A new transgenic mouse line for the visualization of endogenous biotinylated Neuroligin-1

Synapses are established and maintained by adhesion molecules, which include post-synaptic neuroligins (NLGNs). In the absence of highly specific antibodies, the sub-cellular distribution and synaptic function of NLGNs have been extensively manipulated using knock-out, over-expression or rescues approaches, leading to controversial results. In this context, we generated a new transgenic knock-in mouse strain in which endogenous NLGN1 has been N-terminally tagged with a biotin acceptor peptide (bAP) by gene editing using the CRISPR-Cas-9 strategy. Native NLGN1 can be enzymatically biotinylated by the viral delivery or electroporation of biotin ligase, allowing for its subsequent high affinity labeling by streptavidin conjugates. Using complementary assays including behavioral assessment, biochemistry, immunohistochemistry, confocal and electron microscopy, we characterized this new mouse strain and found that addition of the bAP tag on NLGN1 does not affect brain development. Using primary hippocampal neurons cultured from knock-in mice infected with a virus encoding the biotin ligase (BirAER-IRES-GFP), we show that biotinylated bAP-NLGN1 can be pulled-down by streptavidin beads, and selectively labeled with fluorescent streptavidin. Interestingly, by using a co-labelling with intrabodies to PSD-95 or gephyrin, we reveal that bAP-NLGN1 is targeted to both excitatory and inhibitory synapses. Using dSTORM and electron microscopy, we demonstrate that bAP-NLGN1 is organized in a subset of nanomodules in both excitatory and inhibitory post-synapses. Each nanomodule contains only a handful of bAP-NLGN1 molecules, and the number of nanomodules scales with the size of the scaffold. This new transgenic mouse line allows us to revisit the role of native NLGN1 in synapse differentiation.