Promotion of Dendritic Differentiation of Cerebellar Purkinje Cells by Ca2+/calmodulin-dependent Protein Kinase IIα, IIβ and IV and Possible Involvement of CREB Phosphorylation.

• Single or double knockdown of CaMKIIα, IIβ or IV did not inhibit the dendritic differentiation of Purkinje cells. • Triple knockdown of CaMKIIα, IIβ and IV inhibited the dendritic branching of Purkinje cells. • Triple knockdown of CaMKIIα, IIβ and IV reduced the phosphorylation of CREB in Purkinje cells. 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. [ABSTRACT FROM AUTHOR]