Glycine receptor-mediated inhibitory neurotransmission is key for spinal cord function. Recent observations suggested that by largely elusive mechanisms also glycinergic synapses display synaptic plasticity. We imaged receptor fields at ultra-high resolution at freeze-fractured membranes, tracked surface and internalized glycine receptors (GlyR) and studied differential regulations of GlyRβ interactions with the scaffold protein gephyrin and the F-BAR domain protein syndapin I and thereby reveal key principles of this process. S403 phosphorylation of GlyRβ, known to be triggered by synaptic signaling, caused a decoupling from gephyrin scaffolds but simultaneously promoted association of syndapin I with GlyRβ. In line, kainate-treatments used to trigger rearrangements of glycine receptors in murine syndapin I KO spinal cords (mixed sex) showed even more severe receptor field fragmentation than already observed in untreated syndapin I KO spinal cords. Syndapin I KO furthermore resulted in more dispersed receptors and increased receptor mobility also pointing out an important contribution of syndapin I in the organization of GlyRβ fields. Strikingly, syndapin I KO also led to a complete disruption of kainate-induced GlyRβ internalization. Accompanying quantitative ultra-high resolution studies in dissociated spinal cord neurons strongly suggested that the observed defects in GlyR internalization observed in syndapin I KO spinal cords are directly caused by syndapin I deficiency within murine spinal cord neurons. Together our results unveiled important mechanisms organizing and altering glycine receptor fields during both steady-state and particularly upon kainate-induced synaptic rearrangement - principles organizing and fine-tuning synaptic efficacy and plasticity of glycinergic synapses in the spinal cord.SIGNIFICANCE STATEMENTInitial observations suggested that also glycinergic synapses - key for spinal cord and brain stem functions - may display some form of synaptic plasticity. Imaging receptor fields at ultra-high resolution at freeze-fractured membranes, tracking surface and internalized glycine receptors (GlyR) and studying regulations of GlyRβ interactions we here reveal key principles of these kainate-inducible adaptations. A switch from gephyrin-mediated receptor scaffolding to syndapin I-mediated GlyRβ scaffolding and internalization allows for modulating synaptic receptor availability. In line, kainate-induced GlyRβ internalization was completely disrupted and GlyRβ receptor fields were distorted upon syndapin I KO. These results unveiled important mechanisms during both steady-state and kainate-induced alterations of synaptic GlyR fields - principles underlying synaptic efficacy and plasticity of synapses in the spinal cord.
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