| Abstract: | In Purkinje cells (PCs) of the cerebellum, a single “winner” climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9–15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.Monoinnervation of cerebellar Purkinje cells (PCs) by single climbing fibers (CFs) is established in the early postnatal period (1–3). The soma of a neonatal PC is innervated by more than five CFs with similar synaptic strengths, from which a single CF is functionally strengthened (4, 5). The strengthened (“winner”) CF starts dendritic translocation, whereas the other weaker (“loser”) CFs remaining on the soma are eliminated (6–8). In this process, P/Q-type voltage-dependent Ca2+ channels (VDCCs) promote functional differentiation and dendritic translocation of winner CFs, and the early phase of CF synapse elimination (9–11), whereas the late phase of CF synapse elimination is critically dependent on the formation of parallel fiber (PF) synapses and activation of the type 1 metabotropic glutamate receptor (mGluR1)-protein kinase Cγ (PKCγ) pathway (12–17).Segregated dendritic innervation by CFs and PFs is another distinguished feature of the PC synaptic wiring. Although hundreds of thousands of PFs innervate the distal dendritic domain, a single CF monopolizes the proximal dendritic domain, and both innervate a narrow intermediate domain (18). Given that both dendritic translocation of winner CFs and formation of PF synapses proceed upwards from the base of the dendritic tree (6, 19), CFs and PFs must compete with each other to establish their segregated territories. However, the developmental route and the underlying mechanisms of this process are unknown.Our findings indicate that CF and PF territories on PC dendrites are dynamically refined during the early postnatal period, and that the mGluR1-PKCγ signaling pathway regulates segregation by promoting PF synapse elimination. Thus, this signaling cascade plays key roles in sculpting the excitatory synaptic wiring in PCs by eliminating both redundant CF synapses from the soma (3, 20) and competing PF synapses from the proximal dendrites. |
