In control CA1 slices, long-term DOX treatment did not affect dendritic spine morphology and architecture as the strong correlation between spine area and PSD length (Pearson correlation coefficient r = 0.72) was highly similar to the linear correlation obtained from wild-type CA1 spines (Hazai et al., 2013). mainly target dendritic spines, which are small actin-rich protrusions formed along neuronal dendrites. It is now widely accepted that synaptic plasticity and memory formation modify the morphology of dendritic spines, including the appearance of new protrusions, as well as rearrangement of already existing synaptic connections (reviewed in Holtmaat and Svoboda (2009) and Yuste (2010)). The cytoskeleton in spines is formed predominantly by F-actin and serves both as a structural framework to maintain shape and as the principal regulator of protein and vesicular trafficking (Frost et al., 2010; Bosch and Hayashi, 2012). Previous studies have shown that F-actin is highly dynamic in spines (Star et al., 2002; Okamoto et al., 2004) and is regulated by a plethora of signaling pathways (Pontrello and Ethell, 2009; Penzes and Cahill, 2012). Depending on the modulation of input activity or homeostatic regulation, spines can undergo structural changes, e.g., enlargement during long-term potentiation (LTP) or shrinkage during long-term depression, with strict temporal and spatial regulations of actin turnover (Bosch and Hayashi, 2012). The PKD family of serine/threonine kinases comprises three JAM2 isoforms in mammals (PKD1C3). PKDs are activated by members of the novel PKC family and are recruited to the plasma membrane or intracellular membranes via binding DAG to achieve full activation (Steinberg, 2012). Activated PKDs can exert various cellular functions, including the regulation of cell motility and invasion (LaValle et al., 2010b; Olayioye et al., 2013). In nonneuronal tumor cells, PKD activity suppresses cell motility by controlling actin dynamics via Slingshot (SSH1), p21-activated kinase 4 (PAK4), or cortactin (reviewed in Olayioye et al. (2013) and references therein). In the rodent brain, all three PKD isoforms are expressed early in embryonal FIPI development (Oster et al., FIPI 2006). So far, neuronal PKD activity has been shown to affect dendrite development and maintenance, intracellular transport, and Golgi functions, as well as modulation of transmembrane receptors (Cabrera-Poch et al., 2004; Horton et al., 2005; Bisbal et al., 2008; Cz?nd?r FIPI et al., 2009; Wang et al., 2014; Quassollo et al., 2015). In this work, we investigated PKD-mediated effects on dendritic spines and the consequences of altered PKD activity upon evoking different forms of neuronal plasticity and memory formation. We show that endogenous PKDs regulate activity-dependent changes in dendritic spines by regulating F-actin consolidation and provide compelling evidence that PKD activity is required for proper learning and memory formation. Results Endogenous PKD is active in dendritic spines Previously, we have described a PKD activity reporter, which is suitable to visualize endogenous PKD-mediated phosphorylation events in fixed cells (Fig. 1 A; Cz?nd?r et al., 2009; Fuchs et al., 2009) and is present in the dendritic branches and spines of DIV12-13 hippocampal neurons (see the EGFP signal in Fig. 1 B and Fig. S1 A). To compare the extent of reporter phosphorylation in spines, ratiometric images were created by normalizing the a-pS294 to EGFP signal intensities (Fig. 1 B and Fig. S1 A, ratio images). Only mushroom spines with clearly enlarged heads were chosen for the analysis. To confirm the specificity of the pS294 antibody signal, a mutant reporter construct containing alanine instead of the target serine was also investigated (S/A mutant). In all cases, S/A mutant reporter displayed only a negligible ratio signal (Fig. FIPI 1, BCD). Open in a separate window Figure 1. Endogenous PKD is activated within dendritic spines during plasticity-inducing changes in vitro. (A) Schematic representation of the PKD activity reporter, containing the PKD-specific substrate sequence of phosphatidylinositol 4-kinase III (PI4KIII) and the EGFP sequence. The a-pS294 antibody recognizes the phosphorylated Ser294 target site. (B) Inverted fluorescent and a-pS294/EGFP ratio images of tertiary dendritic branches from control or cLTP- or KCl-treated neurons after 30 min. Arrowheads indicate mushroom spines. Bars, 1 m. (C and D) Relative a-pS294/EGFP ratio values in mushroom spines treated with KCl (C) or cLTP (D) for the indicated time. 3 M kbNB 142-70 (kbNB) or 1 M PDBu was applied 1 h before other treatments or for 15 min, respectively. 10 M MK-801, 50 M APV, 1 M nifedipine, and 1 M -conotoxin MVIIC were applied for the indicated time periods. S/A indicates a reporter construct with a nonphosphorylatable alanine mutation. Data were obtained from three to four independent cultures and displayed as mean SEM. The number of analyzed spines is indicated within the graphs..