Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum

Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.