Evaluation of structural and FunctionAl Synaptic plastiCity dynamIcs iN physiopAThologIcal cONditions in in vitro and in vivo experiment models. (FASCINATION)
Progetto FASCINATION will combine interdisciplinary approaches merging in vitro and in vivo state-of-the-art techniques from cellular to imaging, and to molecular neuroscience to assess structural and functional plasticity in preclinical experimental models. In particular, two major aims will be: Aim1: Evaluation of morphological changes in dendritic branching and dendritic spines. We will evaluate morphological changes by AiryScan super-resolution confocal microscopy and 3D volumetric analysis in in vitro neuronal models and ex-vivo samples. For in vitro live-imaging morphological studies, primary neurons will be transfected with EGFP. For ex vivo morphological analyses, brain slices will be labelled with spines specific fluorescent lipophilic dyes. Quantitative analysis will be performed using Fiji software for spine morphology and dendritic branching (Sholl analysis). In parallel, Neurolucida software will be used. Aim2: Evaluation of functional changes by live-imaging approaches. Neuronal morphology is correlated with changes of synaptic activity and calcium levels at dendritic spines.
To evaluate these events, we will use:
1) Novel pCAG_Xph20-GCaMP7f calcium sensor binding the postsynaptic marker PSD-95, to assess calcium transients at dendritic spines;
2) Novel iGluSnFR3.v857.SGZ glutamate sensor, allowing for live-imaging in vitro assessment of neurotransmitter levels at the postsynaptic sites;
3) Constructs encoding for a GFP-based superecliptic pHluorin (SEP) fused to specific subunits of glutamate receptors to evaluate receptors’ endo-exocytosis events;
4) In vivo analysis with fiber photometry and mini endoscopic to measure neuronal activation through recording of calcium oscillations (using calcium sensors, GCaMP7) within specific neuronal populations and with single-cell resolution, respectively;
5) magnetic resonance imaging (MRI) to assess structural connectivity
