Kyoichi Emi

Cbln1 Is a Ligand for an Orphan Glutamate Receptor δ2, a Bidirectional Synapse Organizer

Orphan No More The glutamate receptor δ2 (GluD2), another member of the ionotropic glutamate receptor family, has long been considered to be an orphan receptor because there are no known endogenous ligands. Nevertheless, GluD2 is essential for the normal development of cerebellar circuits. Using immunocytochemistry, binding assays, electrophysiology, and freeze-fracture electron microscopy, Matsuda et al. (p. 363) found that Cbln1, a soluble protein secreted from cerebellar granule cells, binds to the extracellular N terminus of GluD2 on Purkinje cells. Binding has two independent consequences: First, it leads to presynaptic differentiation and second, it causes postsynaptic clustering of several important synapse-specific molecules. Both events are needed for synapse formation between granule cells and Purkinje cells. A signaling complex serves as a synapse organizer that acts bidirectionally on both pre- and postsynaptic components. Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor δ2 (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 regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor

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.

Cbln1 Regulates Rapid Formation and Maintenance of Excitatory Synapses in Mature Cerebellar Purkinje Cells In Vitro and In Vivo

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.

Differential Regulation of Synaptic Plasticity and Cerebellar Motor Learning by the C-Terminal PDZ-Binding Motif of GluRδ2

The δ2 glutamate receptor (GluRδ2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluRδ2−/− 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 GluRδ2−/− Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluRδ2−/− Purkinje cells by the virus-mediated expression of the wild-type GluRδ2 transgene (Tgwt) but not by that of mutant GluRδ2 lacking the C-terminal seven residues to which several PDZ proteins bind (TgΔCT7). These results indicated that the C terminus of GluRδ2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluRδ2−/− 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 GluRδ2 in vivo, we generated transgenic mice that expressed TgΔCT7 on a GluRδ2−/− background. Interestingly, although TgΔCT7 restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluRδ2−/− Purkinje cells. Furthermore, although the gross motor discoordination of GluRδ2−/− 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 GluRδ2, whereas other functions may be differentially regulated by other regions of GluRδ2.

The N-Terminal Domain of GluD2 (GluRδ2) Recruits Presynaptic Terminals and Regulates Synaptogenesis in the Cerebellum In Vivo

The δ2 glutamate receptor (GluRδ2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)–Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2wt) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2wt, but not of a mutant GluD2 lacking the NTD (GluD2ΔNTD), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2NTD–GluK2) did. Similarly, GluD2wt and GluD2NTD–GluK2, but not GluD2ΔNTD, induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF–Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively.

Activity-dependent repression of Cbln1 expression: Mechanism for developmental and homeostatic regulation of synapses in the cerebellum

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is released from cerebellar granule cells and plays a crucial role in forming and maintaining excitatory synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells not only during development but also in the adult cerebellum. Although neuronal activity is known to cause morphological changes at synapses, how Cbln1 signaling is affected by neuronal activity remains unclear. Here, we show that chronic stimulation of neuronal activity by elevating extracellular K+ levels or by adding kainate decreased the expression of cbln1 mRNA within several hours in mature granule cells in a manner dependent on l-type voltage-dependent Ca2+ channels and calcineurin. Chronic activity also reduced Cbln1 protein levels within a few days, during which time the number of excitatory synapses on Purkinje cell dendrites was reduced; this activity-induced reduction of synapses was prevented by the addition of exogenous Cbln1 to the culture medium. Therefore, the activity-dependent downregulation of cbln1 may serve as a new presynaptic mechanism by which PF–Purkinje cell synapses adapt to chronically elevated activity, thereby maintaining homeostasis. In addition, the expression of cbln1 mRNA was prevented when immature granule cells were maintained in high-K+ medium. Since immature granule cells are chronically depolarized before migrating to the internal granule layer, this depolarization-dependent regulation of cbln1 mRNA expression may also serve as a developmental switch to facilitate PF synapse formation in mature granule cells in the internal granule layer.

Reevaluation of the role of parallel fiber synapses in delay eyeblink conditioning in mice using Cbln1 as a tool

The delay eyeblink conditioning (EBC) is a cerebellum-dependent type of associative motor learning. However, the exact roles played by the various cerebellar synapses, as well as the underlying molecular mechanisms, remain to be determined. It is also unclear whether long-term potentiation (LTP) or long-term depression (LTD) at parallel fiber (PF)–Purkinje cell (PC) synapses is involved in EBC. In this study, to clarify the role of PF synapses in the delay EBC, we used mice in which a gene encoding Cbln1 was disrupted (cbln1-/- mice), which display severe reduction of PF–PC synapses. We showed that delay EBC was impaired in cbln1-/- mice. Although PF-LTD was impaired, PF-LTP was normally induced in cbln1-/- mice. A single recombinant Cbln1 injection to the cerebellar cortex in vivo completely, though transiently, restored the morphology and function of PF–PC synapses and delay EBC in cbln1-/- mice. Interestingly, the cbln1-/- mice retained the memory for at least 30 days, after the Cbln1 injection’s effect on PF synapses had abated. Furthermore, delay EBC memory could be extinguished even after the Cbln1 injection’s effect were lost. These results indicate that intact PF–PC synapses and PF-LTD, not PF-LTP, are necessary to acquire delay EBC in mice. In contrast, extracerebellar structures or remaining PF–PC synapses in cbln1-/- mice may be sufficient for the expression, maintenance, and extinction of its memory trace.

A New Rapid Protocol for Eyeblink Conditioning to Assess Cerebellar Motor Learning

Mice with spontaneous and induced mutations causing cerebellar phenotypes have provided key insights into how motor-related memories are stored in cerebellar circuits. Delayed eyeblink conditioning is a form of associative motor learning that depends on the cerebellum. However, neurochemical investigation of the underlying mechanisms has been hampered by the long training period (usually several days) required to establish conditioning. Here, we report a new rapid-training protocol that reliably induced delayed eyeblink conditioning within a single day. The associative memory formation depended on the expression of the δ2 glutamate receptor (GluD2) in cerebellar Purkinje cells. It lasted for several weeks, but could be erased by extinction sessions in a single day. In addition, using the rapid protocol, we found that eyeblink conditioning could be induced in juvenile mice at postnatal day 21, and that the Sindbis-virus-mediated expression of GluD2 could rescue the impaired eyeblink conditioning in GluD2-null mice in vivo.

Impaired delay, but normal trace classical eyeblink conditioning in the Cbln1 mutant mice

P2-b12 Effect of X-irradiation on dendritic spine morphology of hippocampal neurons Katsuyuki Shirai1,2,3, Toshiyuki Mizui1,3, Yukari Yoshida2,3, Masahiko Okamoto1,2,3, Yoshiyuki Suzuki2,3, Kenji Hanamura1, Takashi Nakano2,3, Tomoaki Shirao1 1 Department of Neurobiology & Behavior, Gunma University Graduate School of Medicine, Maebashi, Japan; 2 Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Japan; 3 21st Century COE Program, Japan