), The max activation produced spontaneous firing at rest. B, Sustained-B neurons exhibit accommodation as voltage oscillations are present in all I_H conditions after ∼60 ms following the onset of the stimulus. A2, B2, I_KL current (pA/cm²) as a function of time for A and the shaded region in B. A2, No I_KL current is seen because g_KL is set to 0 for the sustained-A type VGN. B2, The I_KL current at rest and during the AHP grows larger as I_H is activated. Hatched region, To estimate the net charge (Q/cm²) carried by I_KL we took the sum of the area under each minimum, which is proportional to the shaded region. A3, B3, I_H current (pA/cm²) as a function of time for A and the shaded region in B. A3–B3, As expected the I_H current is larger at rest and throughout the stimulus for max activation cases than for the minimum-activation case. Note that because of the slow kinetics of the HCN channel, the current of I_H switches from inward (−) to outward (+) during the upswing of the spike.

Activation of I_H excites faster spiking in the absence of I_KL

Figure 7A1–A3 shows the firing patterns and flow of currents through g_H and g_KL for the sustained-A simulation. Only the beginning of each spike train is shown to emphasize the details of spike shape and timing. Figure 7, A2 and A3, shows the flow of I_H and I_KL currents into and out of the cell (negative and positive currents, respectively). When I_H was minimally activated, very little I_H current flows into the cell before current injection begins (i.e., the resting phase; Fig. 7A3). During current injection, the membrane potential depolarizes and the model produces spikes throughout the current step. Between each spike (as the cell emerges from the afterhyperpolarization), a little I_H current flows into the cell when the membrane potential is more negative than the reversal potential for I_H (−40 mV). When the membrane potential crosses the reversal potential, I_H current flows out of the cell. When the activation range of I_H is shifted in the maximum activation condition (dashed trace), there is significant I_H flowing into the cell during rest and between spikes. As a result of this extra depolarizing current, the model begins to spike spontaneously (Fig. 7A1, black triangles). The flow of inward and outward currents through the I_H conductance is also larger during the current injection resulting in shorter mean intervals between spikes (11.97 ± 0.12 vs 12.32 ± 0.01 ms) and more evoked spikes during the step (139 vs 122 spikes). Also note that the spikes are much shorter in the maximum activation condition because the slow inactivation of I_H means that the channel remains open during the action potential, adding another large repolarizing potassium current past its reversal potential. Note that because of the spontaneous-spiking, the increase in spike rate during the current step is not immediately visible but, becomes more obvious by ∼60 ms after stimulus onset (black arrow). Overall, the excitatory influence of I_H is consistent with the pace-making role of this current in cardiac cells (DiFrancesco, 2010) and with previous suggestions for vestibular afferents (Horwitz et al., 2014; Yoshimoto et al., 2015).

Activation of I_H dampens spiking in the presence of I_KL

The influence of I_H on firing patterns is different in the presence of I_KL. In Figure 7B1–B3, we show a simulated sustained-B firing-pattern by setting g_KL to 0.65 mS/cm². Again, the flow of I_KL and I_H currents during the simulation are shown under the firing pattern (Fig. 7B2,B3). As expected for a model containing I_KL there is some outward current flowing during the resting phase (Fig. 7B2). This translates to a more hyperpolarized resting potential for the sustained-B firing pattern (Fig. 7B) than for the sustained-A firing pattern (Fig. 7A). During current injection, the spike trains exhibited accommodation as the I_KL current prevented the simulation from reaching threshold, as evidenced by the loss of spikes and emergence of voltage oscillations in the later portions of the current step. In the maximum I_H activation condition, there was much more inward I_H current in the resting condition and a resulting depolarization in resting potential. However, this depolarization shifts the membrane potential further into the activation range for g_KL thereby also increasing the amount of outward flowing I_KL. The average outward flowing I_KL is larger in the maximum activation condition than in the minimum activation condition. This illustrates that activation of I_H recruited more I_KL both at rest and during spiking (Fig. 7B2). This recruitment results in spike trains accommodating faster in the maximum-activation condition than in the minimum-activation condition (Fig. 7B1). Thus, in the presence of I_KL, I_H has a net dampening effect on spiking.

The influence of I_H depends on I_KL density and channel composition

In Figure 7, we showed single examples in which I_H could both excite and dampen spiking, depending on the presence of I_KL. In further simulations we varied the size of g_KL from 0 to 0.8 mS/cm² (sustained-A through transient) while keeping all other conductance densities fixed to explore whether the effect on spiking depends on the relative densities of the two conductances. The composition of g_KL was comprised of one of the following: entirely Kv1 (Fig. 8, green), entirely Kv7 (blue), or equal proportions of the two conductances (magenta). We elicited spike trains using a fixed-length (1500 ms), fixed amplitude (55 pA), suprathreshold current step. Simulations were repeated in both the maximum (solid symbols) and minimum (open symbols) activation conditions for I_H to examine the influence of activating I_H. Figure 8A plots the number of spikes evoked by the current step as function of g_KL when comprised entirely by Kv1 type channels_. As g_KL increases, the number of spikes evoked by the current step decreases. Beyond a threshold value, there is a dramatic transition, after which spike number drops precipitously with increasing g_KL. There are effectively two regimes to the response. First, there is a tonic regime where many spikes are evoked during the current injection and firing patterns are largely sustained-A. Second, there is a phasic regime where firing patterns are sustained-B and transient.

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Figure 8.

I_KL is the dominant influence on spike number and spike timing. Model VGN with g_KL values between 0.0 and 0.8 mS/cm², Kv1 to Kv7 ratios for I_KL of 1.0 Kv1(green, ● or ○), 50% Kv1 and Kv7 (magenta, ■ or □), or 1.0 Kv7 (blue, ▴ or Δ) were driven with a 1500 ms depolarizing current step (55 pA). Each model VGN had either minimum (open symbols) and maximum I_H (solid symbols) I_H activation. Trend curves through the data are shown as solid or dashed lines, respectively. A, Only spikes during the current step were used to compute spike number. Spike number as a function of g_KL (1.0 Kv1) for min and max I_H activation. Spike number falls as a g_KL increases and drops sharply once a large enough g_KL is reached and because of rapid adaption of the spike train (data not shown). Max activation of I_H provides a modest boost in spike number over min activation but requires a smaller g_KL value to illicit adaptation. B, Spike number as a function of g_KL for three model VGNs with I_KL ratios of 1.0 Kv1, Kv1/Kv7, and 1.0 Kv7. For all three model VGNs, spike number falls as g_KL increases and drops sharply once a large enough g_KL is reached and adaption occurs. This g_KL value is smallest in 1.0 Kv7 and largest in 1.0 Kv1 model VGNs. Max activation of I_H condition requires a smaller g_KL value to elicit accommodation for both the 1.0 Kv1 and 1.0Kv7 model VGNs. C, Spike number as a function of net charge carried by I_KL (Q I_KL, pC/cm²). Activation of I_H provides a modest increase in spike number regardless of net charge by I_KL or composition of I_KL. D, CV as a function of net charge carried by I_KL for model VGNs that produced at least 10 spikes. CV increased as net charge by I_KL increased. CV values overall were highly regular (CV < 0.05) because of the lack of irregularity imposed by the step stimulus.

Because g_KL can be comprised of different channel subtypes, we also examined the influence of channel composition by repeating the above simulations with 100% Kv7 or equal proportions of Kv1 and Kv7 (Fig. 8B). Recall that the Kv1 and Kv7 channel models have the same voltage-gated activation range but differ in that Kv1 activates quickly and experiences inactivation whereas the Kv7 activates slowly but does not inactivate (Eqs. 6,7). Regardless of channel composition, spike numbers decreased with increasing g_KL. However, the amount of g_KL needed to elicit the transition between the tonic and phasic regimes was much smaller when g_KL included Kv7 components. Also, spike number was more sensitive to increases in Kv7 than to increases in Kv1 (seen as a steeper slope in the spike number vs g_KL curve for the Kv7 and mixed conditions). These differences in the effective potency of Kv7 comes from the differences in the net steady-state current produced by the two models; because Kv7 does not inactivate, it produces more steady-state current that the Kv1 model, which experiences significant inactivation. Differences in the time course of activation also impacts firing pattern. Because Kv1 activates much more quickly than Kv7, the transition between the tonic and phasic regimes is much more abrupt for the Kv1 case. In contrast, for both the Kv7 and mixed Kv7/Kv1 conditions, the spike rate (proportional to total number of spikes) slows downs substantially before the transition to the phasic regime.

When I_H is maximally activated, the influence of g_KL is enhanced, and the transition between the tonic and phasic regimes occurs at smaller g_KL values than in the minimum I_H condition (Fig. 8A,B, compare dashed lines to dotted lines within each g_KL condition). The influence of shifting the activation of I_H is very different in the regimes before and after the transition. In the tonic regime, the maximum activation condition produces more spikes than the minimum activation condition (i.e., the excitatory effect from the previous figure). However, as g_KL increases, the relative gain in spike numbers provided by I_H diminishes. After the transition, the maximum activation condition produces the opposite effect in that for any fixed value of g_KL, there is a decrease in the number of spikes evoked (i.e., the type of dampening seen in Fig. 7B). Together, these results show that the outward flowing potassium currents carried by g_KL counteract the excitatory influence of I_H. Moreover, more g_KL is recruited when the activation range of I_H is depolarized toward the activation range of g_KL.

To examine the influence of I_H in isolation from the complexities of g_KL recruitment and kinetics, we plotted the spike number and the regularity of the spike train as a function of Q_KL, the net subthreshold charge delivered by g_KL (Fig. 8C,D, respectively). We computed Q_KL as the area under the lower envelop of the I_KL versus time curve (Fig. 7B2, hatched area). Note that this is not the same as the net charge delivered by g_KL during the entire spike train, but rather an estimate of the current during the subthreshold portions of the spike-train. As expected, spike number decreases as Q_KL increases. Now, however spike number falls along a single curve rather than three parametric curves for each combination of Kv1/Kv7. In this plot, we see that the influence of I_H alone (when isolated from its ability to recruit I_KL) is excitatory in that it produces more spikes. This is true in both the tonic and phasic regimes (compare open symbols to closed symbols).

To examine the influence of I_H on spike-timing regularity in the simulations, we computed the CV values for the step-evoked spike trains that produced >10 spikes (Fig. 8D); meaning that regularity was only computed within the tonic regime. We make three main observations pertaining to CV. First, all CV values were highly regular (CV 0.005–0.05). We attributed this to the stimulus type, compared with the randomly arriving EPSC trains, steps of current impose relatively little irregularity onto the sustained-A model. Second, overall CV increased with Q_KL. Because the transition from the tonic to phasic regime was most abrupt for the Kv1 dominated g_KL (Fig. 8B), the change in regularity was also most abrupt for this channel composition (Fig. 8D, compare green symbols to blue + magenta). Third, activation of I_H produced only modest changes in CV between 2 and 8 pC/cm² (Fig. 8D, compare solid and dashed trend lines). Spike-timing regularity is much more sensitive to variations in low-voltage-gated potassium currents than to I_H. In other words, highly-regular spiking could not be produced by simply increasing I_H, but only occurred with small Q_KL. Interestingly, CV depends on Q_KL non-monotonically, with a small upturn toward higher CV values at the lowest Q_KL. In this regime, the step-evoked spike trains are non-adapting with spikes arriving with clock-like precision. Because CV is the ratio of the SD to the mean of spike intervals, the decrease in mean intervals combined with invariance in the SD produces the increase in CV. Similarly, maximizing I_H within this regime produces faster spiking but also produces higher CVs.