Fig. 1. Recycled and pre-existing AChRs are removed at different rates from the same synapse. The sternomastoid muscle was labeled first with BTX-biotin/strept-488, then 3-4 days later with strept-594 to selectively label recycled AChRs. (A) High resolution image of a NMJ branch showing that recycled (red and yellow overlay) and pre-existing (retaining their strept-488 after initial labeling; green and yellow overlay) AChRs are intermingled in the postsynaptic membrane. (B) Preexisting and recycled AChRs labeled with different fluorophores were assayed for fluorescence intensities immediately after strept-594 labeling of recycled AChRs (d0), and 2 days later (d2). The total fluorescence intensity of each AChR pool was normalized to 100% at initial imaging. Pseudo-color images provide a linear representation of the density of AChRs. (C) Graph summarizing the results obtained from all junctions by the approach shown in B. Each data point represents the mean percentage of fluorescence intensity ±s.d. Note that recycled receptors are removed significantly faster than pre-existing receptors. Scale bars, 20 µm.