Resurgent sodium current promotes action potential firing in the avian auditory brainstem

Abstract

Key points: Auditory brainstem neurons of all vertebrates fire phase-locked action potentials (APs) at high rates with remarkable fidelity, a process controlled by specialized anatomical and biophysical properties. This is especially true in the avian nucleus magnocellularis (NM) - the analogue of the mammalian anteroventral cochlear nucleus. In addition to high voltage-activated potassium (K_HVA ) channels, we report, using whole cell physiology and modelling, that resurgent sodium current (I_NaR ) of sodium channels (Na_V ) is equally important and operates synergistically with K_HVA channels to enable rapid AP firing in NM. Anatomically, we detected strong Na_V 1.6 expression near hearing maturation, which was less distinct during hearing development despite functional evidence of I_NaR , suggesting that multiple Na_V channel subtypes may contribute to I_NaR . We conclude that I_NaR plays an important role in regulating rapid AP firing for NM neurons, a property that may be evolutionarily conserved for functions related to similar avian and mammalian hearing.

Abstract: Auditory brainstem neurons are functionally primed to fire action potentials (APs) at markedly high rates in order to rapidly encode the acoustic information of sound. This specialization is critical for survival and the comprehension of behaviourally relevant communication functions, including sound localization and distinguishing speech from noise. Here, we investigated underlying ion channel mechanisms essential for high-rate AP firing in neurons of the chicken nucleus magnocellularis (NM) - the avian analogue of bushy cells of the mammalian anteroventral cochlear nucleus. In addition to the established function of high voltage-activated potassium channels, we found that resurgent sodium current (I_NaR ) plays a role in regulating rapid firing activity of late-developing (embryonic (E) days 19-21) NM neurons. I_NaR of late-developing NM neurons showed similar properties to mammalian neurons in that its unique mechanism of an 'open channel block state' facilitated the recovery and increased the availability of sodium (Na_V ) channels after depolarization. Using a computational model of NM neurons, we demonstrated that removal of I_NaR reduced high-rate AP firing. We found weak I_NaR during a prehearing period (E11-12), which transformed to resemble late-developing I_NaR properties around hearing onset (E14-16). Anatomically, we detected strong Na_V 1.6 expression near maturation, which became increasingly less distinct at hearing onset and prehearing periods, suggesting that multiple Na_V channel subtypes may contribute to I_NaR during development. We conclude that I_NaR plays an important role in regulating rapid AP firing for NM neurons, a property that may be evolutionarily conserved for functions related to similar avian and mammalian hearing.

Keywords: action potential; auditory system; development; neuron; nucleus magnocellularis; potassium channel; sodium channel.

Figure 1. Markovian state model of the sodium (Na_V) channel

Reproduced from the model described by Khaliq et al. (2003). C, I, O, and OB denote closed, inactivated, open and open‐blocked states, respectively. The values of the kinetic parameters are shown in Table 2.

Figure 2. Frequency–firing pattern of NM neurons at E19–21

Aa and b, representative voltage responses recorded from an E20 NM neuron to current injections of square pulse trains at 150 Hz, before and during TEA (1 mm) application, respectively. B, overlaid enlargement of representative voltage responses shown in Aa and b. Ca and b, representative voltage responses to current injections of square pulse trains at 200 Hz, before and during TEA application, respectively. Asterisk indicates action potential failure. D, population data showing firing probability of NM neurons as a function of square pulse frequency. The strength of all square pulse trains is 1 nA with a duration of 1 s. Individual square pulse width is 2 ms. Error bar, SEM.

Figure 3. Resurgent sodium current (I _NaR) of NM neurons at E19–21

Aa and b, Ba and b and Ca and b, representative current traces in response to voltage‐clamp protocols that elicit I _NaR shown below traces. The amplitude of the conditioning step is +30 mV in Aa and b, 0 mV in Ba and b and −30 mV in Ca and b. The duration of the conditioning step is 10 ms in Aa–Ca and 100 ms in Ab–Cb. Arrowhead and small arrow in Aa indicate the transient sodium current (I _NaT) and I _NaR, respectively. Ac, Bc and Cc, population data showing the I _NaR amplitude as a function of repolarizing membrane voltage (V _MEMBRANE) in response to the conditioning steps shown in Aa and b, Ba and b and Ca and b, respectively. Error bar, SEM.

Figure 4. I _NaR kinetics of NM neurons at E19–21

A, representative current trace showing the calculation of time to peak and decay time constant (tau) for I _NaR. Decay time constant (tau) is obtained by fitting a single exponential (red trace) to the decay phase of I _NaR. The conditioning step is +30 mV at 10 ms. B, population data showing the decay time constant (tau) of I _NaR as a function of repolarizing membrane voltage (V _MEMBRANE). Error bar, SEM.

Figure 5. I _NaR helps increase Na_V channel availability and facilitates Na_V recovery

A and B, representative current traces in response to voltage‐clamp protocols shown below traces. The conditioning step is +30 mV at 5 ms in A (open channel block state) and −30 mV at 40 ms in B (inactivation state). Δt represents the varying recovery time, increasing from 2 to 50 ms in steps of 2 ms. Arrow in A indicates the generation of I _NaR. C, population data showing the Na_V channel availability (%) as a function of recovery time. In order to calculate Na_V channel availability, a reference pulse to 0 mV was applied to NM neurons (not shown in the figure), and the amplitude of I _NaT after the recovery was normalized to this ‘reference amplitude’. Before the normalization, the amplitude of I _NaT was first adjusted by subtracting the steady‐state current that remained at the end of the conditioning step. The recovery trajectory is fit by a single exponential, in order to obtain recovery time constant (tau) shown in D. D, population data showing the recovery time constant (tau) under two different condition states. ^* P < 0.05. Numbers on bars represent sample size. E, representative current traces shown in A and B are normalized and overlaid for recovery time periods of 2 ms (left), 20 ms (middle) and 40 ms (right). Error bar, SEM.

Figure 6. Simulations of Na_V currents from the model NM neuron

Aa, simulated current traces (upper panel) in response to the voltage‐clamp protocol (lower panel) consisting of a 10 ms conditioning step to +30 mV, followed by step repolarizations to −10, −30, −40, −50 and −70 mV. Arrowhead and small arrow indicate I _NaT and I _NaR, respectively. Ab, current–voltage relationship of simulated I _NaR. B, simulated I _NaT (upper panel) was evoked by depolarizing steps to 0 mV (lower panel). The current obtained under control condition is shown in black. Switching off I _NaR by setting the rate constant for the O→OB transition, ε, to 0 resulted in considerable slowing of I _NaT decay (0‐I _NaR− condition, blue trace). I _NaT was restored under 0‐I _NaR+ condition (ε = 0, O _on = 2.15 ms⁻¹, and O _off = 0.01433 ms⁻¹; red trace). C, simulated I _NaR under control (black trace) and 0‐I _NaR+ condition (red trace). Removal of I _NaR under the 0‐I _NaR+ condition has no effect on persistent Na_V current (I _NaP).

Figure 7. I _NaR promotes frequency firing of model NM neuron

Aa–d, Ba–d and Ca–d, simulated voltage and current responses to square pulse current injections of 200 Hz, under the control, 0‐I _NaR− (i.e. I _NaR removal and slower I _NaT) and 0‐I _NaR+ (i.e. I _NaR removal) conditions, respectively. Aa, Ba and Ca are model output of membrane voltage (V _MEMBRANE); enlarged traces shown in Ac, Bc and Cc. Ab, Bb and Cb are model output of Na_V currents; enlarged traces are shown in Ad, Bd and Cd. Asterisk indicates action potential failure. Arrows indicate the generation (Ad) or elimination (Bd and Cd) of I _NaR.

Figure 8. I _NaR properties of NM neurons at E14–16

A and B, representative current traces in response to voltage‐clamp protocols shown below traces. The strength of the conditioning step is +30 mV (black trace), 0 mV (grey trace) and −30 mV (blue trace) and the duration is 10 ms in A and 100 ms in B. C, population data showing the decay time constant (tau) of I _NaR as a function of repolarizing membrane voltage (V _MEMBRANE). D–F, population data showing the I _NaR amplitude as a function of repolarizing membrane voltage (V _MEMBRANE) in response to different conditioning steps. The strength of the conditioning step is +30 mV in D, 0 mV in E and −30 mV in F, and the duration is 10 and 100 ms. Error bar, SEM.

Figure 9. I _NaR properties of NM neurons at E11–12

A, representative current traces in response to voltage‐clamp protocols shown below traces. The conditioning step is +30 mV at 10 ms. B, population data showing the current amplitude as a function of repolarizing membrane voltage (V _MEMBRANE) in response to conditioning step of +30 mV at 10 ms. C, population data showing the decay time constant (tau) of I _NaR as a function of repolarizing membrane voltage (V _MEMBRANE). D, population data showing time to peak of I _NaR at different embryonic ages. ^* P < 0.05. Numbers on the bars represent sample size. Error bar, SEM.

Figure 10. Frequency–firing pattern of NM neurons at E14–16 and E11–12

Aa and b, representative voltage responses recorded from an E16 NM neuron to current injections of square pulse trains at 150 and 200 Hz, respectively. Ac, population data showing firing probability of E14–16 NM neurons as a function of square pulse frequency. The strength of square pulse trains is 1 nA with a duration of 1 s. Individual square pulse width is 2 ms. Ba and b, representative voltage responses recorded from an E12 NM neuron to current injections of square pulse trains at 70 Hz and 150 Hz, respectively. Bc, population data showing firing probability of E11–12 NM neurons as a function of square pulse frequency. The strength of square pulse trains is 1 nA with a duration of 1 s. Individual square pulse width is 5 ms. Error bar, SEM.

Figure 11. Na_V1.6 distribution in developing NM

Na_V1.6 immunoreactivity at E21 (A), E15 (B) and E11 (C). Left (Aa, Ba and Ca) and right (Ab, Bb and Cb) columns are low‐ and high‐magnification confocal images, respectively. Dashed lines indicate the boundary of NM. NM, nucleus magnocellularis. Scale bars: 50 μm in Ca (left column), 10 μm in Cb (right column).

Figure 12. Axonal localization of Na_V1.6 immunoreactivity at E21

Double staining of Na_V1.6 and neurofilament in E21 NM. Arrows indicate a distinct segment containing Na_V1.6, which overlaps with neurofilament‐stained axon that can be traced back to the cell body. NM, nucleus magnocellularis. Scale bars: 20 μm for all panels.