Fig. 1: Genetic technology for virus-delivered silencing of synaptic transmission.
A Schematic diagram depicting the tetracycline-controlled genetic switches to express a destabilized tetanus toxin light chain (dsTeTxLC) for selective cleavage of a key synaptic vesicle protein, synaptobrevin-2 (Syb2). Virus-1 is equipped with the human synapsin promoter (PhSYN) to express the reverse tetracycline transactivator (rtTA), virus-2 has a bidirectional tetracycline promoter (Ptetbi) to express dsTeTxLC with a short half-life time. Only in the presence of doxycycline (Dox), Ptetbi is switched-on to express dsTeTxLC. Without Dox, the system is switched-off. For validating targeted gene expression in the brain, the virus-3 was used as a tracer to express tdTomato (tdTOM) under control of PhSYN. B Levels of synaptobrevin-2 were determined by a Western blot: without and with dsTeTxLC expression in neurons to validate dsTeTxLC-mediated cleavage of synaptobrevin-2. The beta-tubulin was used for normalization. C Hippocampal slices with stimulating electrode in the dentate gyrus (DG) and recording electrode in CA3 (insert inside the input/output curve). The input/output curves compare the slopes of averaged field potentials (10 repetitions/intensity) with increasing stimulation intensities from control (in black) and TeTxLC-infected (in red) slices (RM-ANOVA test, P < 0.001.
