Identification of a stereotypic molecular arrangement of endogenous glycine receptors at spinal cord synapses

Abstract

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brain stem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in native spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that GlyRs exhibit equal receptor-scaffold occupancy and constant absolute packing densities of about 2000 GlyRs μm-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory PSDs rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

A Quantitative Perspective of Alpha-Synuclein Dynamics – Why Numbers Matter

“…Single molecule localisation microscopy can even be used for absolute quantification, where the numbers of single molecule detections are converted into actual molecule numbers and packing densities (e.g., Maynard et al, 2021). Different approaches have been developed, generally involving some kind of internal calibration standard that can be extrapolated to clusters of detections arising from larger protein complexes or unknown structures (Wu et al, 2020).…”

Event-based vision sensor enables fast and dense single-molecule localization microscopy

“…These advances address some of the remaining challenges in SMLM, including but not limited to imaging thick samples and tissues, reducing phototoxicity and photobleaching for live-cell imaging, but also obtaining quantitative information on the distribution, size, shape, spatial organisation and stoichiometry of macromolecular complexes in order to steer biological interpretation. For a recent review of the field of SMLM, refer to [14,15] While retrieving the positions of single molecules is pivotal in applications as varied as 3D imaging of immunolabelled samples [16,17,18,19], spatial analysis of protein clusters [20,21] or protein 1 dynamics in the cell [22], single-molecule microscopy is also used in a much broader range of applications exploiting other information carried by the point-spread function (PSF). A non exhaustive list of them includes accessing the spectrum of the dyes for multicolor imaging [23,24], retrieving the emitter's orientation via polarization measurements [25], or even probing the local environment of the molecules through modifications of the fluorescence intensity [26,27], or the fluorescent state lifetime [28,29,30,31].…”

“…While retrieving the positions of single molecules is pivotal in applications as varied as 3D imaging of immunolabelled samples [16, 17, 18, 19], spatial analysis of protein clusters [20, 21] or protein dynamics in the cell [22], single-molecule microscopy is also used in a much broader range of applications exploiting other information carried by the point-spread function (PSF). A non exhaustive list of them includes accessing the spectrum of the dyes for multicolor imaging [23, 24], retrieving the emitter’s orientation via polarization measurements [25], or even probing the local environment of the molecules through modifications of the fluorescence intensity [26, 27], or the fluorescent state lifetime [28, 29, 30, 31].…”

“…Significant advances have been possible through the development of new optical techniques [4,5], labeling methods [6,7,8] and the synthesis of brighter dyes compatible with livecell imaging [9,10]. For recent reviews of the field of SMLM, refer to [11,12] Retrieving the positions of single molecules is pivotal in applications as varied as 3D imaging [13,14,15,16], spatial analysis of protein clusters [17,18] or protein dynamics in the cell [19]. Still, the point-spread function (PSF) carries significant additional information, giving access to the spectrum of the dyes for multicolor imaging [20,21], the emitter's orientation [22], the local environment of the molecules through modifications of the fluorescence intensity [23,24], and fluorescent state lifetime [25,26,27,28].…”

“…Retrieving the positions of single molecules is pivotal in applications as varied as 3D imaging [13, 14, 15, 16], spatial analysis of protein clusters [17, 18] or protein dynamics in the cell [19]. Still, the point-spread function (PSF) carries significant additional information, giving access to the spectrum of the dyes for multicolor imaging [20, 21], the emitter’s orientation [22], the local environment of the molecules through modifications of the fluorescence intensity [23, 24], and fluorescent state lifetime [25, 26, 27, 28].…”