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. 2009 Jun 24;29(25):7966–7977. doi: 10.1523/JNEUROSCI.1054-09.2009

Figure 6.

Figure 6.

NKCC1-knockdown phenotype can be compensated by enhanced activation of CREB signaling. Analysis of neuronal protein expression and cellular morphology of animals injected with shNKCC1- and RFP-encoding retroviruses and animals injected with shNKCC1- and CREBYF-encoding retroviruses. a, Confocal images from animals at 7 dpi. Analysis of DCX expression and of the morphology of newborn cells. shNKCC1 (green)- and RFP (red)-injected control animals in left column. shNKCC1 (green)- and CREBYF (red)-injected animals in right column. Many shNKCC1/RFP-cotransduced cells are negative for DCX (blue) (white arrowhead). Yellow arrowheads indicate RFP-only transduced cells expressing DCX. Right column, shNKCC1/CREBFY-cotransduced cells expressing DCX and sending an apical dendrite toward the molecular cell layer (red arrowheads). Yellow arrowhead indicates CREBYF-only transduced immature neuron. Scale bar, 25 μm. b, Confocal images from animals at 7 dpi. Analysis of NeuroD expression and of the morphology of newborn cells. shNKCC1 (green)- and RFP (red)-injected control animals in left column. shNKCC1 (green)- and CREBYF (red)-injected animals in right column. The majority of shNKCC1/CREBFY double-transduced cells express NeuroD (blue) (red arrowheads). White arrowheads indicate shNKCC1-transduced cells which have lost NeuroD expression. Yellow arrowheads indicate RFP- or CREBYF-transduced cells which are NeuroD+. Scale bar, 25 μm. c, Quantification of single-transduced (shNKCC1, RFP, or CREBYF) and double-transduced (shNKCC1/RFP or shNKCC1/CREBYF) cells expressing DCX. CREBYF expression significantly increases the fraction of DCX+ immature neurons among shNKCC1-transduced cells (***p < 0.001). d, Quantification of single- and double-transduced cells expressing NeuroD. CREBYF expression significantly increases the fraction of NeuroD+ cells among shNKCC1-transduced cells (**p < 0.01) e, Quantification of single-transduced and double-transduced immature neurons, which extended an apical dendrite spanning the granule cell layer. CREBYF expression significantly increases the fraction of immature neurons, which have developed a dendrite spanning the granule cell layer among single-transduced cells and shNKCC1-transduced cells (***p < 0.001).