Journal: Frontiers in Cellular Neuroscience

Article Title: VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3

doi: 10.3389/fncel.2017.00324

Figure Lengend Snippet: Vesicular glutamate transporter 2-pH recycling differs from VGLUT1-pH. (A) Left panel: The time course of exo- and endocytosis in response to electrical stimulation at 10 Hz 60 s (bar) is monitored by the increase and decrease in the fluorescence of VGLUT2-pH (black) or VGLUT1-pH (gray) at hippocampal synaptic boutons, acquired every 3 s. Each trace was normalized to the size of the total pool of VGLUT-pH as determined by application of 50 mM NH 4 Cl. The fluorescence of both VGLUT1-pH and VGLUT2-pH increases rapidly upon stimulation and decays with an exponential time course after cessation of the stimulus, consistent with exocytosis followed by endocytosis (left panel). Middle panel: The extent of fluorescence decay from peak [Δ(ΔF/F 0 )] during stimulation is less for VGLUT2-pH (black, 18.26 ± 4.13% from peak, n = 9) than VGLUT1-pH (gray, 41.90 ± 4.31% from peak, n = 11, ∗∗ p < 0.01). Right panel: The post-stimulus endocytic rate of VGLUT2-pH (black, τ decay = 25.20 ± 2.77 s) is also significantly slower than that of VGLUT1-pH (gray, τ = 14.18 ± 1.73 s, ∗∗ p < 0.01). (B) Left panel: To measure exocytosis, hippocampal neurons expressing VGLUT2-pH or VGLUT1-pH were stimulated at 10 Hz for 90 s (bar) in the presence of 0.5 μM bafilomycin (baf). Middle panel: VGLUT2-pH exhibits a slower initial rate of exocytosis than VGLUT1-pH, calculated as the linear rate of fluorescence change, acquired every 3 s, from 0 to 15 s (VGLUT2-pH, 0.0151 ± 0.0008, n = 9; VGLUT1-pH, 0.0209 ± 0.0011, n = 10, ∗∗ p < 0.01). Right panel: Both VGLUT1-pH and VGLUT2-pH fluorescence plateau at a similar level in response to 900 action potentials, indicating that there is no significant difference in the total amount of VGLUT-pH released from the recycling pool (RP, VGLUT1-pH, 53.38 ± 2.11%; VGLUT2-pH, 51.73 ± 1.92% of total pool). (C) Left panel: The fraction of VGLUT2-pH in the readily releasable pool (RRP) is less than VGLUT1-pH, when exocytosis from the RRP is evoked using a stimulus of 20 action potentials at 100 Hz (VGLUT2-pH, 5.32 ± 0.57%, n = 10; VGLUT1-pH, 7.24 ± 0.53%, n = 11, ∗ p < 0.05), or middle panel : by application of hypertonic (300 mM) sucrose in Tyrode’s solution (VGLUT2-pH, 4.81 ± 0.15%, n = 6; VGLUT1-pH, 6.48 ± 0.58%, n = 5, ∗ p < 0.05). Right panel: Similar results are obtained with an alternate stimulus of 30 Hz for 3 s to release RRP. The release of VGLUT2-pH (black, 6.81 ± 0.640% of total pool, n = 8) even from the first second of stimulation (30 action potentials) is significantly smaller than that of VGLUT1-pH (gray, 10.58 ± 1.025%, n = 8, ∗∗ p < 0.01). For RRP measurements, fluorescence intensity measurements were acquired every 1 s. Data are means ± SEM of ΔF/F 0 normalized to total fluorescence over at least 38 boutons per coverslip from 8 to 11 coverslips per construct and at least three independent cultures.

Article Snippet: For measurements of endocytosis after stimulation, the time course of fluorescence decay at each bouton after the initial 3 s was fit with a single exponential (GraphPad Prism) ( ).

Techniques: Fluorescence, Expressing, Construct