FIGURE 1.

His₆-FLAG-YFP-neuroligin-2 (HFY-NL2) localizes to inhibitory postsynaptic sites in cultured hippocampal neurons and binds neurexin. A–C, cultured neurons were transfected with HFY-NL2 expression vector at 9 DIV, then fixed and immunolabeled at 12 DIV. HFY-NL2 imaged by the unamplified YFP signal was restricted to the somatodendritic domain and formed clusters on dendrites labeled for microtubule-associated protein 2 (MAP2). HFY-NL2 clusters were opposed to inhibitory terminals labeled for the vesicular GABA transporter (VGAT) (A), but not to excitatory terminals labeled for the vesicular glutamate transporter (VGluT) (B). HFY-NL2 clusters colocalized with the inhibitory postsynaptic scaffold gephyrin but not with the excitatory postsynaptic scaffold PSD-95; some gephyrin puncta lacking HFY-NL2 are seen in crossing dendrites from untransfected neurons (C). D, COS7 cells expressing HFY-NL2 or YFP-CD4 as a negative control were incubated with the ectodomain of neurexin-1β fused to Myc-alkaline phosphatase, Myc-neurexin-AP. Whereas both constructs expressed well as detected by surface labeling with anti-GFP antibody, only HFY-NL2 mediated neurexin binding as detected with anti-Myc antibody. Scale bars: 10 μm in A (left panels); 5 μm in A (right panels), B and C; 20 μm in D.