Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse.

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 (F(plateau)) does not depend on Ca(2+) manipulations. We manipulated Ca(2+) concentration in the following ways: (1) applying cell permeable chelators BAPTA-AM or EGTA-AM; (2) decreasing Ca(2+) concentration in the extracellular media; (3) enhancing Ca(2+) influx by 4-aminipyridin. We found that neither F(plateau) is decreased by lowering Ca(2+) nor it is increased by enhancing Ca(2+) influx. In contrast, facilitation elicited by a short train of stimuli (F(growth)) was altered by Ca(2+) manipulations. These results suggested that F(plateau) does not result from accumulation of free intracellular Ca(2+). We hypothesized that F(plateau) 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 F(plateau) was induced. Our results suggest that facilitation is mediated by two mechanisms: the increase in the residual Ca(2+) and the increase in RRP. Frequency facilitation during continuous stimulation, F(plateau), is primarily controlled by the increase in RRP.