High-affinity detection of endogenously biotinylated neuroligin-1 at excitatory and inhibitory synapses using a tagged knock-in mouse strain

Neuroligins (NLGNs) are important cell adhesion molecules mediating trans-synaptic contacts between neurons. However, the high-yield biochemical isolation and visualization of endogenous NLGNs have been hampered by the lack of efficient antibodies to these proteins. Thus, to reveal their sub-cellular distribution, binding partners, and synaptic function, NLGNs have been extensively manipulated using knock-down, knock-out, or over-expression approaches, overall leading to controversial results. As an alternative to the manipulation of NLGN expression level, we describe here the generation of a new transgenic mouse strain in which native NLGN1 was N-terminally tagged with a small biotin acceptor peptide (bAP) that can be enzymatically biotinylated by the exogenous delivery of biotin ligase. After showing that knock-in mice exhibit normal behavior as well as similar synaptic number, ultrastructure, transmission properties, and protein expression levels when compared to wild type counterparts, we exploited the fact that biotinylated bAP-NLGN1 can be selectively isolated or visualized using high-affinity streptavidin conjugates. Using immunoblotting and immunofluorescence, we show that bAP-NLGN1 binds both PSD-95 and gephyrin and distributes equally well at excitatory and inhibitory synapses, challenging the historical view that NLGN1 is exclusively localized at excitatory synapses. Using super-resolution fluorescence microscopy and electron microscopy, we further highlight that bAP-NLGN1 forms in the synaptic cleft a subset of nanodomains each containing a few NLGN1 dimers, while the number of nanodomains per synapse positively scales with the post-synapse size. Overall, our study not only provides a novel, extensively characterized transgenic mouse model which will be made available to the scientific community, but also an unprecedented view of the nanoscale organization of endogenous NLGN1.