Immuno-Isolation of Synaptic Vesicles, Western Blot, and [3H]glutamate Uptake Assay

Synaptosomes from hippocampus were prepared, and the LS1 fractions containing synaptic vesicles (SVs) were isolated as described previously (Zander et al., 2010). The LS1 fractions were incubated with ^∗superparamagnetic beads (Dynabeads Pan Mouse IgG, Invitrogen, Life Technologies Inc., Burlington, ON, Canada) coupled to anti-synaptophysin, anti-VGLUT1 or anti-VIAAT antisera (all from Synaptic Systems, Göttingen, Germany). Beads without primary antibodies were used as negative controls. Bead-bound SVs were dissolved in Laemmli buffer, and the resultant protein pattern was analyzed by SDS-PAGE/Western blot (Zander et al., 2010) using anti-VGLUT1, anti-VIAAT, anti-synaptophysin and anti-VGLUT3 (all from Synaptic Systems, Göttingen, Germany). Immuno-signals were visualized by enhanced chemiluminescence. Densitometric analyses were performed using LabImage 1D 2006 (Kapelan Bio-Imaging Solutions) and quantified using standard curves obtained from the initial LS1 fraction.

Vesicular [³H]glutamate uptake analyses were performed as described previously (Zander et al., 2010; Frahm et al., 2015). LS1-immunoisolated SVs were bound to M-280 sheep anti-mouse beads (Invitrogen). Beads coupled with non-specific mouse IgG (Santa Cruz Biotechnologies) were used as negative controls. Uptake assays were performed with immunoisolated SVs and initiated by the addition of L-glutamate (49.5 μM, Sigma–Aldrich) and [³H]-glutamate (0.5 μM, Hartmann Analytic GmbH, Germany). Uptake was stopped after 10 min at 25°C, and unbound radioactivity was removed by centrifugation (435,000 × g, 10 min).