Fig. 4.

Large variations in hypertonic sucrose induced FM2-10 destaining in nascent synapses. A, Destaining pattern of a single bouton illustrating the protocol used in these experiments. Background fluorescence remaining after multiple rounds of high K⁺ destaining was subtracted from each trace.B, Destaining kinetics of four representative boutons from a 6 DIV culture in response to a 30 sec application of hypertonic solution. Boutons were loaded maximally with FM2-10 via high K⁺ stimulation. Remaining fluorescence background after four consecutive rounds of 90 K⁺/2 Ca²⁺ challenge was subtracted from each trace.C, A normalized set of traces from a distinct set of boutons. Individual traces were fit with two decaying exponential functions. The amplitude of the fast component was taken as a measure of the relative size of the RRP. The slow component has a typical time constant slower than 100 sec. Some boutons failed to respond to sucrose application, although they consequently could be destained with 90 K⁺/2 Ca²⁺. D, Graphs depicting the evolution of the ratio of sucrose responses with respect to developmental increase in RP size. Results are pooled from 26 experiments; each symbol represents a single bouton from a particular experiment. The numbers of boutons and mean fraction in RRP on each graph are as follows: 5–6 DIV, 453 boutons and 0.35; 7–8 DIV, 593 boutons and 0.23; 9–10 DIV, 129 boutons and 0.21; 20–22 DIV, 328 boutons and 0.22.