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. 2013 Dec 31;8(12):e83609. doi: 10.1371/journal.pone.0083609

Figure 7. Live-imaging of AMPA-induced cofilinR21Q-mRFP labeled rods and their effect on vesicular transport before and after reversal.

Figure 7

CofilinR21Q-mRFP and amyloid precursor protein (APP)-YFP dual infected neuronal cultures were treated with 25 μM AMPA for 45 min before imaging. Neurons expressing both fluorescent protein chimeras, which formed or did not form rods, were imaged once in the red channel to locate rods and then continuously for 12 sec (50 frames) in the YFP channel. In the AMPA-treated neuron that did not form rods (A), APP-YFP-containing vesicles (B) remain dynamic, as indicated by the retrograde (leftward) and anterograde (rightward) movements of the labeled vesicles in the kymographs (C, D) obtained from the regions of the neurite with red and purple line-scans (colored arrows demarcate the ends of the line scans) corresponding to the colored box on the kymographs. In the AMPA-treated neuron that formed rods (E, yellow arrows), APP-YFP-containing vesicles (F) stopped moving, as indicated by the lack of retrograde (leftward) and anterograde (rightward) movements of the labeled vesicles in the kymographs (G, H) obtained from the regions of the neurite with green and red line-scans (F) corresponding to the colored box on the kymographs. (Scale bars on images A, B, E, F are 15 μm and on kymographs C, D, G, H are 8 μm). In another set of experiments cofilinR21Q-mRFP and APP-YFP expressing neurons were treated 30 min with 25 μM AMPA to induce rods and then washed once with fresh medium and treated with 10 μM DNQX to prevent further AMPA-receptor activity. Neurons with rods were identified in the red channel. One hour after washout rods were still present in many processes (I) and APP-YFP containing vesicles were generally not dynamic, although a few exceptions were observed in kymographs taken along two different line scans (J, K). By three hours after washout (L), most rods have disappeared and APP-YFP vesicles are more dynamic (M, N). Line scans in I and L are not identical due to slight morphological changes that occur during recovery. (Scale bars on I, L = 10 μm and on kymographs J, K, M, N = 5 μm).