Figure 4

Regulation of KCC2 by NL2 is independent of GABA _A receptor function or neuronal activity. A, Representative images showing reduced KCC2 immunostaining (red) in neurons transfected with NL2shRNA (green, arrowheads, 11 DIV), regardless whether treated with BIC or TTX. DMSO (0.1%, Control), BIC (20 μM) or TTX (1 μM) was added into culture medium after transfection at 2 DIV and replenished every two days. Scale bar, 20 μm. B, Bar graphs showing somatic KCC2 immunofluorescence intensity in neurons with (+) and without (-) NL2shRNA transfection (n = 11–12 neurons per group). Cultures were treated with DMSO (Control), BIC or TTX after transfection. ***p < 0.001, unpaired Student’s t-test. C, Representative images showing MAP2 (green) and KCC2 (red) immunostaining at 4 and 12 DIV in neurons treated with DMSO, BIC or TTX, starting at 2 DIV. Scale bar, 20 μm. D, Bar graphs showing developmental increase of the somatic KCC2 immunofluorescence intensity (n = 12 neurons per group). a.u., arbitrary unit. E, Representative immunoblot showing NL2 expression in 4 and 12 DIV neurons treated with DMSO, BIC or TTX, starting at 2 DIV. Actin was used as loading control. F, Quantification of NL2 expression level as measured by immunoblot (n = 3 independent cultures). NL2 expression was normalized to the expression level of 4 DIV control. G, Calcium imaging showing the time courses of GABA functional switch in control neurons and neurons treated with BIC or TTX (3 independent cultures; n = 1141, 717, 737 neurons, respectively).