Research ArticleNEUROSCIENCE

Antiarrhythmics cure brain arrhythmia: The imperativeness of subthalamic ERG K+ channels in parkinsonian discharges

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Science Advances  10 May 2017:
Vol. 3, no. 5, e1602272
DOI: 10.1126/sciadv.1602272
  • Fig. 1 Characterization of ERG K+ currents in acutely dissociated subthalamic neurons.

    (A and B) Representative ERG K+ channel inhibitor E-4031–subtracted (A) and dofetilide-subtracted (B) currents (see original currents in fig. S1). The voltage protocol is shown on (A) (top). The E-4031–subtracted (600 nM) or dofetilide-subtracted (100 nM) K+ currents are elicited by 1-s depolarizing pulses to different voltages from a holding potential of −120 mV with 150 mM K+ in both extracellular and intracellular solutions. Each depolarizing pulse is immediately followed by a repolarization at −80 mV for 1 s to obtain tail current. Scale bars, 500 pA/200 ms. The bottom-left inset shows the currents after subtraction of the currents in drug from those after washout of the drug. Scale bars, 500 pA/200 ms. The bottom-right inset shows enlarged tail currents evoked at −80 mV after prepulse potentials of 0 to 40 mV. Scale bars, 400 pA/60 ms. The dashed lines indicate the zero-current level. (C and D) Activation curves of E-4031–sensitive (C) and dofetilide-sensitive (D) currents. Peak tail currents are measured and normalized to the maximal tail current produced with a preceding depolarization at 40 mV. The normalized peak tail currents (I/Imax) are then plotted against the voltages (Vt) of the preceding depolarization and fitted with a Boltzmann function I/Imax = 1/(1 + exp([V0.5Vt]/k)) with V0.5 (the voltage producing half-maximal activation) of −32 and −29 mV and k (slope factor) of 14 and 15 for E-4031–sensitive (C; n = 6) and dofetilide-sensitive (D; n = 6) currents, respectively. (E and F) Repolarization voltage dependence of deactivation for E-4031– and dofetilide-sensitive K+ currents. Tail currents are elicited at different repolarization potentials immediately after a depolarization at 20 mV. The decay of the tail current is fitted with a monoexponential function. The logarithm of the time constants (τ) is then plotted against the repolarization potential and fitted with a linear function y = 2.57 + 0.01x and y = 3.35 + 0.02x for E-4031–sensitive (E; n = 4) and dofetilide-sensitive (F; n = 6) currents, respectively. The insets show representative tail currents upon repolarization. Scale bars, 200 pA/20 ms (E-4031–sensitive currents) and 400 pA/20 ms (dofetilide-sensitive currents). (G and H) The time constant of the tail current decay at −80 mV is independent of the voltage of the preceding depolarization for E-4031–sensitive (G; n = 5) and dofetilide-sensitive (H; n = 3) K+ currents. Animals used: p9 to p15 rats.

  • Fig. 2 Extracellular recording of spontaneous discharges modulated by ERG inhibitors in acute STN (subthalamic) slices.

    (A to D) Representative cell-attached recordings show that both E-4031 and dofetilide reversibly reduce burst discharges in a dose-dependent fashion (A and B) but do not affect tonic discharges (C and D) (see also fig. S5). (E to H) For the burst mode of discharges, burst rates (average burst counts per minute) (E and F), intraburst spike frequency (G), and the number of spikes per burst (H) before, during, and after application of 5 μM (n = 6) or 10 μM (n = 5) E-4031 are analyzed. *P < 0.05 compared with control, paired two-tailed Student’s t test. N.A., not applicable (no bursts for analysis in that condition). There is no statistically significant difference between “control” and “washout.” *P = 0.13, 0.06, and 0.13 for (F), (G), and (H), respectively, in the study of recovery from 5 μM E-4031 treatment; *P = 0.11, 0.16, and 0.16 for (F), (G), and (H), respectively, in the study of recovery from 10 μM E-4031 treatment. (I to L) For the tonic mode of discharges, single-spike frequency and coefficient of variance (CV) of the interspike intervals (ISIs) before, during, and after E-4031 (I and J; n = 4 in 5 μM and n = 5 in 10 μM) or dofetilide (K and L); n = 3 in 5 μM). Scale bars, 2 s. Animals used: p18 to p26 mice.

  • Fig. 3 Whole-cell patch-clamp recording of spontaneous neuronal activity modulated by different ERG inhibitors in acute STN slices.

    (A) Top: E-4031 (5 and 10 μM) decreases burst discharges and the intraburst spike frequency and even turns bursts into single spikes in a higher concentration (see also fig. S6A). Inset: Superimposed bursts in the absence (black) and the presence (red) of 5 μM E-4031 demonstrate a decreased AHP in the latter condition (see also fig. S9A). Continuous recording of spontaneous activity in a representative neuron is provided in fig. S8. Bottom: 5 μM dofetilide as well as 10 nM astemizole reduce burst discharges. Inset: Superimposed bursts in the absence (black) and presence (red) of 5 μM dofetilide once again demonstrate a decreased AHP by dofetilide. (B) The spontaneous tonic activity is not affected by E-4031, dofetilide, and astemizole. (C to G) For the burst mode, the effects of E-4031 (5 μM) on the burst rate (C), intraburst spike frequency (D), the number of spikes per burst (E), postburst AHP amplitude (F), and the lowest potential (G) are analyzed (each n = 5). (H to K) The effects of E-4031 (5 μM; H and I) or dofetilide (5 μM; J and K) on single-spike frequency and coefficient of variance of the interspike intervals (n = 4 for dofetilide and n = 8 for the others). *P < 0.05 compared to control, paired two-tailed Student’s t test. Scale bars, 20 mV/1 s. Animals used: p18 to p26 mice.

  • Fig. 4 The effect of ERG channel activator on tonic discharges of subthalamic neurons.

    (A to C) Three representative subthalamic neurons that fire in spontaneous tonic activity at a “resting” membrane potential of about −60 mV are shown. The horizontal dashed lines indicate the level of −60 mV. Continuous recording of spontaneous activity in a representative neuron from a rat is provided in fig. S8. (A) PD-118057 (0.5 μM) reversibly hyperpolarizes the membrane (marked by arrows) and turns the tonic spikes into burst discharges (see also Fig. 6A and fig. S4B). (B) The firing mode switching as well as the hyperpolarizing effects of 0.5 μM PD-118057 are abolished by the coapplication of the ERG channel inhibitor E-4031 (5 μM). (C) The tendency toward bursts is correlated with the concentration of PD-118057 as well as its effect on membrane hyperpolarization (marked by arrows). (D to I) There are prominent effects of PD-118057 (0.5 μM) on the burst rate (D), intraburst spike frequency (E), spikes per burst (F), the lowest membrane potential (G), AHP amplitude (H), and the frequency of hyperpolarizing overshoot (I; the hyperpolarization period more negative than the resting membrane potential) (each n = 4). (J) The effects of E-4031 [5 μM; data from Fig. 3 (n = 8)], dofetilide [5 μM; data from Fig. 3 (n = 4)], and PD-118057 (0.5 μM; n = 4) on the interspike intervals and the coefficient of variance of the interspike intervals are compared. *P < 0.05; **P < 0.01 compared to control, paired two-tailed Student’s t test. N.A., not applicable (no bursts for analysis in that condition). Scale bars, 20 mV/2 s. Animals used: p18 to p26 mice.

  • Fig. 5 The effect of the ERG activator on burst discharges of subthalamic neurons recorded at a membrane potential of about −65 mV.

    The horizontal dashed lines indicate the level of −65 mV. (A) A representative neuron that spontaneously fires in bursts with relatively long plateau is shown. PD-118057 (0.5 μM) evidently hyperpolarizes the burst plateau and also shortens burst duration [see analyses in (E) to (J), n = 4; see also figs. S4B and S6B]. (B to D) Three representative neurons that spontaneously fire in relatively short bursts are shown [n = 8, 8, and 4 for (B), (C), and (D), respectively]. Note that the baseline membrane potential is not markedly altered by PD-118057 (0.5 μM) in any case but is slightly depolarized by E-4031 (5 μM). (B) The burst pattern remains in 0.5 μM PD-118057 but is abolished by the addition of 5 μM E-4031. (C and D) PD-118057 (0.5 μM) hyperpolarizes the plateau and abolishes bursts. (E to J) In subthalamic neurons having burst duration >3 s, there are no significant effects of PD-118057 (0.5 μM) on burst rates (E), intraburst spike frequency (F), the lowest potential (I), and AHP amplitude (J). However, PD-118057 significantly shortens the burst duration (G; see also fig. S9B) and decreases the number of spikes per burst (H) (n = 4; *P < 0.05; **P < 0.01 compared to control, paired two-tailed Student’s t test). Scale bars, 20 mV/2 s. Animals used: p18 to p26 mice.

  • Fig. 6 Remedy of locomotor deficits of parkinsonian rats by ERG channel blockers.

    (A) Left: Tyrosine hydroxylase immunohistochemistry shows unilateral dopaminergic deficiency (indicated by arrow heads) in the striatum of parkinsonian (PD) rat coronal brain sections. Middle and right: Whole-cell recording in STN slices from either PD rats [where subthalamic neurons tend to fire in the burst mode (middle); refer to more cases in fig. S4] or normal rats [where subthalamic neurons more frequently fire in the tonic mode (right)] in the absence and presence of 5 μM E-4031 (n = 5; middle) and 0.5 μM PD-118057 (n = 7; right), respectively. Scale bars, 20 mV/2 s. *P < 0.05; **P < 0.01, paired two-tailed Student’s t test. Also refer to in vivo electrophysiological data in fig. S11. (B) Representative locomotor traces of a PD (left) or a normal (right) rat in an arena with administration of normal saline and 200 μM E-4031 (left) or PD-118057 (right), respectively. (C and D) Changes in locomotor activities after direct microinjection of E-4031 or PD-118057 into the STN. The changes in the total distance of movement (left), total duration of movement (middle), and total duration of immobilization (right) in normal or parkinsonian (PD) rats with administration of either 200 μM E-4031 (C; n = 10 for both normal and PD groups) or 200 μM PD-118057 (D; n = 9 for both normal and PD groups) are compared with those injected with normal saline. *P < 0.05, two-way mixed-design analysis of variance (ANOVA). Application of E-4031 markedly improves locomotor activities of PD (but not normal) rats in (C). In contrast, PD-118057 significantly decreases locomotor activities of normal (but not PD) rats in (D). See also fig. S10. Animals used: p37 to p40 rats for brain slice recording; 2- to 3-month-old rats (weighing 260 to 380 g) for other experiments.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602272/DC1

    fig. S1. The original currents of Fig. 1.

    fig. S2. Characterization of ERG K+ currents by channel activator PD-118057 in acutely dissociated subthalamic neurons following similar experimental protocols and analyses of Fig. 1.

    fig. S3. The effect of PD-118057 on E-4031–sensitive currents.

    fig. S4. The effect of the ERG inhibitor and activator on spontaneous firing of subthalamic neurons in acute STN slices from parkinsonian rats.

    fig. S5. The distinct effects of the ERG inhibitor dofetilide on a pair of simultaneously recorded neurons, with one firing in the spontaneous tonic mode and the other in the burst mode.

    fig. S6. The effect of ERG channel inhibitor and activator on burst discharges in rat STN slices.

    fig. S7. The effect of ERG channel inhibitor and activator on the spontaneous firing activity of subthalamic neurons at low and high temperatures.

    fig. S8. Continuous recording of spontaneous firing activity in acute STN slices before, during, and after application of 5 μM E-4031 (A) or 0.5 μM PD-118057 (B).

    fig. S9. The effect of ERG channel inhibitor and activator on the plateau depolarization in the presence of tetrodotoxin.

    fig. S10. The effect of ERG channel modulation on rearing scores and the asymmetric limb use.

    fig. S11. Inhibition of ERG channels ameliorates abnormal burst discharges in parkinsonian rats.

    Methods for figs. S10 (E and F) and S11

    References (100, 101)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. The original currents of Fig. 1.
    • fig. S2. Characterization of ERG K+ currents by channel activator PD-118057 in acutely dissociated subthalamic neurons following similar experimental protocols and analyses of Fig. 1.
    • fig. S3. The effect of PD-118057 on E-4031–sensitive currents.
    • fig. S4. The effect of the ERG inhibitor and activator on spontaneous firing of subthalamic neurons in acute STN slices from parkinsonian rats.
    • fig. S5. The distinct effects of the ERG inhibitor dofetilide on a pair of simultaneously recorded neurons, with one firing in the spontaneous tonic mode and the other in the burst mode.
    • fig. S6. The effect of ERG channel inhibitor and activator on burst discharges in rat STN slices.
    • fig. S7. The effect of ERG channel inhibitor and activator on the spontaneous firing activity of subthalamic neurons at low and high temperatures.
    • fig. S8. Continuous recording of spontaneous firing activity in acute STN slices before, during, and after application of 5-μM E-4031 (A) or 0.5-μM PD-118057 (B).
    • fig. S9. The effect of ERG channel inhibitor and activator on the plateau depolarization in the presence of tetrodotoxin.
    • fig. S10. The effect of ERG channel modulation on rearing scores and the asymmetric limb use.
    • fig. S11. Inhibition of ERG channels ameliorates abnormal burst discharges in parkinsonian rats.
    • Methods for figs. S10 (E and F) and S11
    • References (100, 101)

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