Scale pub, 1 m. maintains both LTP, a sustained enhancement of synaptic transmission (Bliss and Collingridge, 1993), and several forms of long-term memory storage (Ling et al., 2002;Pastalkova et al., 2006;Shema et al., 2007;Sacktor, 2008;Sacktor, 2011;Shema et al., 2011). Both LTP and long-term memory are associated with persistent changes in the morphology of synapses (Kandel, 2001). Postsynaptic structural modifications are most commonly observed in LTP, including increases in spine size (Yuste and Bonhoeffer, 2001;Lang et al., 2004;Yang et al., 2008), in the postsynaptic density (PSD) (Desmond and Levy, 1986;Bourne and Harris, Synephrine (Oxedrine) 2010), and in clusters of PSD-95 (Antonova et al., 2001;El-Husseini Ael et al., 2002;Stein et al., 2003;Ehrlich and Malinow, 2004;Chen et al., Synephrine (Oxedrine) 2007;Ehrlich et al., 2007), the major scaffolding protein of the PSD (Cheng et al., 2006). Whether the persistent activity of PKM that maintains the functional enhancement of synaptic strength in LTP also regulates the structural reorganization of synapses is unknown. To address this question, we examined the role of PKM in the reorganization of PSD-95 during a persistent, protein synthesis-dependent form of chemical LTP (cLTP) induced by forskolin (Gobert et al., 2008), that, like late-LTP induced by afferent tetanic stimulation, activates PKM (Li et al., 2010). == Materials and Methods == == Primary hippocampal culture and treatments == Primary hippocampal neurons were prepared from Sprague-Dawley E18/19 rats (Hilltop), according to the method described by Brewer et al. (Brewer et al., 1993). Cultured neurons were used after 12 to 18 daysin vitro. Cultured neurons were treated with forskolin (50 M, Sigma) to induce a chemical form of late-phase LTP (Gobert et al., 2008). All drugs were stored as 100x concentrated stocks (ZIP in water, forskolin and chelerythrine in DMSO), which were added Synephrine (Oxedrine) directly to culture media to obtain the final concentrations as indicated. == Preparation of Sindbis virus PKM expression vectors == Sindbis virus vectors were used to express PKM according to methods described in the Invitrogen Sindbis Expression Manual. PKM is ligated into a plasmid vector (pSinRep5) under the control of the Sindbis subgenomic promoter using a PCR-based strategy. An insert corresponding to amino acids 184-592 of PKC was obtained using a SOCS-1 plasmid containing the mouse cDNA as a template. The forward primer (5-AAATCTAGAACATGGCATACCCATACGACGTCC-CAGACTACGCTATGGATTCTGTCATGCCTTCC-3) was flanked by an XbaI restriction site followed by a sequence corresponding to Synephrine (Oxedrine) enhanced green fluorescent protein (eGFP). The reverse primer (5-AATTCTAGATCACACGGACTCC-TCAGC-3) was flanked by an XbaI restriction site. Plasmids containing eGFP alone (eGFP-control), eGFP-PKM, or the kinase inactive mutation eGFP-PKM-K281W (Drier et al., 2002;Ling et al., 2002;Shema et al., 2011) were replicated in Baby Hamster Kidney (BHK) cells and collected in Dulbeccos Modified Eagle Media for infection. Primary hippocampal neuronal cultures were infected with 1-5 l (1.2 106 2.3 106infectious units/ml) of eGFP-control, eGFP-PKM, or eGFP-PKM-K281W. Sixteen to 20 hr after the infection, trypan blue staining of the cultures showed no discernible neuronal death (data not shown). In addition to eGFP expression (Fig. 2A, inserts), overexpression of eGFP-PKM and eGFP-PKM-K281W were verified by immunoblot analysis and phosphotransferase assay (data not shown) (Ling et al., 2002). == FIGURE 2. == PKM overexpression increases PSD-95 clustering and postsynaptic GluA2. (A-C) PKM activity is necessary and sufficient for increased PSD-95 clustering. (A) Hippocampal neurons were transfected with Sindbis viral vector expressing eGFP alone (control), eGFP-PKM, the inactive eGFP-PKM-K281W, or eGFP-PKM followed by ZIP. Sixteen-twenty hours after transfection, the neurons (detected by eGFP fluorescence, insets in upper Synephrine (Oxedrine) panels) were fixed and labeled with PSD-95 antiserum (upper panel, whole cells; lower panels, dendrites). Scale bars, 20 m.