C. elegans Dendritic Morphology Analysis

C. elegans was maintained using standard procedures^{48 (link)}. unc-33(ky880); kyIs445 and Pdes-2::unc-33L were described previously^{13 (link)}. Mutations were introduced to the Pdes-2::unc-33L plasmid by PCR-based mutagenesis with KOD-plus high fidelity DNA polymerase (TOYOBO, Tokyo, Japan). Injections were performed as described^{49 (link)}. In short, wild type and mutant unc-33 plasmids (10 ng/μl) and Podr-1::DsRed (50 ng/μl) were injected to unc-33(ky880); KyIs445 mutant worms. DsRed positive worms were transferred to new plates and stable transmission of the extrachromosomal array was determined. For rescue experiments, only DsRed positive worms were observed and analyzed by LSM710 confocal microscope (Carl Zeiss).
For the statistical analysis of the dendritic phenotype, worms were observed in a blind fashion. The observer did not know genotypes until the end of the test. Each worm was classified as “wild type” or “unc-33” by the synaptic vesicle localization. 100 worms were observed for each genotype and plotted as bar graphs. For the quantification of axonal fluorescent intensity, worms were observed in the same manner as the dendritic analysis. The mean fluorescent intensity was measured using Image J (NIH). A 100-μm region of the axon was selected randomly to measure the mean fluorescent intensity.