Essential Role of Somatic Kv2 Channels in High-Frequency Firing in Cartwheel Cells of the Dorsal Cochlear Nucleus

Immunoreactivity of Kv2.1 and Kv2.2 on ankyrin-G-positive AISs and dendrites of cartwheel cells. Ai, Bi, Projected z-stack of optical sections taken from cartwheel cells immunolabeled for ryanodine receptors (RyR; magenta) and ankyrin-G (AnkG; blue). Aii–Av, Bii–Bv, Single optical sections of AISs immunolabeled for ankyrin-G (AnkG; blue), Kv2.1 (green), and Kv2.2 (yellow). The positions of AISs are indicated by arrowheads. The location of Kv2.2 on the edge of AIS is pointed by arrows in Aii–Av. The cell in A is distinct from the one in B. Ci, Projected z-stack of optical sections of AF 647-labeled cartwheel cells. Two cartwheel cells are labeled. Cii–Cv, Single optical sections of a proximal dendrite, which is immunonegative to Kv2.1 (Cii) and Kv2.2 (Civ). Location of the dendrite was marked by arrowheads. Note that a cell is Kv2.2-positive in the panels (arrow), indicating that the antibodies used reached the tissue.

Biophysical properties of GxTX-sensitive Kv2 current in cartwheel cells. A, Outward current evoked by voltage steps. Pulse protocol is indicated at the bottom of A. The recordings were made at room temperature (23–24°C) using ACSF supplemented with NBQX, MK-801, strychnine, picrotoxin, TTX, apamin (a SK channel blocker), and penitrem A (a BK channel blocker). To remove inward current by voltage-gated calcium channels, CaCl₂ in the ACSF was excluded and replaced with equimolar MgCl₂, and 0.25 mm EGTA-Na was added. GxTX-sensitive current was obtained by subtraction. B, The current–voltage relationship of the outward current in the absence (control) or presence of 100 nm GxTX (GxTX). The amplitude of steady-state was used for the plotting. Here and the following figures, error bars indicate SEM, numbers in parentheses indicate the number of replications (cells). Statistical significance was tested using two-way repeated measure ANOVA and Bonferroni post hoc tests (significance at p < 0.05). n.s.: not significant; ***p < 0.001. C, GxTX-sensitive tail current evoked by voltage steps. The voltage pulse protocol is shown in the left of C. D, GxTX-sensitive, normalized conductance (G) plotted as a function of voltage (n = 13 in each point). The activation curves were fit with the Boltzmann function with a V_1/2 of –11.84 ± 0.86 mV (n = 13) and a slope factor (k) of 7.90 ± 0.76 mV (n = 13). Ei, Activation kinetics of Kv2 current isolated by GxTX subtraction as in A. Activation was fit with a single exponential function, and τ is the time constant. Fits are shown as red lines. Eii, τ as a function of voltage. Fi, Deactivation kinetics of Kv2 current obtained by GxTX subtraction. Deactivation was fit with a single exponential function, and fits are shown as red lines. Fii, τ as a function of voltage.

The effects of GxTX on simple or complex spikes elicited by brief current injection. Ai, Simple spike evoked by current injection (1-ms duration). Recordings were made in the ACSF at 33–34°C supplemented with NBQX, MK-801, strychnine, and picrotoxin. GxTX broadened the action potential width (inset) and depolarized the potential of ADP. Aii, Summary of the change ADP measured at 10 ms after the beginning of depolarizing current injection (dashed line in Ai, overlay). ***p < 0.001. Aii, Summary of the half-width of action potentials; ***p < 0.001. Bi, The simple spike converted into complex spikes by GxTX. Bii, Summary of the change of spiking pattern by GxTX. Out of 24 simple-spiking cells, seven cells started showing complex spiking in GxTX. Ci, Complex spikes evoked by current injection. In GxTX, fAHP became less negative (inset) and ADP became more prominent. Cii–Civ, Summary of fAHP, ADP, and half-width of first action potential in complex spikes. ADP was measured at 10 ms after the beginning of depolarizing current injection (dashed line in Ci, overlay). Statistical significance was tested using paired t tests; *p < 0.05, **p < 0.01, ***p < 0.001.

The effects of GxTX on sustained firing induced by depolarizing current injection. A, The effects of GxTX on a simple-spiking cell. Tonic firing induced by square-wave current injection in the absence (control) or presence (GxTX) of GxTX. In GxTX, the cell showed phasic response when the cells were excited by 400- or 700-pA current injection. B, The effects of GxTX on a complex spiking cell. The generation of trains of complex spikes disappear in the middle of current injection (GxTX, 350 and 700 pA). C–E, Summary of the input-output relationship of simple-spiking cells (C), complex-spiking cells (D), and both simple-spiking and complex-spiking cells (E, combined). Statistical significance was tested using two-way repeated measure ANOVA and Bonferroni post hoc tests. n.s.: not significant; *p < 0.05, ***p < 0.001.

A train of parallel fiber-EPSCs evoked by various stimulus frequencies. A, A train of EPSCs induced by repetitive stimulation of parallel fibers. The membrane potential was held at –80 mV in the presence of strychnine and picrotoxin. Averages of five traces were used. Note that the amplitude of the last EPSCs is still large compared with the first EPSC in every stimulus frequency. B–D, Summary of relative amplitude of EPSCs plotted against stimulus numbers. Ei, A train of EPSCs in the absence (control) or presence (GxTX) of GxTX. Eii, Summary of the effects of GxTX on a train of EPSCs evoked at 100 Hz. Statistical significance was tested using two-way repeated measure ANOVA and Bonferroni post hoc tests. n.s.: not significant.