A NOVEL REPORTER FOR THE ANALYSIS OF SYNAPTIC ACTIVITY IN VIVO

At the present time the analysis of synaptic transmission in the in vivo brain can only be approached very indirectly. We developed a quantitative method that is based on the capture of a highly diffusive small molecule from the extracellular space which permits the identification of active terminals and the characterization o their level of activity. The methodology is based on a synaptic vesicle construct that has been engineered starting from the scaffold of the synaptic vesicle transmembrane protein VAMP2. We fused eGFP at the cytosolic end and introduced at the lumenal C-terminal domain a "bait" sequence that binds a cognate small diffusible fluorescent peptide. This method was initially tested in an eukaryotic cell line (Hela cells). The construct was correctly expressed in a microvesicular compartments with the correct orientation. The vesicular compartment expressing our construct was viable and found to capture the cognate fluorescent peptide from the extracellular environment. We characterized binding kinetics and the degree of saturation of this pair in different experimental conditions. Similar results were obtained in neuronal cells where the construct was found to localize specifically in presynaptic boutons with an activity dependent uptake of the tracer whose degree correlated with the number of action potentials invading the terminal. These esperiments were run both in vitro (primary hippocampal cultures and organotypic slices) and in vivo (visual thalamus or dLGN and visual cortex). In the latter condition activity-dependent synaptic stimulation was achieved by a sequence of light pulses.