Protein kinase C-dependent and independent signaling pathways regulate synaptic GluR1 and GluR4 AMPAR subunits during in vitro classical conditioning.

Protein kinase C (PKC) signal transduction pathways have been implicated in mechanisms of synaptic plasticity and learning, however, the roles of the different PKC family isoforms remain to be clarified. Previous studies showed that NMDAR-mediated trafficking of GluR4-containing AMPARs supports conditioning and that the mitogen-activated protein kinases (MAPKs) have a central role in the synaptic delivery of GluR4 subunits. Here, an in vitro model of classical conditioning in pond turtles, Pseudemys scripta elegans, was used to assess the role of PKC isoforms in mechanisms underlying this form of learning. We show that the PKC antagonists chelerythrine and bisindolylmaleimide I attenuated conditioned response (CR) acquisition and expression, as did the PKCzeta pseudosubstrate peptide inhibitor ZIP. Analysis of protein expression revealed that PKCzeta is activated in early stages of conditioning followed shortly afterward by increased levels of PKCalpha/beta and activation of ERK MAPK. Data also suggest that PKCzeta is upstream from and activates ERK. Finally, protein localization studies using confocal imaging indicate that inhibitors of ERK, but not PKC, suppress colocalization of GluR1 with synaptophysin while inhibitors of PKC and ERK attenuate colocalization of GluR4 with synaptophysin. Together, these data suggest that acquisition of conditioning proceeds by two stages of AMPAR trafficking. The first is PKC-independent and ERK-dependent synaptic delivery of GluR1 subunits to activate silent synapses. This is followed by PKC-dependent and ERK-dependent synthesis and delivery of GluR4 subunits that supports the acquisition of CRs. Therefore, there is a selective role for PKC and MAPK signaling pathways in multistep AMPAR trafficking that mediates acquisition of classical conditioning.