Figure 4.

FOXO knockdown disrupts the establishment of neuronal polarity in the cerebellar cortex in vivo. (A,B) P3 rat pups were injected in the cerebellum with a GFP expression plasmid and then subjected to electroporation. Five days later, at P8, pups were sacrificed and coronal sections of cerebella were subjected to immunohistochemistry with a monoclonal antibody to GFP (green) and a rabbit polyclonal antibody to calbindin (red), the latter to label Purkinje cells. Transfected GFP-positive cerebellar granule neurons bear dendrites and have associated parallel fibers (PF) along the ML. Bars, 50 μm. (C) Coronal sections of cerebella electroporated as in A with the control U6-cmvGFP or U6/foxo-cmvGFP RNAi plasmid were subjected to immunohistochemistry with the GFP antibody (green) and the calbindin antibody (red). The bottom panels show a higher magnification of the numbered cells. In control animals (U6), granule neurons in the IGL were typically associated with parallel fibers. In contrast, FOXO knockdown (U6/foxo) led to loss of associated parallel fibers. Concomitant with the decrease in parallel fiber abundance, the length of secondary processes in the IGL was increased in granule neurons in FOXO knockdown animals as compared with granule neurons in control transfected animals. Arrows and arrowheads indicate secondary processes in the IGL (dendrites in control animals) and parallel fibers, respectively. (D) Quantification of total length of secondary processes in the IGL of granule neurons in animals electroporated and analyzed as in C. FOXO knockdown significantly increased the length of secondary processes in the IGL in granule neurons (P < 0.001; t-test, 335 neurons measured). (E) Quantification of parallel fiber phenotype upon FOXO knockdown in vivo. The percentage of granule neuron somas in the IGL that were associated with parallel fibers was significantly reduced in FOXO knockdown animals as compared with control transfected animals (P < 0.001; t-test, n = 3, 811 neurons measured). (F,G) P8 rat pups electroporated at P3 with the control U6-cmvGFP or U6/foxo-cmvGFP RNAi plasmid together with the FOXO6-Res expression plasmid or its control vector were analyzed as in A–E. Expression of FOXO6-Res in the background of FOXO knockdown in vivo significantly reduced the length of secondary processes in the IGL (dendrites in control animals) (P < 0.01; ANOVA, n = 3, 216 neurons measured) and significantly increased the number of parallel fibers associated with IGL granule neurons (P < 0.05; ANOVA, n = 3, 2655 neurons measured) as compared with FOXO knockdown animals.