Demonstration of presynaptic protein kinase C activation following long-term potentiation in rat hippocampal slices.

Pharmacological and biochemical evidence implicate the Ca2+ and phospholipid-dependent protein kinase C in long-term potentiation. The in vitro hippocampal slice preparation was used to demonstrate redistribution of protein kinase C from cytosol to membrane and protein kinase C-dependent phosphorylation of the presynaptic growth-associated protein-43 substrate following long-term potentiation induction in area CA1. Protein kinase C translocation was assessed using both quantitative immunoblotting with a monoclonal antibody recognizing a common epitope in the alpha and beta isoforms of protein kinase C and Ca2+ and phospholipid-dependent phosphorylation of exogenous histone substrate. Slices examined 5 min after tetanus-induced spike potentiation showed no change in protein kinase C redistribution, whereas slices examined at 15-, 30- and 60-min intervals all showed a similar degree of protein kinase C translocation to membrane, although only at 15 min was the effect statistically significant. Additionally, an increase in protein kinase C-dependent growth-associated protein 43 phosphorylation was observed 10 min after high-frequency stimulation. The translocation of protein kinase C and phosphorylation of growth-associated protein 43 were dependent upon high-frequency (repetitive 400 Hz) afferent stimulation, as no effects were observed in slices receiving low-frequency (1 Hz) or no stimulation. The N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovaleric acid (50 microM), inhibited induction of long-term potentiation, redistribution of protein kinase C and phosphorylation of growth-associated protein 43. A significant redistribution of the predominantly presynaptic protein kinase C isoform, protein kinase C-alpha, was also detected 15 min after induction of long-term potentiation using an alpha-isoform-specific monoclonal antibody. These observations support a presynaptic role for protein kinase C and growth-associated protein 43 in the early maintenance phase of LTP, and further suggest that a retrograde messenger produced postsynaptically following N-methyl-D-aspartate receptor activation mediates these effects.