Molecular mechanism of parallel fiber-Purkinje cell synapse formation

Abstract

The cerebellum receives two excitatory afferents, the climbing fiber (CF) and the mossy fiber-parallel fiber (PF) pathway, both converging onto Purkinje cells (PCs) that are the sole neurons sending outputs from the cerebellar cortex. Glutamate receptor δ2 (GluRδ2) is expressed selectively in cerebellar PCs and localized exclusively at the PF-PC synapses. We found that a significant number of PC spines lack synaptic contacts with PF terminals and some of residual PF-PC synapses show mismatching between pre- and postsynaptic specializations in conventional and conditional GluRδ2 knockout mice. Studies with mutant mice revealed that in addition to PF-PC synapse formation, GluRδ2 is essential for synaptic plasticity, motor learning, and the restriction of CF territory. GluRδ2 regulates synapse formation through the amino-terminal domain, while the control of synaptic plasticity, motor learning, and CF territory is mediated through the carboxyl-terminal domain. Thus, GluRδ2 is the molecule that bridges synapse formation and motor learning. We found that the trans-synaptic interaction of postsynaptic GluRδ2 and presynaptic neurexins (NRXNs) through cerebellin 1 (Cbln1) mediates PF-PC synapse formation. The synaptogenic triad is composed of one molecule of tetrameric GluRδ2, two molecules of hexameric Cbln1 and four molecules of monomeric NRXN. Thus, GluRδ2 triggers synapse formation by clustering four NRXNs. These findings provide a molecular insight into the mechanism of synapse formation in the brain.

Keywords: Purkinje cell; glutamate receptor δ2; motor learning; neurexin; parallel fiber; synapse formation.

Figure 1

Multiple roles of PC-specific GluR δ2 in cerebellar wiring and function. (A) A cerebellar PC receives a large numbers of PF innervation at distal dendrites and a single CF innervation at proximal dendrites. GluRδ2 is selectively expressed in cerebellar PCs and exclusively localized at the PF-PC synapse. GluRδ2 at the PF synapse regulates PF-PC synapse formation, LTD induction, motor learning, and CF territory. (B) A cerebellar PC visualizes by EGFP. (C) Immunohistochemical staining of a mouse brain with anti-GluRδ2 antibody.

Figure 2

Close relationship between the amount of GluRδ2 protein and the size of the active zone. (A) Ablation of GluRδ2, when induced in the adult brain, resulted in the disruption of synaptic connections with PF terminals in a significant number of PC spines. In addition, some of residual PF-PC synapses show mismatching between pre- and postsynaptic specializations (Takeuchi et al., 2005). White and black arrowheads indicate the edges of active zone and PSD, respectively. (B) Schematic presentation of the relationships between the amount of GluRδ2 protein and the sizes of presynaptic active zone (hatched) and PSD (cross-hatched). The length of active zone became shorter in the order of normal, matched, and mismatched synapses according to the decrease of the density of GluRδ2-immunogold labeling at postsynaptic sites (Takeuchi et al., 2005). Based on the direct relationship between the density of postsynaptic GluRδ2 and the size of presynaptic active zones in GluRδ2 mutant mice, we proposed that GluRδ2 makes a physical linkage between the active zone and PSD by interaction with an active zone component. Normal, normal synapse of wild-type mice; matched, matched synapse of induced GluRδ2 KO mice; mismatched, mismatched synapse of induced GluRδ2 KO mice; free, free spine of induced GluRδ2 KO mice.

Figure 3

Induction of presynaptic differentiation by GluR δ 2. (A) HEK293T cells transfected with expression vectors for GluRδ2 and tagRFP (red) were seeded on top of cultured cerebellar neurons transfected with an expression vector for vesicle-associated membrane protein-2 (VAMP-2) fused with EGFP at its N terminus (EGFP-VAMP2) (green). After 2 days of co-culture, cells were immunostained for EGFP. Note that numerous EGFP-VAMP2 signals accumulated on the surface of HEK293T cells expressing GluRδ2. Scale bar represents 10 μm. (B) Schematic presentation of the accumulation of GC axon terminals on the surface of HEK293T cells expressing GluRδ2.

Figure 4

The GluR δ2-Cbln1-NRXN trans-synaptic triad mediates synapse formation (modified from Uemura et al., 2010). (A) Suppression of Cbln1 synaptogenic activity by the extracellular domain of NRXN1β (NRXN1β-ECD) and the N-terminal domain of GluRδ2 (GluRδ2-NTD) in cultured cerebellar neurons. In primary cultures of cerebellar neurons, numerous punctate staining signals for VGluT1 were found on the dendrites of PCs from wild-type mice. VGluT1 signals were significantly reduced in PCs from Cbln1 KO mice. Addition of Cbln1 restored the intensity of VGluT1 signals. The restoring activity of Cbln1 was suppressed by addition of NRXN1β-ECD and GluRδ2-NTD. (B) Suppression of Cbln1 synaptogenic activity by NRXN1β-ECD and GluRδ2-NTD in vivo. Electron micrographs of cerebella from wild-type and Cbln1 KO mice and those from Cbln1 KO mice injected with Cbln1 together with or without NRXN1β-ECD and GluRδ2-NTD. In wild-type mice, all PC spines formed synaptic contacts with PFs. In Cbln1 KO mice, many PC spines lacked synaptic contacts (free spines). Injection of Cbln1 restored PF-PC connections in Cbln1 KO mice. The in vivo synatogenic activity of Cbln1 was suppressed by co-injection of NRXN1β-ECD and GluRδ2-NTD. n, normal synapses; f, free spines. Scale bars represent 5 μm in (A) and 0.5 μm in (B).

Figure 5

Molecular mechanism of PF-PC synapse formation. Before PF-PC synapse formation, Cbln1 secreted from cerebellar GCs may interact with presynaptic NRXNs. Cbln1-induced NRXN dimerization is not sufficient to trigger presynaptic differentiation. When the contact between the PF terminal and PC spine takes place, GluRδ2 triggers synapse formation by clustering four NRXNs through triad formation.