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.

Recent applications of superresolution microscopy in neurobiology

“…On average, 200 copies of gephyrin molecules were found per synapse, though the absolute figures and cluster size were highly dependent on the type of sample. These authors also showed that the other major inhibitory receptors in brain (GABA A receptors) colocalized with gephyrin [23]. In a very recent extension of that study, gephyrin was shown to form scaffolds which act as shallow energy traps ( 3 kBT) for glycine neurotransmitter receptors [24].…”

“…They could also follow dynamic changes undergone by the nanoclusters. Similarly, Specht et al [23] used a combination of PALM/STORM microscopies to quantify the abundance of the peripheral, non-receptor protein gephyrin, labeled with the fluorescent proteins Dendra2 and mEos2, in inhibitory synapses of a knock-in mouse expressing mRFP-gephyrin [23]. On average, 200 copies of gephyrin molecules were found per synapse, though the absolute figures and cluster size were highly dependent on the type of sample.…”

Gephyrin: a central GABAergic synapse organizer

“…34 Recently, quantitative nanoscopy was used to calculate the number of gephyrin molecules and inhibitory synaptic receptor binding sites. 35 Purely GABAergic synapses were found to harbor~40-500 gephyrin molecules and 30-200 GABA A receptors organized in microclusters. In contrast, spinal cord synapses, which contain both GlyRs and GABA A receptors, showed a twofold higher packing density of gephyrin, suggesting that the binding to GlyRs affected the compactness of the hexagonal gephyrin lattice.…”

“…In contrast, spinal cord synapses, which contain both GlyRs and GABA A receptors, showed a twofold higher packing density of gephyrin, suggesting that the binding to GlyRs affected the compactness of the hexagonal gephyrin lattice. 35 Moreover, although the phosphorylation of gephyrin is known to modulate its binding affinity, clustering and receptor association, it is not yet known whether increased gephyrin cluster stability correlates with tighter packaging within the lattice. 35 Overall, these data suggest that gephyrin is tightly packed beneath the GlyR-containing postsynaptic membrane.…”

“…35 Moreover, although the phosphorylation of gephyrin is known to modulate its binding affinity, clustering and receptor association, it is not yet known whether increased gephyrin cluster stability correlates with tighter packaging within the lattice. 35 Overall, these data suggest that gephyrin is tightly packed beneath the GlyR-containing postsynaptic membrane.…”