To directly test our hypothesis, we developed a genetic means to extend the period of GABA-induced depolarization specifically in newborn neurons during early postnatal neurogenesis. Previous studies have shown that KCC2 up-regulation is a primary factor underlying the functional switch of GABAergic responses from depolarization to hyperpolarization during neuronal maturation (Rivera et al., 1999). We designed several shRNAs against mouse kcc2 (shRNA-K2) and identified effective ones with in vitro analysis (Figure S4). To test the shRNA efficacy in vivo, we injected retroviruses into the dentate gyrus of P10 animals. Ca2+ imaging analysis showed that shRNA-K2+ new neurons at 7 dpi exhibited a significantly larger Ca2+ rise compared to those expressing shRNA-C1 (Figures 4C to 4D). Functionally, expression of shRNA-K2 itself led to increased total dendritic length and complexity of new neurons at 7 dpi in the early postnatal hippocampus (Figures 5A to 5C), supporting a conserved role of depolarizing GABA signaling in promoting dendritic growth of immature neurons during neuronal development.
