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. 2017 May 23;4(3):ENEURO.0377-16.2017. doi: 10.1523/ENEURO.0377-16.2017

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Copyright © 2017 Hermanstyne et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 2. — Acute knockdown of Kv4.1 decreases A-type K⁺ currents (I_A) in SCN neurons during the day and at night. A–D, Representative whole-cell Kv current recordings, obtained during the day (ZT7–ZT12) or at night (ZT19–ZT24) from eGFP-positive SCN neurons in acute slices prepared from adult animals 14–30 d following injections of the nontargeted shRNA-expressing (A, C) or the Kv4.1-targeted shRNA-expressing (B, D) AAV8, are shown. Whole-cell Kv currents, evoked during (2 s) voltage steps to potentials ranging from −40 to +30 mV (in 10-mV increments) from a holding potential of −70 mV, were recorded from nontargeted shRNA- and Kv4.1-targeted shRNA-expressing SCN neurons during the day (A1–B1) and at night (C1–D1). A second voltage-clamp paradigm, that included a brief (60 ms) prepulse to −10 mV to inactivate I_A, was then presented, and whole-cell Kv currents in each cell were recorded again (A2–D2). The voltage-clamp paradigms are illustrated (light gray) below the current records. Digital offline subtraction of the recordings with the prepulse (A2–D2) from those without the prepulse (A1–D1) isolated I_A (A3–D3); the subtracted records are also shown on an expanded time scale. E, I_A densities at each test potential in each cell were calculated and mean ± SEM values are plotted as a function of test potential. F, Mean ± SEM I_A densities at +30 mV were significantly lower in Kv4.1-targeted shRNA-expressing (n = 10–27), than in nontargeted shRNA-expressing (n = 8–10) SCN neurons during the day and at night; p values (one-way ANOVA) are indicated. G, The activation curves for Kv4.1-targeted shRNA-expressing SCN neurons (open red squares, n = 8) and nontargeted shRNA-expressing SCN neurons (open black squares, n = 12) are not significantly (p = 0.052, Student’s t test) different (V_1/2 = −1.8 ± 3.2 mV, k = 13.5 ± 1.1 and −9.3 mV ± 1.3 mV, k = 12.7 ± 0.8, respectively). The inactivation curves plotted as the mean ± SEM normalized current for Kv4.1-targeted shRNA-expressing (filled red squares, n = 8) and nontargeted shRNA-expressing (filled black squares, n = 12) SCN neurons are also shown. The inactivation curve for Kv4.1-targeted shRNA-expressing SCN neurons (V_1/2 = −60.3 mV ± 1.7 mV and k = 7.0 ± 0.6) is not significantly (p > 0.05, Student’s t test) different from the nontargeted shRNA-expressing SCN neurons (V_1/2 = −59.4 mV ± 2.1 mV and k = 7.9 ± 0.7).