In confirmation, we also found increased levels of phosphorylated p130-CAS, a downstream substrate of FAK, in mutants ( Figure S3C). To establish a system in which we could manipulate neurons pharmacologically and genetically, we cultured dissociated neurons from Pcdh-γdel/del and control cortex. To label the morphology of individual neurons at random, we lipofected cultures (∼1%–5% efficiency) with a construct encoding YFP and measured dendrite

arborization by Sholl analysis at 2, 8, and 14 days in vitro (DIV). click here Consistent with in vivo analyses, we found that dendrite complexity was significantly reduced in mutant neurons ( Figures 4A, 4B, and 4I; Figures S4A–S4F). As expected for homophilic adhesion molecules, loss of the γ-Pcdhs affected dendrite arborization in a cell-autonomous manner, as

shown by Cre transfection of individual Pcdh-γfcon3/fcon3 neurons ( Figures S4G and S4H). Next, we cultured BGB324 molecular weight cortical neurons in the presence of three pharmacological inhibitors. We broadly inhibited PKC isoforms with Gö6983 (Gschwendt et al., 1996), which significantly rescued dendrite arborization in mutant neurons toward control levels (Figures 4E and 4I), as did the addition of U73122, a potent inhibitor of PLC activity (Bleasdale et al., 1989) (Figures 4G and 4I). A recently characterized inhibitor of FAK, PF-573228 (referred to as PF-228; Slack-Davis et al., 2007), completely rescued the phenotype, increasing arborization in mutant neurons such that they were indistinguishable from controls (Figures 4A, 4C, 4F, and 4I). MARCKS associates with the plasma membrane in part through a basic effector domain (ED). PKC phosphorylation

of four serines in the ED causes MARCKS to lose its associations with actin and the membrane (Swierczynski and Blackshear, 1995 and Hartwig et al., 1992). In hippocampal neurons, transfection of MARCKS or a nonphosphorylatable version with all four ED serines mutated to asparagines (N/S-MARCKS), but not a pseudophosphorylated mutant with these serines replaced by aspartates (D/S-MARCKS) (Spizz and Blackshear, 2001), led to significant increases in dendrite arborization (Li et al., 2008). We tested these constructs for their ability to rescue arborization in Pcdh-γ mutant neurons. Cultures were Parvulin transfected at 1 DIV and fixed for Sholl analysis at 8 DIV; all MARCKS constructs were efficiently expressed in transfected neurons ( Figures S4I–S4K). Compared to controls, both MARCKS-GFP and N/S-MARCKS-GFP (but not D/S-MARCKS-GFP) greatly increased arborization in mutant neurons to levels even above those of untransfected control neurons ( Figures 4D, 4H, and 4I). Although these experiments alone cannot exclude the possibility that γ-Pcdhs affect dendrite branching through a distinct pathway parallel to the PKC pathway, this is unlikely.