TeNT binds to central neurons via recycling synaptic vesicles.

<p>(A) Cultured rat hippocampal neurons were incubated with the receptor-binding domain of TeNT (HCR/T, 50 nM) either in non-depolarizing conditions (TTX: 5 mM KCl, 0 mM Ca²⁺, 1 µM TTX) or depolarizing conditions (high K⁺ buffer: 55 mM KCl, 2 mM Ca²⁺). Cells were processed for immunocytochemistry and HCR/T was detected using a mouse anti-FLAG tag monoclonal antibody. Vesicular glutamate transporter 1 (vGLUT1) antibodies were used to mark excitatory synapses and antibodies against vesicular GABA transporter (vGAT) were used to mark inhibitory terminals. The binding of HCR/T to neurons was markedly enhanced under depolarizing conditions. Scale bars in all figures are 10 µm unless otherwise indicated. (B) Quantification of HCR/T staining intensity at excitatory/inhibitory terminals normalized to VGLUT1 or vGAT intensity. Error bars represent SD, n = 9, ***p≤0.001. (C) Dissociated spinal cord neurons were incubated with HCR/T (50 nM) in TTX buffer or high K+ buffer. Binding of HCR/T was markedly increased under depolarizing conditions. (D) Quantification of HCR/T to vGAT staining intensity ratio. Error bars represent SD, n = 18, ***p≤0.001.