Fig. 3: N-acetyl-L-cysteine (NAC) treatment prevents oxidative stress and hypofunction of T-Ca²⁺ channels in TRN neurons of young GBR-treated Gclm-KO mice.

A Micrographs showing immunofluorescent labeling for 8-oxo-dG (oxidative stress marker, green), parvalbumin (PV, red), and WFA/PNN (blue) in the TRN of PND40 WT, KO GBR, and KO GBR-NAC mice. WFA/PNN: perineuronal net labeled with Wisteria floribunda agglutinin (WFA). NAC treatment in KO GBR mice decreases 8-oxo-dG labeling (B), increases the number of PV-IR neurons (C) and PNN + PV-IR neurons (D). Note that the numbers of PV-IR and PNN + PV-IR neurons are even higher in KO GBR-NAC as compared to WT mice. Data are from 5 mice per group. Threshold for the initial membrane potential required to induce single (E), or repetitive bursting (F) upon depolarization, with no significant differences between groups (WT n = 11; KO GBR n = 10; KO GBR-NAC n = 6). G Density of T-Ca²⁺ currents activated at resting membrane potential (RMP), with higher current density in KO GBR-NAC as compared to WT and KO GBR mice (p = 0.026 and p = 0.012, respectively, Kruskal–Wallis test followed by paired-wise comparisons and Bonferroni corrections) (WT n = 24; KO GBR n = 7; KO GBR-NAC n = 12). Density of T-Ca²⁺ (H) and SK (I) currents activated from each of the initial membrane potentials, with significant overall difference between groups for T-Ca²⁺ (F = 60.96 DFn = 2 DFd = 731; p < 0.0001) and SK currents (F = 75.16 DFn = 2 DFd = 697; p < 0.0001). The maximal currents activated from membrane potentials ≤ −80 mV are recovered by NAC treatment, with similar current density in TRN neurons of KO GBR-NAC and WT mice. Note however that T-Ca²⁺ and subsequently SK channels are more activated from depolarized membrane potentials (≥−65 mV) in TRN neurons of KO GBR-NAC as compared to both WT and KO GBR mice (shift in steady-state inactivation of T-Ca²⁺ channels). J Correlation between T-Ca²⁺ and SK current densities (Spearman r = −0.98 for WT; −0.97 for KO GBR; −0.91 for KO GBR-NAC; p < 0.0001). K–M Superfusion of 100 µM GSH in aCSF rapidly increases T-Ca²⁺ currents upon depolarization, particularly when the initial membrane potential of TRN neurons is ≤ −70 mV (shift in steady-state inactivation of T-Ca²⁺ channels). This effect of GSH is more pronounced in KO GBR (M) than in WT mice (L), suggesting more oxidized extracellular redox-sensitive sites of CaV3.2 channels in KO GBR as compared to WT mice. Horizontal dotted lines on (L) and (M) indicate maximal average current density for WT mice. Representative recordings of maximal T-Ca²⁺ currents before and after 5 min GSH application in aCSF are given in (K). aCSF: artificial cerebrospinal fluid. Two-way ANOVAs with Bonferroni post-tests; *p < 0.05 **p < 0.01 ***p < 0.001. Data are presented as means ± s.e.m.