title: Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites.
abstract: 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/μm(2). 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.
authors.affiliation: Biologie Cellulaire de la Synapse, Inserm U1024, Institute of Biology, École Normale Supérieure ENS, 46 rue d'Ulm, Paris 75005, France.
authors.forename: Christian G
authors.initials: CG
authors.lastname: Specht
authors.forename: Ignacio
authors.initials: I
authors.lastname: Izeddin
authors.forename: Pamela C
authors.initials: PC
authors.lastname: Rodriguez
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Beheiry
authors.forename: Philippe
authors.initials: P
authors.lastname: Rostaing
authors.forename: Xavier
authors.initials: X
authors.lastname: Darzacq
authors.forename: Maxime
authors.initials: M
authors.lastname: Dahan
authors.forename: Antoine
authors.initials: A
authors.lastname: Triller
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry / Q000648:ultrastructure*; D002478:Cells, Cultured; D008565:Membrane Proteins / Q000737:chemistry / Q000648:ultrastructure*; D057054:Molecular Imaging / Q000379:methods; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure*; D009433:Neural Inhibition / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D018009:Receptors, Glycine / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D013569:Synapses / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D011963:Receptors, GABA-A; D018009:Receptors, Glycine; C073441:gephyrin