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 brainstem. 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 spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant 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 postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

“…Yet, the common pan‐inhibitory synapse marker is the postsynaptic scaffold protein gephyrin that is present at glycinergic and GABAergic synapses alike [7] . Gephyrin is an integral protein that stabilizes GlyRs and GABA A Rs at the postsynaptic density, [8, 9] and its concentration closely correlates with the number of inhibitory receptors and synaptic strength [2, 10–12] …”

“…To circumvent these drawbacks, two alternative methods of genetic tagging were applied. The first method involves knock‐in mice expressing mRFP‐gephyrin where expression levels, subcellular distribution of the endogenous protein and synaptic function are largely preserved [6, 12] . The second method consists in intracellular expression of eGFP fused anti‐gephyrin nanobody (recombinant antibody‐like light‐weight protein) that visualizes the inhibitory synapses in fixed and live cells [18] .…”

“…[7] Gephyrin is an integral protein that stabilizes GlyRs and GABA A Rs at the postsynaptic density, [8,9] and its concentration closely correlates with the number of inhibitory receptors and synaptic strength. [2,[10][11][12] Gephyrin is often visualized using genetic tagging. eGFP, mCherry, mEos2 and other fluorescent proteins can be fused with gephyrin and expressed in cells via transfection or infection.…”

“…The first method involves knock-in mice expressing mRFP-gephyrin where expression levels, subcellular distribution of the endogenous protein and synaptic function are largely preserved. [6,12] The second method consists in intracellular expression of eGFP fused antigephyrin nanobody (recombinant antibody-like light-weight protein) that visualizes the inhibitory synapses in fixed and live cells. [18] This approach can be especially effective when combined with a transcriptional control system that matches the expression level of the protein tag with that of its endogenous target, minimizing off-target labeling and unwanted functional effects.…”

Glycine Receptor Subtypes and Their Roles in Nociception and Chronic Pain

“…As the β GlyR is modified by post-translational modifications that adjust its subcellular localization ( Specht et al, 2011 ; Grünewald et al, 2018 ) and function ( Caraiscos et al, 2002 ; Muñoz et al, 2021 ), it is likely that pain-related plasticity phenomena associated with glycinergic neurotransmission are linked with modifications of the β subunits. Future studies and novel models ( Maynard et al, 2021 ) may contribute to clarify the specific roles of β GlyR subunit on pain signaling.…”