• cre recombinase
• dendritic spines
• glutamate indicators
• neuroscience
• viral vectors

The field of connectomics aims to map the complex network of specialized synaptic connections that coordinate the flow of information at different resolution levels. The microscopic level investigates the organization of single synapses; the mesoscopic one analyses the circuit-level architecture of neural populations and the macroscopic one takes into consideration the functional connectivity between large brain regions. In the past two decades considerable methodological advances have been made thanks to the synergy of molecular biology, genetics, virology, and microscopy in the development of viral vectors. They allow the investigation of complex network of specialized synaptic connections that coordinate the flow of information. These effective tools are bound to provide new insights on the neural basis of dysfunctional synaptic signaling and on brain dysconnectivity which both characterize developmental diseases such as autism spectrum disorder, schizophrenia and other disorders with developmental origins. Given the hypothesis that dynamics at synaptic level can reflect responses at whole brain level, I engineered viral vectors both to map, to manipulate and to monitor neural circuits, as well as to induce region-, time- and cell type-specific fluctuations. During my Ph.D. I started two projects, and I took part to a third ongoing project in collaboration with Dr. Alessandro Gozzi’s lab. These are three independent projects which will be described in separate chapters. To this aim I used three experimental strategies and evaluate different levels of investigation: i) site-specific recombinase-expressing vectors to specifically map and manipulate target neuronal populations at mesoscopic level; ii) viral vectors with Tet-On system to remotely control synaptic activity at microscopic level; iii) constitutively GFP-expressing vector to sparse labelling the whole brain and to correlate macroscopic functional connectivity to microscopic synaptic structural rearrangements.
i) With the first project, I performed a functional assay of progressive amino-terminal deleted Cre recombinase isoforms to implement recombinase-based technologies to study the structure and subsequently the function of a defined neuronal population avoiding unwanted somatic recombination due to “leakage” activity.
ii) In parallel, I studied the perturbations at microscopic synaptic level to test the hypothesis that cellular mechanisms of synaptic plasticity are link to the generation of activity patterns. To investigate the connection between synaptic dysregulation and functional dysconnectivity in mental disorders, I focused my attention on the thalamo-prefrontal projecting neurons of adult mice, since this circuitry dysfunction has been observed to lead to severe impairments in mental disorders such as autism, schizophrenia, and depression. To do so, I engineered a viral approach based on Dox-controlled genetic switches for remotely and reversibly promoting bidirectional control of synaptic strength at basal condition by the viral-encoding modulation of AMPA receptor subunits at the level of thalamo-prefrontal cortex circuit.
iii) Eventually, taking advantage of a mouse model of 22q11.2 connectopathy I probed mechanistic links between synaptic pathology and altered macroscale functional. A reporter-expressing viral vector has been used to highlight synaptic anatomic structures such as dendritic spines to correlate it with global activity analysis during development in 22q11.2 connectopathy. The observations about the correlation between synaptic activity and global functional activity complements microscale modeling and advances our understanding of 22q11.2 connectopathy, suggesting that disrupted synaptic homeostasis can lead to multiscale alterations in neural connectivity.