Role of synaptically stimulated phospholipase A(2) and arachidonic acid on the prolongation of protein kinase C-mediated activity-dependent neuronal facilitation in Hermissenda B cells.

摘要

In Hermissenda, associative pairings of light and rotation produce an increase in cell excitability in the B photoreceptors of the animal's eye that contributes to several light-elicited behaviors. This increase in cell excitability requires a light-induced increase in intracellular Ca 2+ contingent with activation of G-protein coupled receptors as a result of presynaptic vestibular hair cell stimulation. The convergence of biochemical pathways that arises from this activity-dependent mechanism induces a sustained activation of PKC which is crucial for the induction of associative conditioning. Here, we first investigated the acquisition function that results after associative conditioning at the biophysical and biochemical level. Using an in vitro procedure that mimics training of the intact animal, we found an incremental increase in the excitability of the B photoreceptors with successive pairings of light and presynaptic stimulation of hair cells that was asymtotic with 4--5 pairings. Analysis of protein kinase C (PKC) in the animal's photoreceptors indicated a training-induced redistribution of PKC form the cytosolic to the membrane compartment. These results suggest that the intracellular redistribution of a protein kinase contributes critically to the kinetics of new learning. In a second series of experiments we investigated the role of arachidonic acid as a cofactor necessary for sustained PKC activation and its mechanisms of activation using the fluorescent probe ADIFAB. To this end, we applied GABA, the endogenous transmitter that mediates the interaction between the B cells and the hair cells, melittin, a phospholipase A2 (PLA2) activator, AACOCF3, a specific PLA2 inhibitor, and artificial sea water onto isolated eyes incubated in ADIFAB. The results showed that in these cells GABA releases arachidonic acid mainly through the activation of PLA2. Finally, we determined the time course of PKC-mediated facilitation by exposing isolated nervous systems to pairings of discrete lights in the presence of fatty acids. Convergence of high Ca2+ and fatty acids produced a PKC-mediated increase in cell excitability that lasted for at least 90 min and it was protein synthesis independent. In total, fatty acids paired with light induce a prolonged facilitation in the B photoreceptors that underlies an intermediate period of memory consolidation.