Figure 3. Display events are attributable to fast retrieval of receptors after exocytosis. A, Principle of pH changes to detect internalized receptors. Acidic solution (pH 5.5) is yellow, fluorescent receptors are green, and nonfluorescent receptors are black. At pH 7.4, receptors are fluorescent after exocytosis (1), but if they remain on the plasma membrane they will not be fluorescent at pH 5.5 (2). Alternatively, if they are internalized again at pH 7.4 (3), they will remain fluorescent when extracellular pH is switched to pH 5.5 (4). If they are internalized at pH 5.5, they will remain invisible (5). B, Three types of events recorded in neurons expressing TfR–SEP at 0.5 Hz with alternate extracellular pH changes. Right, Images at pH 5.5 at 2× contrast for clarity. Exocytosis occurs at time 0 (green arrows). In a, a cluster is visible at pH 5.5, 2 s after exocytosis (yellow arrow). In b, no cluster is visible at pH 5.5. However, a second event is seen 20 s after the first. It could report a second exocytosis after closure at pH 5.5. In c, a cluster is visible 6 s after exocytosis (yellow arrow). Note the difference in the kinetics of fluorescence decrease between a and c. Left, Images at pH 5.5 (2, 2, and 6 s for events 1–3, respectively) are presented at the same contrast as the pH 7.4 images. Scale bar, 1 μm. C, Histogram of events of the three types described in B, with estimated opening time, i.e., the minimal and maximum interval between exocytosis and internalization. Dotted line shows the lower limit for the proportion of events of type b being kiss-and-run events. D, Patch-clamp recording of a neuron during the application of solution at pH 5.5. The current evoked (peak amplitude of 560 ± 90 pA, n = 3) is blocked by 500 μm amiloride (92 ± 8%, same cells). E, Examples of events detected as in Figure 2B showing pH 5.5 resistant fluorescent clusters (yellow arrows) 2 s (a) and 6 s (b) after exocytosis is detected (green arrows) in the presence of amiloride (500 μm). Fifty-eight such events were detected in eight cells. Images at pH 5.5 are shown at 2× contrast. Scale bar, 1 μm. F, Neuron transfected with TfR–SEP, before (left) and 2 s after application of trypan purple (5 mm). Most of the SEP fluorescence is quenched, similar to pH 5.5 perfusion. A dendritic region is highlighted, showing two clusters, one completely quenched (blue arrow) and another one still visible, revealing internalized TfR–SEP (yellow arrow). Scale bar, 10 μm. G, Examples of kiss-and-run events with a protocol similar to ppH but in which the pH 5.5 solution is replaced by a solution containing trypan purple. Clusters are visible in trypan purple 2 s (a) or 6 s (b) after exocytosis. A total of 163 such events were detected in seven cells. Images with trypan purple are shown at 2× contrast. Scale bar, 1 μm.