, 1995, Lundblad et al., 1995 and Bannister and Kouzarides, 1995) had no effect on cFos regulation or VEGFD transcription ( Figure 2G). We also used RNA interference (RNAi) to specifically decrease CBP mRNA levels in hippocampal neurons ( Figure 2H). This caused a significant reduction of VEGFD mRNA levels ( Figure 2H) confirming the role of CBP in modulating VEGFD transcription. A morphometric analysis revealed that hippocampal neurons expressing E1A have shorter and simplified

dendritic trees compared to neurons expressing E1AΔCR1 ( Figures 2I and 2J). These results indicate that CBP acts downstream of nuclear calcium-CaMKIV signaling to regulate VEGFD expression in hippocampal neurons. INCB28060 To investigate whether VEGFD is involved in mediating the effects of nuclear calcium-CaMKIV signaling on neuronal structure, we either transfected or infected hippocampal neurons, respectively, with an rAAV plasmid (pAAV-VEGFD) or an rAAV (rAAV-VEGFD) containing an expression cassette for HA-tagged VEGFD or we treated the neurons with recombinant VEGFD (rVEGFD). Expression of HA-tagged VEGFD was detected immunocytochemically and by

immunoblotting in rAAV-VEGFD-infected hippocampal neurons and in the culture media ( Figures S1G and S1H). Although VEGFD-HA expression or exogenously applied rVEGFD had no detectable effect on neuronal morphology, both treatments rescued the reduction in dendrite length and complexity caused by expression of CaMBP4 or CaMKIVK75E ( Figures 3A–3C, 3E, and 3F). In contrast, VEGFD-HA and rVEGFD INK1197 molecular weight failed to restore normal spine density in CaMBP4 or CaMKIVK75E-expressing neurons ( Figures 3D and 3G), indicating that the mechanisms through which nuclear calcium-CaMKIV signaling regulate dendrite geometry

and spine density are distinct. Because much VEGFD belongs to a family of closely related factors that in part share the receptors ( Achen and Stacker, 2008), we tested whether VEGF or VEGFC also affect dendrite arborization. However, neither recombinant VEGF (rVEGF) nor recombinant VEGFC (rVEGFC) was able to rescue the reduction in dendrite length and complexity caused by CaMBP4 or CaMKIVK75E expression ( Figure S2), indicating a specific role for VEGFD in the control of dendrite arborization by nuclear calcium-CaMKIV signaling. To determine whether the observed reduction in VEGFD expression that followed blockade of nuclear calcium-CaMKIV signaling is sufficient to alter dendritic architecture, we used RNAi to lower VEGFD expression in hippocampal neurons. DNA sequences encoding short hairpin RNAs (shRNAs) designed to target the mouse VEGFD mRNA were inserted downstream of the U6 promoter of an rAAV vector. The resulting rAAV, rAAV-shVEGFD, also harbors a calcium/calmodulin-dependent protein kinase II (CaMKII) promoter-containing expression cassette for the red fluorescent protein, mCherry ( Figure 4A).