Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses

Glycine receptors (GlyRs) are heteropentameric chloride channels that mediate fast inhibitory neurotransmission in the brainstem and spinal cord, where they regulate motor and sensory processes. GlyRs are clustered in the post-synaptic membrane by strong interactions of the β subunit with the scaffold protein gephyrin. Even though GlyRβ mRNA is highly expressed throughout the brain, the existence of synaptic GlyRs remains controversial as there is little conclusive evidence using conventional fluorescence microscopy and electrophysiological recordings. Here we exploit the high sensitivity and spatial resolution of single molecule localisation microscopy (SMLM) to investigate the presence of GlyRs at inhibitory synapses in the brain, focusing on several areas of the telencephalon. Making use of a knock-in mouse model expressing endogenous mEos4b-tagged GlyRβ, we identified few GlyRs in sub-regions of the hippocampus. Dual-colour SMLM revealed that these sparse receptors are integrated within the post-synaptic gephyrin domain, pointing to a possible role in maintaining the structural integrity of inhibitory synapses. In contrast, we found functionally relevant numbers of synaptic GlyRs at inhibitory synapses in the ventral striatum. Our results highlight the strength of SMLM to detect few and sparsely distributed synaptic molecules in complex samples and to analyse their organisation with high spatial precision.