Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse

Abstract

At the crustacean neuromuscular junction, facilitation elicited by a repetitive stimulation reaches a plateau level that is proportional to the stimulation frequency. In the present study we demonstrated that plateau facilitation (Fplateau) does not depend on Ca2+ manipulations. We manipulated Ca2+ concentration in the following ways: (1) applying cell permeable chelators BAPTA-AM or EGTA-AM; (2) decreasing Ca2+ concentration in the extracellular media; (3) enhancing Ca2+ influx by 4-aminipyridin. We found that neither Fplateau is decreased by lowering Ca2+ nor it is increased by enhancing Ca2+ influx. In contrast, facilitation elicited by a short train of stimuli (Fgrowth) was altered by Ca2+ manipulations. These results suggested that Fplateau does not result from accumulation of free intracellular Ca2+. We hypothesized that Fplateau results from the accumulation of synaptic vesicles properly activated for transmitter release, the readily releasable pool (RRP). To test this hypothesis, we measured the increase in RRP employing local applications of hypertonic solutions (HS). We found that the size of RRP was significantly increased after Fplateau was induced. Our results suggest that facilitation is mediated by two mechanisms: the increase in the residual Ca2+ and the increase in RRP. Frequency facilitation during continuous stimulation, Fplateau, is primarily controlled by the increase in RRP.