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[Preprint]. 2024 Jun 19:2023.02.01.525891. [Version 2] doi: 10.1101/2023.02.01.525891

Figure 8. Ank-G-GFP+ AIS exhibit structural plasticity in conditions of altered network activity in vitro.

Figure 8.

A Representative images of ank-G-GFP labeled AIS in OTCs. Lower left panel: AIS after exposure to 6 mM KCl. Lower right panel: AIS after exposure to 10 mM MgSO4, both for 20 DIV. Scale bar = 10 μm. Experimental procedures are outlined in the cartoon, indicating treatment duration and DIV. B Left in graph: AIS elongation after exposure of OTC to 10 mM MgSO4 for 20 days. Mean AIS length was increased by 4.2 μm compared to controls (n = 6 OTC, 50 AIS per OTC). Right in graph: AIS remain unchanged after exposure of OTC to 6 mM KCl for 20 days (n = 6 OTC, 50 AIS per OTC, one-way ANOVA and Tukey’s test). C Under conditions of decreased network activity (10 mM MgSO4 for 10 days), AIS are elongated (unpaired t-test, n = 6 OTC, 50 AIS per OTC). Rescue conditions (normal growth medium) from DIV 10 to 20 resulted in a return to baseline AIS length (unpaired t-test, n = 6 OTC, 50 AIS per OTC). D Under conditions of increased network activity (6 mM KCl for 10 days), no changes in AIS length were observed (unpaired t-test, n = 6 OTC, 50 AIS per OTC). Consequently, no AIS length changes were seen after applying rescue conditions from DIV 10 to 20 (normal growth medium; unpaired t-test, n = 6 OTC, 50 AIS per OTC). E Rapid AIS shortening in hippocampal acute slices. The GFP construct was activated by injection of an AAV5-CaMKIIa-Cre virus and acute slices were prepared and maintained in ACSF. KCl (15 mM) was applied to a selected soma, which showed a rapid decline of GFP fluorescence along the AIS within 10 min of the application (green arrowhead also in inverted images). Neurons that were outside of the KCl application showed no significant AIS length changes (magenta arrowheads, also in inverted images). Change in the relative length of individual AIS targeted by high potassium (green) and surrounding AIS (grey) is plotted against the duration of the application (4 pairs of sample and control AIS, unpaired t-tests, scale bar = 10 μm).