To facilitate KPT-330 analysis of PF structure, we took advantage of the strong synaptogenic effect of Cbln1, which induces functional PF-PC synapses in cbln1-null slices within 8 hr ( Ito-Ishida et al., 2008). Time-lapse images of slices at 6–8 days in vitro (DIV) were obtained at 1 hr intervals for 6–9 hr using a confocal microscope. Without recombinant Cbln1, PFs in the cbln1-null slices

showed few morphological changes ( Figure 1B). Addition of recombinant WT-Cbln1 to the medium induced dynamic structural changes in the PFs ( Figures 1C–1E and S1; Movie S1 available online). Interestingly, active axonal protrusions emerged from PFs. The protrusions changed shape in every imaging frame and subsequently transformed into axonal boutons, which indicated emergence of new presynaptic terminals. We categorized these protrusions as simple protrusions (SPs) and circular protrusions (CPs) according to the morphological criteria ( Figure 1C). Single SPs were defined as filopodia-like

protrusions whose tips were located away from the main axons. CPs were defined as circular structures with dark central areas ( Figure 1C). CPs appeared either as complete rings without gaps ( Figures 1F and S1) or as winding filopodia whose tips were located in the proximity to the main axon ( Movie S1). Both SPs and CPs were located in the proximity of the GFP-positive PC dendrites ( Figures 1D and 1E), suggesting that they may interact with PCs. SPs were often observed to undergo morphological changes that lead to the formation of CPs ( Figure 1F). To examine further Selleck Selumetinib the relationship between PF protrusions and postsynaptic sites, we expressed chimeric GluD2 fused to GFP at the N terminus (GFP-GluD2) in PCs in cbln1-null slices. Similar to endogenous GluD2, GFP-GluD2 accumulated

as multiple clusters which suggested locations of PC postsynaptic sites ( Figure 1F). We found that the axonal changes occurred at sites where PFs were in the proximity to GluD2-positive clusters ( Figures 1F and S1A–S1C). Multiple Tryptophan synthase SPs, which were observed 6 hr after the addition of WT-Cbln1, merged to form a CP whose central dark area matched the position of GFP-GluD2 clusters ( Figure 1F). Among 13 CPs which were formed adjacent to the PC dendrites expressing GFP-GluD2, 9 CPs contained GFP-GluD2 signals in the central region ( Figure S1C). These findings indicate that the PF protrusions are formed in association with the postsynaptic sites. To examine the relationship between PF protrusions and synaptic boutons, we next examined the history of newly formed PF boutons. We analyzed the synaptogenic events that occurred synchronously after the addition of recombinant WT-Cbln1 to cbln1-null slices. We first identified the synaptogenic events by identifying boutons that were newly formed within 5 hr after the addition of WT-Cbln1 and lasted for 4 hr or longer.