Acute reduction of cholinergic transmission during adulthood produces reversible decreases in GABA synapse size. (A) Diagram indicating predicted timeline of wild-type C. elegans development at 15°C and temperature shifts employed. Approximate time of hatch is indicated. Temperature shifts to 25°C (red) and time of imaging (green) are indicated. E, embryo. (B,C) Merged confocal images of unc-47p::mCherry::RAB-3 and UNC-49::GFP labeling in the ventral nerve cord region of adult wild-type animals (B) or cha-1(y226) mutants (C) following a shift to 25°C for the durations indicated. Scale bar: 20 µm. (D,E) Average number of GABA presynaptic (D) and postsynaptic (E) clusters per 50 µm for wild type (black) and cha-1(y226) mutants (gray). (F,G) Average size of GABA presynaptic (F) and postsynaptic (G) clusters for wild type (black) and cha-1(y226) mutants (gray). *P<0.05, **P<0.01, ***P<0.0001; ANOVA with Dunnett's multiple comparisons test.