Skip to main content
. 2011 Nov 2;4:34. doi: 10.3389/fnmol.2011.00034

Figure 1.

Figure 1

Development of an mOrange2-based reporter of VGLUT1 vesicle recycling. (A) Schematic of VGLUT1-2XmOr2 depicts two copies of mOr2 inserted into the first luminal loop of rat VGLUT1. (B) The fluorescence of VGLUT1-2XmOr2 colocalizes with synaptophysin staining at varicosities. Hippocampal neurons transfected with VGLUT1-2XmOr2 were stained with antibody to synaptophysin, followed by Cy5-conjugated secondary antibody. Inset shows a 4× magnification of the designated box. Size bar, 10 μm. (C) The rate of FM4-64 destaining at 10 Hz for 90 s is not significantly different between boutons from untransfected (black) and transfected (red) neurons. (D) Time-lapse images show the fluorescence change of VGLUT1-2XmOr2 in response to neural activity. Hippocampal neurons transfected with VGLUT1-2XmOr2 were stimulated at 10 Hz for 60 s. After onset of the stimulus, exocytosis of VGLUT1-2XmOr2 results in a rapid increase in fluorescence (15, 30, and 60 s), followed by a decay after the stimulus (90, 120, and 300 s) as the vesicle reacidifies. Color scale is shown to the right. Scale bar, 2 μm. (E) The change in fluorescence intensity over baseline (ΔF/F0) at boutons expressing VGLUT1-pH (blue) is approximately twice that of VGLUT1-2XmOr2 (red) during stimulation at 10 Hz for 60 s (bar). Photobleaching over the time course of the experiment was measured by imaging in the absence of stimulation at a range of pH values (black symbols). Photobleaching of VGLUT1-2XmOr2 (open diamonds) was similar to that of VGLUT1-pH (+ symbol) at pH 7.4. Inset: normalized fluorescence change of VGLUT1-2XmOr2 over the range of pH 4.0 to 10.0 in permeabilized cells indicates VGLUT1-2XmOr2 exhibits a pKa of 6.71. (F) Kinetics of fluorescence changes normalized to total fluorescence signal in the presence of NH4Cl are similar for VGLUT1-pH (blue) and VGLUT1-2XmOr2 (red). Data in (C, E, and F) are means ± SEM of the change in fluorescence (ΔF) normalized to initial fluorescence (average of the first five data points, F0) over at least 20 boutons per coverslip from 12 to 16 coverslips and at least three independent cultures.