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Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25-28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid-related orphan receptor α (RORα1), were recapitulated by electroporating a dominant-negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells' physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells.

Cerebellar long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is thought to play an essential role in certain forms of motor learning. Like hippocampal LTD, cerebellar LTD is mediated by the endocytosis of AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors at postsynaptic sites. However, similar sets of kinases and phosphatases have opposite regulatory effects on hippocampal and cerebellar LTD, although the mechanisms responsible for this difference remain largely unclear. Activity-dependent dephosphorylation of stargazin (an AMPA receptor auxiliary protein) by calcineurin regulates hippocampal LTD, but whether and how stargazin is involved in cerebellar LTD is unknown. In this study, we showed that stargazin is highly phosphorylated at basal states and is dephosphorylated by the application of high KCl plus glutamate (K-glu) or of a metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), both of which chemically induced LTD in cerebellar slices. This chemically induced dephosphorylation of stargazin was specifically blocked by a calcineurin inhibitor. Indeed, inclusion of the calcineurin auto-inhibitory peptide in the patch pipette solution completely inhibited the LTD induced by the conjunctive stimulation of PFs and Purkinje cells. Furthermore, in Purkinje cells expressing stargazin-9D, in which all nine serine residues are mutated to aspartate, neither conjunctive stimulus nor DHPG treatment induced LTD. Finally, immunohistochemical analyses revealed that neither K-glu nor DHPG induced the endocytosis of AMPA receptors in Purkinje cells expressing stargazin-9D. Together, these results indicate that hippocampal and cerebellar LTD share a common pathway, namely dephosphorylation of stargazin by calcineurin.