Cerebellar Purkinje cells develop the most elaborate dendritic trees among neurons in the brain. To examine the role of Ca²⁺/calmodulin-dependent protein kinase (CaMK) IIα, IIβ and IV in the dendritic differentiation of Purkinje cells, we introduced siRNA against these CaMKs into Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. Single-cell electroporation enables us to transfer siRNA into specific cells within a heterogeneous cell population. In addition, we can easily and reliably transfer multiple types of siRNA into a cell simply by loading them together in one micropipette. Any one of the siRNA against CaMKIIα, IIβ and IV (single knockdown) or any combinations of two of the siRNA against these CaMKs (double knockdown) had no significant effects on the dendritic differentiation of Purkinje cells. However, the combination of all three siRNA against these CaMKs (triple knockdown) inhibited the branching of Purkinje cell dendrites. Furthermore, the triple knockdown reduced the phosphorylation of CREB in Purkinje cells. These findings suggest the promotion of dendritic differentiation of Purkinje cells by CaMKIIα, IIβ and IV and the possible involvement of phosphorylation of CREB as a common substrate of these CaMKs.

小脑浦肯野细胞发育出大脑神经元中最精美的树突树。为了检查Ca的作用²⁺ /钙调蛋白依赖性蛋白激酶（蛋白激酶）IIα，IIβ和IV在浦肯野细胞的树突分化，我们介绍的siRNA针对这些CaMKs成使用单个细胞电穿孔技术浦肯野细胞中小脑细胞培养物。单细胞电穿孔使我们能够将siRNA转移到异质细胞群内的特定细胞中。此外，只需将它们一起装在一个微量移液器中，我们就可以轻松可靠地将多种类型的siRNA转移到细胞中。siRNA的反任一项的CaMKII α ，II β和IV（单敲除）或两种针对这些CaMK的siRNA的任意组合（双敲除）对浦肯野细胞的树突状分化均无明显影响。但是，针对这些CaMK的所有三种siRNA的结合（三联体敲低）抑制了Purkinje细胞树突的分支。此外，三联击倒降低了浦肯野细胞中CREB的磷酸化。这些发现表明CaMKIIα，IIβ和IV促进了浦肯野细胞的树突分化，并且可能将CREB磷酸化作为这些CaMK的常见底物。