Quantitative Analysis of Anchoring Proteins of the Inhibitory Synapse through Single Molecule Localization Techniques

Abstract

The post-synaptic area of the inhibitory synapse is characterized by sub-resolved clusters of the scaffold protein Gephyrin [1] that anchors GABAA receptor (GABAAR). Super-resolution techniques [2] based on Individual Molecule Localization (IML) [3] provide the capability to quantitatively study these subcellular structures, giving access to its protein distribution at the molecular level.In this work the highly heterogeneous spatial distribution of Gephyrin cluster along the 3D neuronal network has been investigated through dual color STORM (Stochastic Optical Reconstruction Microscopy) to characterize the post-synaptic area, for example reveling the interaction between Gephyrin and GABAARs.To monitor the response of Gephyrin cluster to Long-Term Potentiation of inhibition (iLTP) their functional link with the synaptic terminals has been highlighted. The insight provided at the molecular level makes IML techniques the suitable tool for a quantitative study of Gephyrin distribution at synaptic level. Quantitative approach based on clustering analysis [4] provided access to a more comprehensive set of parameters able to define the response to iLTP, such as the area and the density of the scaffold protein. At the fluorescent tag level, to fulfill the requests of the quantitative analysis, an irreversibly photoactivatable fluorescent protein has been used (mEos), knowing its photophysical properties [5].[1] Petrini E. M. et al, Nat. Comm., 2014.[2] Deschout H. et al., Nat. Methods, 2014.[3] Specht C.G. et al. Neuron, 2013.[4] Pavan M. et al. IEEE Trans. Pattern Analysis and Machine Intelligence, 2007.[5] Durisic N. et al., Nat. Methods, 2014.