High-Threshold K⁺ Current Increases Gain by Offsetting a Frequency-Dependent Increase in Low-Threshold K⁺ Current

Voltage- and current-clamp simulations

All simulations were constructed in MatLab 6.5 using a fourth-order Runge-Kutta algorithm with a time step dt of 0.01 ms. We used a point model described by an electrode current (I_E), IK_LT, IK_HT (described above), a single-leak current, and an Na⁺ current. The voltage-dependent currents evolved using the following equation:

For IK_LT and IK_HT (n and k), the steady-state current X_∞(V) and the time constant τ(V) evolved according to the equations above; for the Na⁺ current, the activation gate m and the inactivation gate h evolved according to the following:

and

with α_m(V) = 97.97e^[0.082(V)], β_m(V) = 0.0555e^[-0.093(V)], α_h(V) = 0.00013e^[-0.1016(V)], and β_h(V) = 2.4e^[0.0384(V)]. Maximum conductance for the currents, when present, were g_maxNa = 350 μS/cm², g_maxKLT = 15 μS/cm², g_maxKHT = 50 μS/cm², and g_maxleak = 1 μS/cm². Densities were chosen to replicate the F-I characteristics and to preserve the ratio of g_KvLT: g_KHT found in the neuron. Reversal values for the currents were E_Na⁺ = 50 mV, E_K⁺ = -88.5 mV, and E_leak = -70 mV with capacitance (C) = 10 nF/cm². Voltage was integrated according to the following:

Kinetics of K⁺ currents in ELL pyramidal cells

Pyramidal cells in the ELL of weakly electric fish express high levels of TEA-sensitive Kv3 K⁺ channels that contribute to spike repolarization (Rashid et al., 2001a,b; Noonan et al., 2003). These cells are capable of firing at frequencies above 200 Hz with a relatively wide firing frequency range (80-250 Hz). To gain an understanding of how high-threshold K⁺ currents contribute to excitability, we determined the kinetics of the TEA-sensitive and TEA-insensitive currents in pyramidal cells. Current kinetics was determined in whole-cell voltage clamp in the presence of TTX, Cd²⁺, and synaptic blockers to block synaptic transmission and Ca²⁺-dependent K⁺ currents. In general, cells expressed ∼1-3 nA of total outward K⁺ current that became visible between -60 and -50 mV and increased linearly between -70 and 50 mV. Application of 0.5-1 mm TEA blocked nearly 75% of the outward current and revealed a TEA-insensitive low-threshold K⁺ current (Fig. 1A). Subtracting the post-TEA from control records revealed a faster high-threshold K⁺ current (Fig. 1A). In both cases, little inactivation was observed over a 100 ms time frame. In the high-voltage range (0-50 mV), both currents shared relatively similar activation rate values. In contrast, at lower voltages and those more relevant to the spike waveform (-70 to 0 mV), there was a clear difference in the activation and deactivation rates between the currents.

The V_1/2 of activation of IK_LT was -36.5 ± 1.2 mV with a slope factor of 9.1 mV (Fig. 1B) (n = 5). The time constants (τ) for activation ranged between 0.55 ± 0.18 ms (50 mV) and 1.66 ± 0.21 ms (-20 mV), whereas deactivation time constants ranged between 4.2 ± 0.53 ms (-40 mV) and 0.88 ± 0.17 ms (-70 mV) (Fig. 1C) (n = 5). The V_1/2 of activation of IK_HT was more positive at -15.3 ± 0.6 mV with a slope factor of 10.4 (Fig. 1B) (n = 5). The time constants for IK_HT activation and deactivation were faster than IK_LT, with activation τ ranging between 0.38 ± 0.07 ms (50 mV) and 0.85 ± 0.08 ms (-20 mV), whereas deactivation time constants ranged between 1.98 ± 0.26 ms (-40 mV) and 0.68 ± 0.09 ms (-70 mV) (Fig. 1C) (n = 5).

To later test the interactions between low- and high-threshold K⁺ currents, we used the above kinetics to reproduce these currents in a voltage-clamp simulation. Because the kinetics of neither current was adequately described by Hodgkin and Huxley formalism (Hodgkin and Huxley, 1952), the time constant-voltage relationships were fit and modeled using an arbitrary function (Lorentzian). In particular, IK_HT showed a relatively slow deactivation rate with respect to the midpoint of activation. In a simple Hodgkin and Huxley two-state model, the slowest time constant value occurs at or near the midpoint of the steady-state activation. For IK_HT, the slowest time constant value occurred at approximately -40 mV (1.98 ms) despite having a V_1/2 value of -15 mV, suggesting a more complex gating mechanism. Using a Boltzmann fit for steady-state activation and a Lorentzian fit for the time constant-voltage relationship, we accurately reproduced IK_LT and IK_HT in a voltage-clamp simulation (Fig. 1A, right).

We anticipate that the TEA-sensitive IK_HT was primarily composed of Kv3 current, because pyramidal cell spike responses are insensitive to α-dendrotoxin (Noonan et al., 2003), a blocker of TEA-sensitive Kv1.1, 1.2, and 1.6 K⁺ channels. We did not further investigate the molecular identity of TEA-insensitive current(s) at this time to focus on the net effects of high-threshold currents on spike firing.

IK_HT has a frequency-dependent effect on the F-I relationship and spike waveform

Under normal conditions, square wave current injections to pyramidal cells evoked a minimum firing frequency of 60-80 Hz. Increasing current evoked greater firing frequencies that saturated at 260-280 Hz (Fig. 2A). Neurons showed no adaptation in firing rate within the first 100 ms and very little after 500 ms current steps. The gain of the F-I relationship decreased with greater current injections at firing frequencies beyond ∼200 Hz (Fig. 2A). Driving the cell beyond 260-280 Hz resulted in a characteristic damped oscillation (spike failure) associated with spike broadening and a decrease in spike height.

To understand the precise role of IK_HT in the output of pyramidal cells, we bath applied low concentrations (500 μm) of TEA. In general, blocking IK_HT with TEA reduced the firing frequency range and the amount of current required to cause spike failure. Like the control condition, spike firing started at 60-80 Hz but saturated at the lower level of 210-240 Hz, with greater current injection causing spike failure in the form of a damped oscillation. The difference in firing rate between control and TEA conditions increased with larger current injections (Fig. 2A, middle panel), increasing from 13 ± 8.0 Hz at low frequencies (defined as 80-120 Hz) to 32 ± 5.8 Hz at high frequencies (200-240 Hz) (p < 0.05; n = 4). Because of the differential effects of IK_HT removal on the F-I relationship, we hypothesized that an increased contribution of IK_HT at high firing frequencies should be apparent in the effects of TEA on spike waveform parameters. This prediction was confirmed by analysis of the spike waveforms recorded at high and low frequencies in the presence or absence of TEA (Fig. 2B, right panel). Removal of IK_HT had little effect on spike waveform when the cell was firing at low frequencies (∼70 Hz) but had a clear effect at high frequencies (∼220 Hz) (Fig. 2B, right panel). We quantified the spike parameters associated with K⁺ current, including the AHP size, spike decay rate, spike half-width, and spike height as a function of firing frequency (Fig. 3A-D). Application of TEA resulted in a small baseline shift in these parameters relative to control and revealed a clear frequency dependency in the contribution of IK_HT to all four parameters with much larger and statistically significant effects in the high-frequency firing region (Fig. 3A-D, right panels). Specifically, blocking IK_HT preferentially attenuated AHP size, spike decay rate, and spike height and increased spike half-width at high frequencies.

It has been established that AHP-associated K⁺ currents are critical for sustained firing by preventing cumulative inactivation of Na⁺ current, thereby preventing failure of the spike-generating mechanism (Erisir et al., 1999). Because IK_HT contributes in a frequency-dependent manner to the F-I relationship and spike parameters, removal of IK_HT should have a frequency-dependent effect on the availability of Na⁺ current. The availability of Na⁺ current can be inferred from the rate of spike rise. Plotting the rate of spike rise at different firing frequencies revealed that under control conditions, availability of Na⁺ current remained relatively constant and was not significantly different between the low- and high-frequency firing ranges (Fig. 4A). Conversely, in the absence of IK_HT, there was a frequency-dependent decrease in the rate of spike rise that was significantly different between low- and high-frequency firing regions (Fig. 4A). Under control conditions, the rate of spike rise was highly correlated to spike height and AHP size (Fig. 4B). In the absence of IK_HT, spike height and AHP size spread over a greater range but were highly correlated with the rate of spike rise, in agreement with the idea that the AHP is critical for preventing Na⁺ current inactivation (Fig. 4B). Other spike parameters associated with K⁺ current (spike half-width, decay rate, and height) also correlated strongly with rate of spike rise (r ≥ 0.75; data not shown). Therefore, in the absence of IK_HT, a cumulative inactivation of Na⁺ current reduced the rate of spike rise and spike height. The slower rate of spike rise and reduced spike height presumably activate less K⁺ current, which acts to increase spike width and reduce AHP size. The data thus show that IK_HT contributes in a frequency-dependent manner to the spike-generating mechanism and stabilizes the high-frequency range by minimizing Na⁺ current inactivation.

IK_HT function is not fully reproduced by preventing Na⁺ current inactivation

To examine the role of IK_HT and IK_LT in our system, we built a firing model using both types of K⁺ currents described above and a modified Hodgkin-Huxley modeled Na⁺ current (see Materials and Methods). To be consistent with pyramidal cell recordings, we set the conductance density of IK_HT approximately three times greater than IK_LT. The firing model accurately reproduced the F-I relationship in exhibiting a minimum firing rate of ∼80 Hz and a maximum rate of ∼250 Hz (Fig. 5A). Removing IK_HT in the model further uncovered a frequency-dependent effect on the F-I relationship that closely resembled that found in pyramidal cells (compare Figs. 5A and 2A). Without IK_HT, the model started firing at ∼90 Hz but had a maximum rate of 190 Hz. At low frequencies, the spikes were similar in timing and shape with or without IK_HT despite a loss of ∼65% of the total K⁺ current activated by the spike waveform (Fig. 5B), suggesting that at low frequencies, IK_LT is the primary conductance repolarizing the action potential. Conversely, at high frequencies, spike parameters and the F-I relationship diverged. Like the experimental results, spike failure was associated with progressively shorter and wider spikes with or without IK_HT but occurred at a lower frequency when IK_HT was absent (Fig. 5B). Spike failure during prolonged current pulses was attributable to the Na⁺ current inactivation variable (h) decreasing dramatically compared with the control condition that incorporated IK_HT. The model then reproduced the preferential effects of IK_HT on the high-frequency region, indicating that IK_HT is required to maintain a narrow spike and a fast AHP to slow Na⁺ current inactivation at high frequencies.

If the primary role of IK_HT is to offset Na⁺ current inactivation, then preventing inactivation through some other mechanism should stabilize high-frequency firing and reproduce the F-I relationship in the absence of IK_HT. Under this condition, one can determine how much IK_HT contributes to sustaining high-frequency firing by offsetting Na⁺ current inactivation. To test this, we used a “resetting” function in the model lacking IK_HT to reduce Na⁺ current inactivation after each spike to a level equal to that maintained in the control model at 180 Hz (h = 0.09). Note that this value of h is more than sufficient to support high-frequency firing, because lower levels (h = 0.07) of h are capable of sustaining 250 Hz in the control case. Resetting the h variable in the absence of IK_HT, however, restored only a portion of the F-I relationship relative to the control (Fig. 5C). Although the system did not fail at higher firing frequencies (as expected with a fixed reset of the h variable), the gain of the F-I relationship was preferentially reduced at high frequencies. We considered the possibility that a lack of full recovery of the F-I relationship with an h reset condition could arise from the lower level of outward repolarizing current when IK_HT was removed. To overcome this, we increased the amount of IK_LT in the h-reset condition but found that this did not facilitate high-frequency firing. Rather, it produced a subtractive effect on the F-I relationship, as shown by an increased threshold for firing and inability to fire at high frequencies within the current range used (Fig. 5C).

Like the F-I relationship, providing an h reset in the absence of IK_HT only partially recovered the spike waveform in a frequency-dependent manner (Fig. 5D). In the low-frequency region, the h reset had no effect, whereas at high frequencies, it completely rescued spike height and rate of rise, confirming a reflection of the state of Na⁺ channel availability. This would suggest that an h-reset function can effectively mimic IK_HT actions on these two specific spike parameters at high frequencies (Fig. 5D). However, the rate of spike repolarization, spike half-width, and AHP amplitude were only partially affected by reducing Na⁺ inactivation with the h-reset function (Fig. 5D, right inset). This indicates that IK_HT can normally exert an effect on other currents at high frequencies, which might add to the extent of Na⁺ channel inactivation. Notably, we found that if spikes were driven at high frequency by a series of short (1 ms) depolarizing pulses instead of constant current injection, an h-reset function could fully restore all aspects of spike shape and the F-I relationship when IK_HT was removed. This result suggests that the level of depolarization necessary to drive spike discharge at high frequencies creates an additional offset in the repolarizing phase of spikes that would ordinarily be reduced by IK_HT.

IK_LT shows a frequency-dependent increase between spikes in the absence of IK_HT

Because maintaining the availability of Na⁺ current with the h reset did not restore the F-I relationship and the high-frequency firing range of the model neuron compared with the control, we examined the time course of IK_HT and IK_LT during spiking at different firing frequencies (Fig. 6A,B). The fast activation and deactivation time constants of IK_HT allowed it to closely track voltage and deactivate sufficiently fast between spikes to maintain interspike outward current levels at zero (Fig. 6A,B). Although the IK_LT did not track voltage as closely as IK_HT, in the presence of IK_HT it was also able to deactivate at a sufficient rate to maintain interspike current levels near zero throughout the frequency range tested (90-240 Hz) (Fig. 6A,B).

However, removal of IK_HT revealed a very different behavior for IK_LT during the interspike interval compared with control. In the absence of IK_HT and a low-firing frequency, IK_LT produced very little outward current during the interspike interval. In the case of high-frequency firing (180 Hz), however, there was a dramatic increase in the amount of IK_LT during the interspike interval (Fig. 6B). We reasoned that this could arise when the removal of IK_HT increased spike half-width and reduced the AHP, resulting in a more positive interspike voltage (never falling below -60 mV) that more effectively activated the lower threshold IK_LT. The increase in IK_LT could be caused by its slow deactivation time falling within the interspike interval or its low threshold for activation. We found that making the IK_LT deactivation rate as fast as IK_HT had no effect on its level between spikes, whereas changing its activation voltage to more positive values (equivalent to the activation voltage of IK_HT; a 21.2 mV shift) greatly decreased the interspike current. Thus, as the firing frequency increases in the absence of IK_HT, the interspike voltage increases in a frequency-dependent manner and causes a corresponding increase in IK_LT levels because of its relatively negative activation voltage.

To test whether IK_LT showed a frequency-dependent increase during the interspike interval in the neuron, we constructed spike commands similar to that of the firing system without IK_HT, with an interspike voltage of -60 mV, and a fixed half-width of 1 ms to deliver at frequencies of 100 and 200 Hz. The use of these parameters provided a spike train similar to both neuron and model in which spikes had a larger mean interspike voltage at higher frequencies. These spike waveforms were then used as voltage-clamp commands to assess the behavior of IK_HT and IK_LT in the pyramidal neuron. As predicted from the model, IK_LT showed a strong frequency-dependent increase during the interspike interval (Fig. 6C). In contrast, IK_HT maintained a near-zero level of outward current during the interspike interval regardless of frequency (Fig. 6C).

To test the hypothesis that the baseline level of IK_LT contributes to the selective effects of IK_HT removal on the F-I relationship at high firing frequencies, we used a similar technique to the h reset described above. In the absence of IK_HT, the interspike interval value of the IK_LT conductance variable (n) rose to values from 0.55 to above 0.7 at high frequencies (>170 Hz). To effectively prevent the increase in IK_LT and not affect the kinetics through the development of a spike (as changing steady-state or kinetic properties would), we reset the n variable after each spike to the level obtained during the interspike interval in the presence of IK_HT (n = 0.56) (Fig. 7A). We found that although using the n reset did not fully recover the F-I relationship, there was a significant increase in the gain and ability to sustain high-frequency firing (Fig. 7B). In fact, the F-I relationship with the n reset closely matched the condition with the h reset alone (Fig. 7C). With just the n reset, however, the firing system failed at ∼200 Hz because of a decrease in the availability of Na⁺ current through cumulative inactivation.

The data above suggest that IK_HT plays an important role in maintaining both a low level of interspike IK_LT and a high level of Na⁺ current availability. If so, using the h and n resets together in the absence of IK_HT should recover the F-I relationship completely. Indeed, using both resets generated an F-I relationship almost identical to the control case, with stable spike firing at 250 Hz (Fig. 7C). Thus, the function of IK_HT is to slow the accumulation of Na⁺ current inactivation and prevent the IK_LT increase between spikes (Fig. 7D,E). Additionally, by preventing the increase of IK_LT, IK_HT permits a more negatively activated IK_LT to exist in a fast-firing neuron, enabling the firing system to benefit from the effects of IK_LT at low frequencies without dampening high-frequency firing (Fig. 5C).

The increase in interspike IK_LT observed at high firing frequencies might be expected to help restore spike repolarization. However, in our system, a higher level of interspike IK_LT had no effect on spike waveform but instead reduced the gain of the F-I relationship. We therefore sought to determine the mechanism by which the frequency-dependent increase in IK_LT dampened cell excitability during high-frequency discharge. Because the increase of IK_LT between spikes is frequency dependent, an increase in outward current flow mediated by IK_LT could effectively produce a leak current to decrease gain by opposing the excitatory input current that drives the neuron to fire at high frequencies.

To test the mechanism, we incorporated a frequency-dependent coefficient that fit the interspike IK_LT-frequency relationship in the absence of IK_HT (Fig. 8A). The conductance increase was added between spikes, lasting 1.5 ms from the point where the derivative of n crossed the -0.045 ms^-1 point in a positive direction. This point corresponded to the beginning of the AHP across the entire frequency range. To test the effects of a lasting K⁺ current, we set the reversal of the frequency-dependent leak equal to the reversal potential for K⁺.

The addition of a frequency-dependent leak that provided outward current produced an F-I relationship that resembled the condition without IK_HT (Fig. 8B). To test whether the frequency-dependent interspike outward current behaved like a simple subtractive current to that applied through the electrode (I_E), we subtracted the current directly from I_E (Fig. 8B). This test resulted in an F-I relationship with the same loss in gain at high firing frequencies as when a leak current was incorporated separate from I_E (above case)_. It is also important to note that the frequency-dependent leak current between spikes had almost no effect on the availability of Na⁺ current (Fig. 8C). This shows that the effects of IK_LT on the F-I relationship are mediated by the outward current flow that opposes the driving excitatory current in the high-frequency region. The role of IK_HT is thus normally to increase the gain of the F-I relationship and firing frequency range by preventing a cumulative inactivation of Na⁺ current as well as an increase in a low-threshold K⁺ current at high frequencies of spiking.

IK_HT contribution to spike waveform is frequency dependent

The ability of IK_HT to function primarily in the high-frequency range of the F-I relationship resulted from a frequency-dependent contribution of IK_HT to spike parameters. At low frequencies, the removal of IK_HT had a relatively small effect on spike parameters, whereas at higher frequencies the impact was much larger. The difference in contribution that becomes apparent when IK_HT is removed is the result of the firing system being selectively stressed by Na⁺ current inactivation at high frequencies. With Na⁺ current inactivation, there is a corresponding drop in spike height, which reduces the activation of the IK_LT current. Under these conditions, the additional outward current provided by IK_HT is critical to maintaining a minimal amount of repolarizing force to guarantee the recovery of Na⁺ current and maintain the spike waveform. We directly tested the relationship between IK_HT and Na⁺ inactivation in a model by forcing an h reset after each spike in the absence of IK_HT. We found that preventing Na⁺ channel inactivation did not fully recover several parameters of the spike waveform, including spike repolarization rate, half-width, and AHP size. Rather, we found that these spike parameters were partially affected by the injected current, which is inherently large at higher frequencies. IK_HT thus helps retain the spike waveform at high frequencies in part by offsetting the underlying driving current.

Frequency-dependent changes in spike waveform affect the F-I relationship

The gain of an F-I relationship is affected by at least two processes that shape the spike waveform. The first involves recovery of Na⁺ current, in which a decrease in Na⁺ current availability decreases the gain of the F-I relationship. The spike waveform also affects the activation of IK_LT. Spike waveforms with a more depolarized interspike voltage dramatically increase IK_LT activation because of its low threshold of activation. An increase in IK_LT reduces the gain of the F-I relationship and thus has a similar effect to the removal of Na⁺ current. Because IK_LT and Na⁺ current are being affected by a frequency-dependent spike waveform, their effects on the F-I relationship in the absence of IK_HT is also frequency dependent.

In the absence of IK_HT, the ability of IK_LT to reduce the gain of the F-I relationship in the high-frequency region was mediated by a frequency-dependent increase in the flow of outward current between spikes. Furthermore, the interspike outward current increase did not affect the availability of Na⁺ current compared with control. More importantly, the interspike IK_LT acts to oppose the driving excitatory current. Normally, this mechanism would produce a subtractive effect on the F-I relationship. In the case of IK_LT, however, the frequency-dependent increase of outward current between spikes results in a preferential subtractive effect in the high-frequency firing region (i.e., divisive effect).

A frequency-dependent increase in IK_LT results from a low-voltage threshold for activation

The low-threshold K⁺ current differed from IK_HT in two respects: a more negative steady-state voltage for activation and a slower deactivation time constant. Hence, the increase in IK_LT activation during the interspike period in the absence of IK_HT could be the result of two different mechanisms. The first possibility would involve a temporal summation of IK_LT resulting from a deactivation time that lies within the interspike interval. In the absence of IK_HT, the summation of IK_LT would intensify because of a more positive interspike voltage and a slower deactivation time constant. We found that changing the activation time constants of IK_LT in the model to match those of IK_HT produced no difference in the F-I relationship and had no effect on interspike IK_LT levels. Second, it is possible that the low voltage for activation causes a significant increase in IK_LT activation with more positive interspike voltages. In support of this, with a positive change in the voltage for activation, the ability of IK_LT levels to increase between spikes was eliminated. This implies that the primary kinetic distinction that differentiates the functional role of IK_HT and IK_LT in our cells is the steady-state voltage relationship.

Molecular identity and function of IK_HT and IK_LT

Previous work has established that ELL pyramidal cells express Kv3 channels (Rashid et al., 2001a,b). Thus, IK_HT is likely to derive almost entirely from the Kv3 subfamily, especially given its high sensitivity to TEA. Although the voltage dependence for the IK_HT reported here is more negative than that from Kv3 currents in expression systems (Weiser et al., 1994; Kanemasa et al., 1995; Hernandez-Pineda et al., 1999; Rudy et al., 1999; Fernandez et al., 2003), it is similar to those reported for Kv3 currents in some mammalian central neurons (Martina et al., 1998, 2003; Baranauskas et al., 2003; McKay and Turner, 2004). Additionally, Kv3 channels are known to be modulated by numerous intracellular factors that may contribute to the discrepancy between expression and neuronal data (Macica and Kaczmarek, 2001; Moreno et al., 2001; Macica et al., 2003; Lewis et al., 2004). The identity of the IK_LT is unknown. IK_LT was insensitive to Cd²⁺ or TEA, and the spike waveform of ELL pyramidal cells is not affected by dendrotoxin, a blocker of the Kv1.1 and 1.6 K⁺ channels that are also TEA sensitive (Coetzee et al., 1999; Noonan et al., 2003). It therefore seems unlikely that Kv1.1, Kv1.6, Kv1.7, or Ca²⁺-sensitive K⁺ channels contribute to the IK_LT of pyramidal cells. A more likely candidate is Kv1.5, which is insensitive to TEA and dendrotoxin, can be noninactivating, and has a fast activation time constant. Given the kinetic distinctiveness of IK_LT and IK_HT and the steepness of the slope factor of the Boltzmann fit, it is likely that both currents correspond to distinct groups of channels in terms of molecular identity and that the makeup of each current likely derives from a relatively homogenous population of channels.

Many high-frequency firing cells are known to express high-threshold Kv3 K⁺ channel subtypes in conjunction with other low-voltage-activated K⁺ channels (Wang et al., 1998; Erisir et al., 1999; Rothman and Manis, 2003c). The frequency-dependent effects we observe for IK_HT in relation to other channels may then influence the input-output characteristics of many other cells. Indeed, a divisive effect on the F-I relationship resulting from Kv3 block has been observed in other high-frequency firing neurons. Genetic and pharmacological studies have shown that removal of Kv3 currents reduces the gain of the F-I relationship preferentially in the high-frequency region of neocortical interneurons and reticular thalamic neurons (Erisir et al., 1999; Porcello et al., 2002). Synaptic transmission in parallel fibers of the cerebellum in Kv3.1-3.3 knock-out animals is impaired primarily during high-frequency spiking (Matsukawa et al., 2003). In medial nucleus of the trapezoid body cells, the ability to follow stimulation pulses in the absence of Kv3 current was also found to be frequency dependent, impairing spike firing only in the high-frequency range (Wang et al., 1998). Thus, the ability for IK_HT to interact with low-threshold K⁺ and Na⁺ channels to extend the firing range and frequency-following capability may be a common mechanism in many high-frequency firing cells.