Figure 4

E_GABA shift is dependent on intracellular Zn²⁺ rise. (a) Co-culture-induced E_GABA depolarization depends on Zn²⁺-mediated KCC2 inhibition. Left, TPEN application reverts co-culture induced E_GABA shift (co-culture/TPEN, −60.8±1.9 mV; −79.7±3 mV; n=16, P<0.01; control/TPEN, −72.8±1.9 mV; −77.4±3.8 mV; n=14; P=0.76; paired t-test), but fails to rescue E_GABA depolarization when KCC2 is blocked by DIOA (20 μM; DIOA −53.5±2.4 mV; plus TPEN −55.3±4.3 mV; n=6; P=0.78). Right, traces from a single co-cultured neuron before and following TPEN (20 μM) treatment. (b) Co-cultured neurons have higher basal Zn²⁺. Fluorescence monitoring from FluoZin-3 loaded control (purple circles; n=125) and co-cultured (green squares; n=158) neurons exposed to TPEN. (c) Co-culture-induced intracellular Zn²⁺ rise (control, purple column, n=67; co-culture, green column, n=98) is prevented by GluRs blockers (APV 20 μM; NBQX 10 μM, n=24, yellow column ordinate as in b). (d) Effects of Zn²⁺ chelation on co-culture-induced E_GABA depolarization. E_GABA shift was absent when neurons were co-cultured in the presence of the Zn²⁺-sensitive dye FluoZin (5 μM), chelating intracellular Zn²⁺ (control, −77±3.8 mV; n=8; 4 h co-culture −47.7±4.6 mV; n=13; 4h co-culture plus FluoZin −75±3.5 mV; n= 13; P<0.01), while tricine (1 mM, added to the co-culture medium, and washed during recordings, to avoid acute KCC2 inhibition mediated by extracellular Zn²⁺ removal¹⁹) was ineffective (control, −72.1±3.4 mV; n=13; 4h co-culture −52.2±4.4 mV; n=17; 4 h co-culture plus tricine −56.8±3.3 mV; n= 6; P=0.65; ^##P<0.01 versus control cultures treated with tricine)