Figure 3.

Examining the effects of ablating LMTK3 on KCC2 expression levels and activity

(A) Brain extracts from WT (+/+), heterozygotes (−/+), and homozygous (−/−) mice were immunoblotted with LMTK3, KCC2, and GAPDH antibodies. The levels of LMTK3 and KCC2 expression were then compared with those in WT (100%) mice; n = 3; ∗p < 0.05.

(B) Brain sections from WT (+/+) and KO (LMTK3-KO) mice were stained with LMTK3 and MAP2 antibodies followed by confocal microscopy, and a representative image of CA1; scale bar: 20 μm.

(C) High-magnification confocal images of the stratum pyramidale (s.p.) of WT (+/+) and LMTK3-KO (−/−) stained with KCC2 antibody; scale bar: 10 μm. KCC2 fluorescence intensity and total stained area were then compared with those seen in WT (100%), 8–10 slices from 3 mice.

(D) Representative traces and I–V plots are shown for the polarity of currents induced by rapid application of muscimol in DGGCs in slices from WT and LMTK3-KO mice loaded with 32-mM Cl⁻ at differing voltages.

(E) E_GABA values and [Cl⁻]_i were determined from the voltage ramps and then compared in DGGCs between genotypes; ∗p < 0.01; t test; n = 7–9 mice.

(F) Individual shifts in E_GABA are shown for DGGCs from WT and KO mice following a 15-min exposure to the KCC2 inhibitor 11K. The magnitude in the E_GABA shift (ΔE_GABA) was then compared between genotypes. ∗p < 0.05; n = 7–9 mice. In all panels data represent mean ± SEM. Voltages are adjusted with a liquid junction potential value of −13 mV.