Quantitative Nanoscopy of Inhibitory Synapses: Counting Gephyrin Molecules and Receptor Binding Sites

• Published in: Neuron (Cambridge, Mass.) Mass.), 2013-07, Vol.79 (2), p.308-321
• Main Authors: Specht, Christian G., Izeddin, Ignacio, Rodriguez, Pamela C., El Beheiry, Mohamed, Rostaing, Philippe, Darzacq, Xavier, Dahan, Maxime, Triller, Antoine
• Format: Article
• Language: English
• Subjects: Animals; Binding sites; Binding Sites - physiology; Carrier Proteins - chemistry; Carrier Proteins - ultrastructure; Cells, Cultured; Experiments; Life Sciences; Membrane Proteins - chemistry; Membrane Proteins - ultrastructure; Microscopy; Molecular Imaging - methods; Nanostructures - chemistry; Nanostructures - ultrastructure; Neural Inhibition - physiology; Neurobiology; Neurons; Neurons and Cognition; Phosphorylation; Protein Binding - physiology; Proteins; Rats; Rats, Sprague-Dawley; Receptors, GABA-A - chemistry; Receptors, GABA-A - metabolism; Receptors, GABA-A - ultrastructure; Receptors, Glycine - chemistry; Receptors, Glycine - metabolism; Receptors, Glycine - ultrastructure; Synapses - chemistry; Synapses - metabolism; Synapses - ultrastructure
• ISSN: 0896-6273; 1097-4199
• DOI: 10.1016/j.neuron.2013.05.013

The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultrastructure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000–10,000 molecules/μm2. The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale. •Molecule counting using quantitative single-fluorophore imaging•Dual-color three-dimensional PALM/STORM using adaptive optics•Number and density of gephyrin scaffolds at inhibitory PSDs•Topology and density of receptor binding sites at inhibitory synapses The molecular organization of synapses regulates the efficacy of synaptic transmission. However, little is known about the absolute protein numbers at synapses. Specht et al. implement single-molecule imaging to count synaptic scaffold proteins and receptor binding sites.