Fig. 3

Developmental change of CLC-3 Cl⁻ channels in the mouse brain. a Representative traces of voltage-dependent Cl⁻ currents recorded in CA3 pyramidal neurons at different ages of animals. b Quantified Cl⁻ current density (HP = + 90 mV) illustrating age-dependent decline during early brain development. c NaGluc inhibition of Cl⁻ currents in WT neonatal neurons (P10–12). d Dose-response curve of NaGluc inhibition on Cl⁻ currents in hippocampal slices. e Characterization of CLC-3 knockout mice. PCR analysis confirmed a lack of the exon 7 of Clcn3 gene. Immunostaining also confirmed a lack of CLC-3 expression in hippocampal CA3 region of the Clcn3^−/− mice. Note the smaller size of the Clcn3^−/− mice at P12 compared to WT mice. Scale bar = 10 μm. f Brain slice recordings revealed a voltage-dependent outward rectifying Cl⁻ current in WT hippocampal CA3 pyramidal neurons, but not in CLC-3 KO neurons (P8–12). g I-V plot of voltage-dependent Cl⁻ currents in WT (black) and CLC-3 KO (gray) neurons. The green curve shows no further inhibition of NaGluc on the remaining Cl⁻ currents in CLC-3 KO neurons. h Typical immuno-fluorescent images showing different expression level of CLC-3 Cl⁻ channels in the hippocampus of neonatal (P11, left panel) and adult mice (3.5 months, right panel). The high magnification images of CA3 were placed in the up-left corner. Low magnification image scale bar = 200 μm; Inset scale bar = 10 μm. i Quantified data showing CLC-3 immunostaining intensity in neonatal and adult CA3 regions (unpaired Student’s t-test, P < 0.001). j, k Western blot revealed a significant decrease of surface CLC-3 Cl⁻ channels in the adult hippocampus (Mann-Whitney test, P < 0.01). Data are shown as mean ± s.e.m., ** P < 0.01, *** P < 0.001