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. 2017 Mar 23;7:370. doi: 10.1038/s41598-017-00325-2

Figure 2.

Figure 2

Expression of human APP in cortical cultures decreases KCC2 expression and changes GABA-induced responses. (a) Left panel: Comparative RT-qPCR analyses of Slc12a2 (coding for NKCC1) and Slc12a5 (coding for KCC2) mRNA levels in primary rat cortical cultures infected or not (control, Co) at 6 DIV with an adenovirus coding for human APP (hAPP). Results were obtained between 13 and 17 DIV (n = 20 for each condition in 9 independent experiments), normalized to GAPDH mRNA and expressed as percentage (mean ± s.e.m.) of Co (***P < 0.001, Student’s t-test). Middle panel: Representative Western blot of cell lysates from Co and hAPP cultures. hAPP and total APP, were detected between 13 and 17 DIV with the WO2 and anti-APP C-terminal antibody, respectively. Blots were further probed using anti-NKCC1, -KCC2, and -α tubulin antibodies. Right panel: quantification of total APP, NKCC1 and KCC2/α tubulin ratios expressed as percentage (mean ± s.e.m.) of Co (Co n = 28 and hAPP n = 30 in 16 independent experiments, *P < 0.05, ***P < 0.001, Mann-Whitney test (total APP) and Student’s t-test (NKCC1 and KCC2)). (b) GABA (100 µM)-induced calcium response in Co and hAPP cultures measured between 13 and 17 DIV incubated with or without 10 µM bumetanide (bum.) for 1 h. Insets: representative traces of ten cell recordings. Traces are expressed as ΔF/F0, where ΔF corresponds to a change in F340/F380 ratio and F0 is the basal fluorescence value. Percentage (mean ± s.e.m.) of cells responding to GABA (left panel) and amplitude of this response (right panel) (Co n = 138, hAPP n = 122, hAPP+ bum. n = 208 cells in 4 independent experiments), ANOVA test followed by Bonferroni’s multiple-comparison post-test (compared to controls: *P < 0.05, ***P < 0.001; to untreated hAPP: ### P < 0.001). Frequency and representative traces of action potentials depending on the current injected (c) and difference in membrane potential (ΔV) (d) in Co and hAPP expressing cortical neurons before or after GABA (100 µM) application measured between 13 and 19 DIV (Co n = 17 and hAPP n = 19 cells in 6 independent experiments, in (c) *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test applied for each current step).