Figure 5.

Chronic Depolarization Elevated Basal Ca²⁺ Level Similarly Irrespective of Tonotopic Regions

(A–C) Effects of Cav channel blockers on increase of Kv current during depolarization in high-CF neurons. [K⁺]_o in culture medium was elevated by 1.5 times, while Cav channel blockers were added for 11–14DIV (A). Tail current without blockers (left), after the treatment of nifedipine (10 μM, left middle), ω-conotoxin GVIA (2 μM, right middle), and cocktail of blockers (right) (B). Arrows indicate tail current at −40 mV, and roughly represents Kv1 current. Kv1 current calculated at +20 mV in high-CF neurons (C). Data in control (black) and 1.5K treatment (red) were from Figures 2J and 3G. Numbers in parentheses are the number of cells. **p < 0.01 compared with control.

(D) Neurons were cultured in normal medium, and two-photon imaging was made at 11DIV.

(E and F) Ca²⁺ signals in high- (E) and low-CF (F) neurons. Cells were loaded with a high-affinity indicator (OGB-1, G) and Alexa 594 (R) through a patch pipette, and line scans were made at the soma (top, red line). ΔG/R increased in both neurons (bottom), when a depolarizing voltage pulse (10 s) was applied under voltage clamp (middle).

(G) ΔG/R at corresponding voltage.

(H) [Ca²⁺]_i measured with a low-affinity indicator (Fluo-5F) under voltage clamp.

(I) Dependence of [Ca²⁺]_i on membrane depolarization. Values were similar between high- and low-CF neurons (p > 0.1), and data from both neurons were combined. *p < 0.05, **p < 0.01 compared with −60 mV. Note that [Ca²⁺]_i was still within nanomolar range (about 200 nM) even at membrane potential comparable with that in 2K medium (−40 mV). The increase of [Ca²⁺]_i was occluded by nifedipine (10 μM, light blue). Cocktail of blockers in (B) showed similar suppression of [Ca²⁺]_i; [Ca²⁺]_i in the cocktail was 117.3 ± 22.4 nM at −60 mV, 133.9 ± 34.5 nM at −40 mV, and 213.0 ± 70.8 nM at −20 mV (n = 5) (p > 0.1 compared with that of nifedipine at each voltage).

See also Figures S4–S6.