Taisuke Miyazaki

Subtype switching of vesicular glutamate transporters at parallel fibre–Purkinje cell synapses in developing mouse cerebellum

Two subtypes of the vesicular glutamate transporter are expressed differentially in two excitatory afferents synapsing on to Purkinje cells: VGluT1 (BNPI) in axon terminals of cerebellar granule cells (i.e. parallel fibres; PFs) and VGluT2 (DNPI) in those of the inferior olivary neurons (climbing fibres; CFs). In the present study, we examined their expression in the developing mouse cerebellum. By in situ hybridization, the inferior olivary nucleus selectively expressed VGluT2 mRNA through postnatal life. In the cerebellum, both subtypes were transcribed in the external and internal granular layers during the first postnatal week. Thereafter, VGluT1 mRNA showed marked upregulation in the internal granular layer, whereas VGluT2 mRNA disappeared from the external and internal granular layers by the end of the third postnatal week. By immunohistochemistry, CF terminals consistently exhibited VGluT2 immunoreactivity in the postnatal cerebellum. By contrast, in the first 10 days of postnatal life, VGluT2 predominated in PF terminals, despite the transcription of both transporters in developing granule cells. During the second 10 days, VGluT2 in PF terminals was replaced with VGluT1 from deep regions of the molecular layer upwards, correlating with dendritic translocation of CFs. This replacement was accomplished by postnatal day 30. Taking that late‐borne PFs are laid down successively on earlier ones in the molecular layer, the deep‐to‐superficial replacement represents maturation‐linked switching from VGluT2 to VGluT1 in individual PFs, and is likely to be regulated at both the transcription and translation levels.

Cbln1 is essential for synaptic integrity and plasticity in the cerebellum

Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber–Purkinje cell synapses, the establishment of the proper pattern of climbing fiber–Purkinje cell innervation, and induction of long-term depression at parallel fiber–Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluRδ2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity.

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.

Distal Extension of Climbing Fiber Territory and Multiple Innervation Caused by Aberrant Wiring to Adjacent Spiny Branchlets in Cerebellar Purkinje Cells Lacking Glutamate Receptor δ2

Organized synapse formation on to Purkinje cell (PC) dendrites by parallel fibers (PFs) and climbing fibers (CFs) is crucial for cerebellar function. In PCs lacking glutamate receptor δ2 (GluRδ2), PF synapses are reduced in number, numerous free spines emerge, and multiple CF innervation persists to adulthood. In the present study, we conducted anterograde and immunohistochemical labelings to investigate how CFs innervate PC dendrites under weakened synaptogenesis by PFs. In the GluRδ2 knock-out mouse, CFs were distributed in the molecular layer more closely to the pial surface compared with the wild-type mouse. Serial electron microscopy demonstrated that CFs in the knock-out mouse innervated all spines protruding from proximal dendrites of PCs, as did those in the wild-type mouse. In the knock-out mouse, however, CF innervation extended distally to spiny branchlets, where nearly half of the spines were free of innervation in contrast to complete synapse formation by PFs in the wild-type mouse. Furthermore, from the end point of innervation, CFs aberrantly jumped to form ectopic synapses on adjacent spiny branchlets, whose proximal portions were often innervated by different CFs. Without GluRδ2, CFs are thus able to expand their territory along and beyond dendritic trees of the target PC, resulting in persistent surplus CFs by innervating the distal dendritic segment. We conclude that GluRδ2 is essential to restrict CF innervation to the proximal dendritic segment, by which territorized innervation by PFs and CFs is properly structured and the formation of excess CF wiring to adjacent PCs is suppressed.

P/Q-type Ca2+ channel alpha1A regulates synaptic competition on developing cerebellar Purkinje cells.

Synapse formation depends critically on the competition among inputs of multiple sources to individual neurons. Cerebellar Purkinje cells have highly organized synaptic wiring from two distinct sources of excitatory afferents. Single climbing fibers innervate proximal dendrites of Purkinje cells, whereas numerous parallel fibers converge on their distal dendrites. Here, we demonstrate that the P/Q-type Ca2+ channel α1A, a major Ca2+ channel subtype in Purkinje cells, is crucial for this organized synapse formation. In the α1A knock-out mouse, many ectopic spines were protruded from proximal dendrites and somata of Purkinje cells. Innervation territory of parallel fibers was expanded proximally to innervate the ectopic spines, whereas that of climbing fibers was regressed to the basal portion of proximal dendrites and somata. Furthermore, multiple climbing fibers consisting of a strong climbing fiber and one or a few weaker climbing fibers, persisted in the majority of Purkinje cells and were cowired to the same somata, proximal dendrites, or both. Therefore, the lack of α1A results in the persistence of parallel fibers and surplus climbing fibers, which should normally be expelled from the compartment innervated by the main climbing fiber. These results suggest that a P/Q-type Ca2+ channel α1A fuels heterosynaptic competition between climbing fibers and parallel fibers and also fuels homosynaptic competition among multiple climbing fibers. This molecular function facilitates the distal extension of climbing fiber innervation along the dendritic tree of the Purkinje cell and also establishes climbing fiber monoinnervation of individual Purkinje cells.

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (CaV) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of CaV2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca684Q) developed progressive motor impairment and aggregation of mutant CaV2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca2+ channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca684Q neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant CaV2.1 channels.

Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking μ3B, a subunit of AP-3B. μ3B−/− mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of γ-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in μ3B−/− mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

Control of Synaptic Connection by Glutamate Receptor δ2 in the Adult Cerebellum

Precise topological matching of presynaptic and postsynaptic specializations is essential for efficient synaptic transmission. Furthermore, synaptic connections are subjected to rearrangements throughout life. Here we examined the role of glutamate receptor (GluR) δ2 in the adult brain by inducible and cerebellar Purkinje cell (PC)-specific gene targeting under the pure C57BL/6 genetic background. Concomitant with the decrease of postsynaptic GluRδ2 proteins, presynaptic active zones shrank progressively and postsynaptic density (PSD) expanded, resulting in mismatching between presynaptic and postsynaptic specializations at parallel fiber-PC synapses. Furthermore, GluRδ2 and PSD-93 proteins were concentrated at the contacted portion of mismatched synapses, whereas AMPA receptors were distributed in both the contacted and dissociated portions. When GluRδ2 proteins were diminished, PC spines lost their synaptic contacts. We thus identified postsynaptic GluRδ2 as a key regulator of the presynaptic active zone and PSD organization at parallel fiber-PC synapses in the adult brain.

Differential Roles of Glial and Neuronal Glutamate Transporters in Purkinje Cell Synapses

Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate–aspartate transporter (GLAST) and excitatory amino acid transporter 4 (EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.

Functional Coupling between mGluR1 and Cav3.1 T-Type Calcium Channels Contributes to Parallel Fiber-Induced Fast Calcium Signaling within Purkinje Cell Dendritic Spines

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Cav3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Cav3.1 gene knock-out animals show that Cav3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Cav3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.