Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor delta2 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cbln1 binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.

D-serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

The ␦2 glutamate receptor (GluR␦2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluR␦2 Ϫ/Ϫ mice display ataxia and impaired motor learning. In addition, long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is abrogated, and synapse formation with PFs and climbing fibers (CFs) is severely disturbed in GluR␦2 Ϫ/Ϫ Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluR␦2 Ϫ/Ϫ Purkinje cells by the virus-mediated expression of the wild-type GluR␦2 transgene (Tg wt ) but not by that of mutant GluR␦2 lacking the C-terminal seven residues to which several PDZ proteins bind (Tg ⌬CT7 ). These results indicated that the C terminus of GluR␦2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluR␦2 Ϫ/Ϫ cerebellum, especially morphological abnormalities at PF and CF synapses, could not be rescued by virus-mediated transient expression. Thus, whether these phenotypes are mediated by the same signaling pathway remains unclear. To address these issues and to further delineate the function of GluR␦2 in vivo, we generated transgenic mice that expressed Tg ⌬CT7 on a GluR␦2 Ϫ/Ϫ background. Interestingly, although Tg ⌬CT7 restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluR␦2 Ϫ/Ϫ Purkinje cells. Furthermore, although the gross motor discoordination of GluR␦2 Ϫ/Ϫ mice was restored, the cerebellar motor learning underlying delayed eyeblink conditioning remained impaired. These results indicate that LTD induction and motor learning are regulated by signaling via the C-terminal end of GluR␦2, whereas other functions may be differentially regulated by other regions of GluR␦2.