We analyzed all postsynaptic partners (labeled and unlabeled, as shown in Figures 3E and 3F) of axonal boutons that contact mHRP-labeled dendritic processes, and found that presynaptic boutons contacting stable dendritic branches had fewer postsynaptic partners than those contacting extended branches (stable: 1.38 ± 0.06, extended: 2.19 ± 0.12 postsynaptic profiles/presynaptic bouton, n = 78 and 47, respectively, p < 0.001; Figure 3I). Furthermore, 79% of synapses on extending dendrites contacted MSBs whereas 38% of synapses on stable dendrites

contacted MSBs. Our previous studies showed that mechanisms that increased synaptic strength and maturation also stabilize dendritic branches (Haas et al., 2006), suggesting that synapses on stable branches may be more mature PLX3397 order than those on dynamic branches. DAPT We previously reported that the proportion of the presynaptic terminal area that is occupied by clustered synaptic vesicles increased during development when synapses mature and termed this metric the maturation index (Li and Cline, 2010). Here, we mapped the maturation index of synapses on stable, extended, and retracted branches (Figures 4A–4C). We found that synapses on stable dendrites had a higher maturation index compared to those on

extended dendrites (stable: 45.2 ± 1.7, n = 78; extended: 35.5 ± 2.5, n = 47, p < 0.001; Figure 4D). We also found that synapses Rolziracetam on retracted dendrites had a low maturation index (17.7 ± 10.2, n = 4 synapses), suggesting that disassembly of synaptic components occurs

prior to branch retraction, consistent with our previous in vivo imaging studies (Ruthazer et al., 2006) and studies in the neuromuscular junction (Colman et al., 1997). This analysis demonstrates that synapses on stable dendrites were significantly more mature than those on extended or retracted dendrites. Data presented above showed that synapses on extended branches tended to be clustered within 1 μm of each other. Analysis of synapse maturation relative to synapse distribution on extended branches showed that synapses that were clustered within 1 μm of each other were less mature, with an average maturation index of 28.9 ± 2.9 (n = 33), while synapses spaced further apart than 1 μm were more mature, with an average maturation index of 42.6 ± 5.0 (n = 12, p < 0.05; Figure 4E). By contrast, synapses on stable branches were relatively mature and their maturation indices were independent of the distance between synapses (maturation index of synapses within 1μm and larger than 1 μm: 45.6 ± 2.3 versus 44.7 ± 2.8, n = 47 and 28, respectively). This analysis indicates that extending branches tend to have clustered immature synapses, whereas synapses on stable branches are more mature and more sparsely spaced. The MSBs that contact mHRP-positive dendrites also contact unlabeled dendrites (Figures 3E and 3F).