Quantitative Single Molecule Analysis of the Nanoscale Organization of the Inhibitory Synapse

Abstract

Super-resolution techniques based on Single Molecule Localization (SML) provide the capability to study sub-cellular structures at the molecular scale and to access quantitative information about proteins distribution in biological systems [1].In this work we exploit PALM (Photo-Activatable Localization Microscopy) in order to decipher the functional organization and distribution of gephyrin, a scaffold protein of the inhibitory synapse that mediates the anchoring of GABAA receptor [2, 3]. We mapped the spatial distribution of gephyrin clusters along the 3D organization of neuronal dendrites, highlighting the synaptic gephyrin clusters identified by their matching with specific marker of pre-synaptic terminals. The choice of a photoactivatable fluorescent protein with a fully characterized photophysics (mEos) [4] combined with a quantitative approach based on clustering analysis coming from graph theory [5] allow us to quantitatively monitor the rearrangement of the gephyrin clusters in response to a chemically induced form of long-term potentiation of inhibitory synapses (chem-iLTP) [2].This approach suggested that, during the expression of chem-iLTP, gephyrin is redistributed from extra-synaptic to synaptic compartments. Interestingly, we observe that the increase of synaptic gephyrin is strictly linked to an increase of heterogeneity in the inner organization of the scaffold protein at the synapse that rearranges in nanodomains.[1] Deschout H. et al., Nat. Methods, 2014.[2] Petrini E. M. et al, Nat. Comm., 2014.[3] Specht C.G. et al. Neuron, 2013.[4] Durisic N. et al., Nat. Methods, 2014.[5] Pavan M. et al. IEEE Trans. Pattern Analysis and Machine Intelligence, 2007.