, 2010; Matsuda and Yuzaki, 2011; Uemura et al., 2010). The tripartite Nrx-Cbln1-GluD2 synaptic organizer is unique because it is indispensable for the formation and maintenance of PF-PC synapses in vivo (Ito-Ishida et al., 2008; Kakegawa et al., 2009; Yuzaki, 2011). Indeed, mice lacking Cbln1 or GluD2 (cbln1-null or glud2-null mice) exhibit significant reductions in normal synapses A-1210477 order and numerous naked
spines lacking their presynaptic partners ( Guastavino et al., 1990; Hirai et al., 2005; Kurihara et al., 1997). Naked spines are also present in other mutant mice, such as nodding ( Sotelo, 1990) and tottering mice ( Rhyu et al., 1999), and wild-type mice when PFs are reduced by irradiation or certain drugs ( Baloyannis and Kim, 1979; Hartkop and Jones, 1977). Although the remaining PF terminals in these animals sprout and enlarge to increase the number of PF synaptic contacts over time, such compensatory responses do not occur in cbln1-null or glud2-null mice ( Guastavino et al., 1990; Hirai et al., 2005; Kurihara MLN0128 research buy et al., 1997). These findings suggest that Nrx-Cbln1-GluD2 signaling plays a crucial role in PF differentiation. Nevertheless, whether and how Nrx-Cbln1-GluD2 interaction contributes to the structural modification of PFs has not been analyzed. In the present study, we performed morphological analysis of PFs during PF-PC synapse formation in vitro and in vivo. Taking advantage of
the strong synaptogenic effect of Cbln1 (Ito-Ishida et al., 2008), we performed dual time-lapse imaging of PFs and PCs to observe the sequential events of synaptogenesis in organotypic culture. Activation of Cbln1-GluD2 signaling induced axonal protrusions from PFs prior to presynaptic bouton formation. Interestingly, PF protrusions frequently formed circular
structures that encapsulated PC spines. Immunohistochemical and electron microscopic analyses confirmed that such PF protrusions, which often completely encapsulated spines, were observed in the immature mouse cerebellum in vivo. Furthermore, we found that such PF protrusive changes were dependent on Cbln1, GluD2, and Nrx. Finally, live imaging revealed that formation of PF protrusions and their encapsulation of PC spines either were followed by the accumulation of postsynaptic GluD2 and presynaptic vesicles. These results indicate that Nrx-Cbln1-GluD2 signaling induces presynaptic morphological changes, which may further accumulate pre- and postsynaptic components to promote bidirectional maturation of PF-PC synapses by a positive feedback mechanism. To observe dynamic structural changes in PF-PC synapse formation during development, we performed dual time-lapse imaging of PFs and PCs in cerebellar slice cultures prepared from mice at postnatal day (P) 8. Granule cells were transfected with DsRed2-encoding complementary DNAs (cDNAs) by electroporation (Yang et al.
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