A NOVEL METHOD FOR FUNCTIONAL ANALYSIS OF SYNAPSES:ASSAY VALIDATION AND DESIGN OF EXPRESSION SYSTEMS FOR IN VIVO EXPERIMENTS ON BRAIN CIRCUITS.

An important avenue in neuroscience is represented by a more in depth analysis of cortical activity, the expectation being to find novel correlations between specific animal behaviours or cognitive functions and unique patterns of activity in neurons and synaptic networks. This goal can be reached thanks to the development of novel methodologies that ideally should be sensitive enough to provide quantitative information about single elements but also providing a view of the activity in the entire cortical network. In my laboratory in the last few years we have developed a series of biosensors for the investigation of synaptic activity both in vitro and in the animal in vivo. In order to develop a genetically encoded indicator of synaptic network activity, we have generated a series of reporters of synaptic vesicle re-use. These sensors have been named as the GreenZip family. These indicators report synaptic activation through the uptake of small fluorescent peptidic markers during cycles of exo-endocytosis, whose frequency is greatly enhanced by synaptic transmission and neuro-transmitter release. These new tools have been engineered by modifying the scaffold of the vesicular protein VAMP2 (Synaptobrevin2) through the insertion, at the intraluminal ending, of a "bait" domain with binding activity for a 4 kD peptide dubbed Synbond. The latter is conjugated with a fluorophore or with other detectable molecules. This pair of binders was selected for their high binding affinity (in the nM range) and the reporter gene was named GreenZip (the prefix Green indicates the presence of a GFP molecule at the N-terminal, cytosolic domain). These constructs have been shown to work in cultured neuronal networks (dissociated cultures of hippocampal neurons). The activity-dependent uptake of Synbond was characterized in detail and found to correlate well with synaptic efficacy and with the frequency of stimulation of presynaptic cells. The sensitivity is indeed very high, allowing individual active synapses to be easily visualized after just a few action potentials. To test the feasibility of this method for in vivo analysis, this family of molecules was expressed by electroporation of cDNA in brain slices (cortical, cerebellar and hippocampal cultured slices) and in vivo in the LGN thalamic nuclei (by cDNA electroporation in retinal ganglion cells). In these experiments, we demonstrated that Synbond, diffuses quickly across brain tissue and reaches synapses and an activity-dependent labeling of GreenZip-expressing that synapses can be achieved. This activity-dependent labeling of presynaptic boutons correlates with stimulus duration and light intensity and is already detectable after just a few light pulses. Therefore, this technique permits unprecedented in vivo recordings from large synaptic networks with very high spatial and temporal resolution. These experiments were run in living animals and the detection of GreenZip-expressing synapses (by GFP) and of Synbond uptake was obtained retrospectively after sacrificing the animal, because the thalamus is located too deeply inside the brain to be reached by available optical technologies. To overcome this limitation and express greenzip molecules also at the brain surface, and potentially in any brain area of our interest, and to run out the validation process I completed the development of a transgenic animal model capable of overexpressing GreenZip in all tissues and at any time point in the development, exploiting the technology of recombinant DNA and using the genomic locus Rosa26 in association with the Cre recombinase system. In addition, I also achieved the generation of a family of lentivirus constructs, based on a modified HIV virus that aims to be able to express GreenZip in many areas, and in many synaptic networks, more selectively using stereotaxic injections of the viral vectors inside specific subgroups of cortical cells and cortical regions including the visual areas.