Fig. 7

Activity- and CPEB2-dependent axonal VGLUT2 synthesis increases functional VGLUT2⁺ vesicles. A DIV17-23 neurons were stimulated without (basal) or with 4X HFS and incubated for 2 h before activity (10 Hz for 90 s)-stimulated FM4-64FX loading. The FM4-64FX puncta signals in VGLUT2⁺ vesicles were quantified in 10 image fields from 3 independent cultures and expressed as relative ratios. The region of interest at 4X magnification is shown in the insert. Scales, 10 μm and 1 μm in magnified images. B-C The experimental procedure was illustrated in the upper panel. CPEB2-WT and -KO neurons were seeded in microfluidic chambers and cultured for 18 to 19 DIV. After axotomy, the same stimulation and loading protocol were applied without (control) or with 50 μg/ml cycloheximide (CHX). The axonal region was outlined with an axonal marker, βIII-tubulin (marked by white dashed lines). The signal intensity of FM4-64FX and VGLUT2 was color-coded. The FM4-64FX puncta signals in VGLUT2⁺ and VGLUT2⁻ vesicles were quantified in 20 image fields from 5 independent cultures and expressed as a relative ratio. Data are mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, two-way ANOVA with Fisher’s LSD post-hoc test. Scale, 5 μm