Phospholipase Cβ4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum

Abstract

Elimination of excess climbing fiber (CF)-Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, G␣q, and the ␥ isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLC␤4, one of two major isoforms expressed in Purkinje cells. PLC␤4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLC␤4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLC␤4 mRNA but reciprocally high levels of PLC␤3 mRNA are found. These results indicate that PLC␤4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.The mRNA for phospholipase C (PLC) ␤4 has been reported to be particularly enriched in cerebellar Purkinje cells (PCs) (1, 2, 3). PCs express high levels of type 1 metabotropic glutamate receptor (mGluR1) (4, 5), which stimulates PLC through the activation of heterotrimeric G-proteins of the G␣q͞11 family (6, 7). PCs are also very rich in the ␥ isoform of protein kinase C (PKC␥) (8, 9, 10) and in inositol 1,4,5-trisphosphate receptors (11), both of which are activated after hydrolysis of phosphatidylinositol 4,5-bisphosphate by the PLC␤ enzymes. G␣q has been shown to colocalize with mGluR1 in PC dendritic spines (S. Nakagawa, J. Tanaka, M.W., M.K., M.I.S., and Y.I., unpublished data). It is, therefore, likely that the signal transduction cascade from mGluR1 to PKC␥ and inositol 1,4,5-trisphosphate receptor activation proceeds via G␣q and PLC␤4 and that this cascade specifically plays an important role in PC function.PCs receive distinct types of excitatory inputs from parallel fibers (PFs) and climbing fibers (CFs) (12, 13). Each PF synapse is weak, but one PC receives inputs from many (Ϸ10 5 ) PF synapses. In contrast, CFs originate from the inferior olive and form strong excitatory synapses on proximal dendrites of PCs. In an adult mouse, Ͼ85% of PCs are innervated by single CFs. Massive elimination of supernumerary CFs occurs during the second and third postnatal weeks until a one-to-one relation between CFs and PCs is attained at approximately postnatal day 21 (P21). This relationship then is maintained through adult life. It was reported previously that mutant mice deficient in PKC␥, mGluR1, or G␣q retain persistent multiple CFs into adulthood and display motor discoordination (14,15,16). These results suggest that G␣q mediates signals from mGluR1 that are necessary for regression of multiple CFs during cerebellar development.To identify the isoform(s) of PLC␤ that transduces these signals, we used mutant mice deficient in PLC␤4. These mice were used previously to investigate the physiological significance o...

Calbindin in Cerebellar Purkinje Cells Is a Critical Determinant of the Precision of Motor Coordination

“…Further confirmation and extension of these findings came from studies showing that a perturbation of the mGluR1-activated signaling cascade at virtually every level [e.g., G-protein G␣q (Offermanns et al, 1997), phospholipase C␤ (Kano et al, 1998), and IP 3 (Matsumoto et al, 1996;Miyata et al, 2000)] results in impaired LTD and disturbed cerebellar motor control. IP 3 causes Ca 2ϩ release from intracellular stores (Mikoshiba et al, 1994;Finch and Augustine, 1998;Takechi et al, 1998), and synaptically induced dendritic Ca 2ϩ signaling has been known for a long time to be a critical step in LTD induction (Sakurai, 1990;Konnerth et al, 1992).…”

Type-1 metabotropic glutamate receptor in cerebellar Purkinje cells: a key molecule responsible for long-term depression, endocannabinoid signalling and synapse elimination

“…In early post-natal days, all Purkinje cells are innervated by multiple climbing fibres (multiple innervation) Crepel 1982;Lohof et al 1996). These surplus climbing fibres are eliminated eventually with the progress of post-natal development, and most Purkinje cells become innervated by single climbing fibres (mono-innervation) by the end of the third post-natal week in mice (Kano et al , 1998Offermanns et al 1997).…”

“…Kano, Hashimoto and co-workers show that the mutant mice deficient in mGluR1, G aq , PLCb4 or PKCg are all impaired in climbing fibre synapse elimination (Kano et al , 1998Offermanns et al 1997;Hashimoto et al 2000Hashimoto et al , 2001bIchise et al 2000). Levenes et al (1997) also reported incomplete synapse elimination in the mGluR1 knockout mice.…”

“…In situ hybridization and immunohistochemical studies indicate that Purkinje cells in the rostral cerebellum mainly express PLCb4, whereas those in the caudal cerebellum mainly have PLCb3 (Kano et al 1998;Nakamura et al 2004). Miyata et al (2001) analysed the PLCb4 knockout mice and found that both mGluR1-mediated Ca 2C mobilization and LTD were deficient in the rostral cerebellum, whereas these responses were intact in the caudal cerebellum.…”

Postsynaptic P/Q-type Ca 2+ channel in Purkinje cell mediates synaptic competition and elimination in developing cerebellum

“…CF elimination consists of two distinct phases in mice, the early phase from P7 and the late phase from around P12 (12), which involve distinct mechanisms (9,12). Whereas several factors crucial for the late phase of CF synapse elimination have been disclosed (13)(14)(15)(16)(17)(18)(19)(20), mechanisms of biased strengthening of single CFs and the early phase of CF synapse elimination are unknown. Because synapse refinement critically depends on neuronal activity (1,4,5,16,21,22), we studied whether the P/Q-type voltage-dependent Ca 2+ channel (VDCC), the major high-threshold VDCC in PCs (23), is involved in the development of CF to PC synapses.…”