To directly visualize the DD synaptic remodeling process, including synapse elimination and synapse formation, we labeled DD presynapses by expressing GFP::RAB-3 (Mahoney et al., 2006 and Klassen and Shen, 2007) under the DD-specific flp-13 promoter ( Kim and Li, 2004). In synchronized cultures, the distribution of RAB-3 fluorescence in the dorsal and ventral processes of DD neurons was examined at various time points including 11, 16, 18, CHIR-99021 19.5, 22, 26,

and 28 hr after egg laying (see Figure S1 available online). A cytoplasmic mCHERRY marker was used to accurately identify the DD processes. Before synaptic remodeling, all GFP::RAB-3 puncta are located ventrally ( Figure S1B, B1). Upon the start of remodeling, ventral puncta gradually VX-770 nmr become smaller ( Figure S1B, B2 and B3), weaker ( Figure S1B, B4), and eventually disappear ( Figure S1B, B5). Concurrently, RAB-3 puncta

appear in the dorsal processes and become more intense over time ( Figure S1B, B3–B5). DD synaptic remodeling process was quantified by counting animals containing GFP::RAB-3 puncta only in the ventral processes (only V), both in ventral and dorsal processes (V+D), or only in the dorsal processes (only D), as shown in Figure S1C. Indeed, we observed a steady decrease in “only V” animals and a concomitant increase in the “only D” animals throughout the remodeling process, indicative of the gradual elimination of ventral synapses

and the concurrent formation of dorsal synapses ( Figure S1C). We chose to focus the present study on the time points 16, 18, 19.5, 22, and 26 hr after egg laying, during which the majority of the remodeling process takes place ( Figure S1C). We have recently identified that a protein, CYY-1, which contains a cyclin-like domain, and CDK-5 are important for the correct localization of presynaptic components in C. elegans ( Ou et al., 2010). Ketanserin Since the remodeling of DD synapses involves the formation of new synapses in distal axons, it is likely that regulation of axonal transport is an important step during this structural plasticity process. Therefore, we tested if these two molecules, CYY-1 and CDK-5, affect synaptic remodeling of DD neurons. To do this, we utilized the putative null alleles cyy-1(wy302) and cdk-5(ok626). In L1-staged cyy-1 or cdk-5 animals, RAB-3 fluorescence is distributed “only V” ( Figure 1A, A3 and A5) just as in wild-type animals ( Figure 1A, A1). However, in the L4 or young adult-staged cyy-1 or cdk-5 animals, RAB-3 fluorescence still remains in the ventral process ( Figure 1A, A4 and A6) unlike in the wild-type controls, where RAB-3 is only found in the dorsal processes ( Figure 1A, A2).