Figure 3.

Suppression of activity-driven, membrane insertion of AMPARs in neurons lacking KCC2. A, Neurons were transfected with GluA1-SEP. Left, Summary graph of mean SEP fluorescence (normalized to control) at pH 5.5 (n = 11) and upon NH₄Cl application (n = 26 cells). ***p < 0.005. SEP fluorescence was detected in neurons imaged at pH 7.4 (Aa) but was near-completely quenched at pH 5.5 (Ab). Upon collapse of pH gradients using 50 mm NH₄Cl at pH 7.4 (Ac), additional fluorescence was detected reflecting an intracellular pool of SEP-GluA1. Right, Spinning disc confocal fluorescence micrographs of SEP-GluA1-expressing hippocampal neurons at time points a–c. Scale bar, 2 μm. B, Overlay of mCherry (red) and SEP (pseudocolors) fluorescence micrographs of dendritic sections of neurons before (Control) and 35 min after cLTP induction. Filled arrowheads indicate cLTP-induced SEP fluorescence spots. Open arrowheads indicate spines with unchanged SEP fluorescence. KCC2 suppression by RNA interference, but not blockade by the antagonist VU0240551 (6 μm), abolished cLTP-induced increase in SEP fluorescence. Scale bar, 2 μm. C, SEP fluorescence 35 min after cLTP induction normalized to control in neurons expressing SEP-GluA1 or SEP-GluA2 and either nontarget (shNT) or KCC2-directed (shKCC2) shRNA, KCC2 shRNA together with shRNA-proof recombinant KCC2 (rescue) or neurons exposed to KCC2 antagonist VU0240551 (6 μm) or vehicle only. **p < 0.01. ***p < 0.005. n = 18–29 neurons in each condition. D, Left, SEP fluorescence before cLTP induction in neurons expressing SEP-GluA1 and either nontarget (n = 47) or KCC2-directed (n = 56) shRNA, normalized to the mean SEP fluorescence in shNT-expressing neurons. *p < 0.05. Right, Change in SEP fluorescence (p of control) upon 5 min perfusion of NH₄Cl solution, pH 7.4, to reveal intracellular SEP-GluA1 pool (shNT, n = 23; shKCC2, n = 28). ***p < 0.005.