Neuroligin-2 Localizes Primarily to GABA Synapses but Can Be Recruited to Glutamate Synapses
(A, B) Immunoreactivity for endogenous neuroligin-2 in hippocampal neurons clusters at GABA synapses with gephyrin and GAD, but not at glutamate synapses with PSD-95 or VGlut.
(C) YFP-neuroligin-1 expressed at low level is partially diffuse and partially concentrated at glutamate synapses labeled with PSD-95 (arrow), but not at GABA synapses labeled with gephyrin.
(D) Coexpression of PSD-95-CFP enhances the localization of YFP-neuroligin-1 to glutamate synapses (arrow).
(E) YFP-neuroligin-2 expressed at low level is highly punctate and colocalizes with gephyrin at GABA synapses (arrowhead), but is not detected with PSD-95 at glutamate synapses.
(F) Coexpression of PSD-95-CFP recruits YFP-neuroligin-2 to glutamate synapses (arrow) in addition to its localization with gephyrin at GABA synapses (arrowhead).
Scale bars (A) and (B) 10 μm, (C)–(F) 5 μm.

Purified Neurexin Coclusters Neuroligins with Postsynaptic Proteins
(A–C) Purified neurexin-Fc attached to beads induced clusters of gephyrin, PSD-95 (A), GABA receptor (B), and NR1 (C) at contact sites with dendrites (arrows in phase contrast images). Postsynaptic protein clusters induced by beads bearing neurexin-Fc lacked immunoreactivity for presynaptic proteins synapsin or GAD.
(D, E) In neurons expressing low levels of YFP-neuroligin-1 or YFP-neuroligin-2, purified neurexin-Fc attached to beads induced coclustering of gephyrin and PSD-95 with neuroligins. Dendrite-bead contacts either exhibited strong aggregation of all of YFP-neuroligin, gephyrin, and PSD-95 (arrows) or no detectable aggregation of any of these proteins (arrowheads).
Scale bars 10 μm.

Neuroligin Aggregation Alone Is Synaptogenic
Neurons expressing low levels of YFP-neuroligins were exposed to beads bearing antibodies against YFP to aggregate the neuroligins on dendrites independently of neurexin. Aggregation of YFP-neuroligin-1 resulted in coaggregation of PSD-95 but not gephyrin (A), whereas aggregation of YFP-neuroligin-2 resulted in coaggregation of both PSD-95 and gephyrin (B). Aggregates of the postsynaptic proteins induced by neuroligin aggregation lacked immunoreactivity for the presynaptic protein synapsin. Arrows indicate sites of induced aggregation; other such sites are enlarged below (Nlg1, YFP-neuroligin-1; Geph, gephyrin; PSD, PSD-95; Syn, synapsin; Nlg2, YFP-neuroligin-2). Scale bar 5 μm.

Neurexin Clusters Neuroligins but Not Dystroglycan
(A) Coculture of fibroblasts expressing neurexin-1β-CFP with neurons resulted in only endogenous synaptic clusters (arrows) but no induced clusters of β-dystroglycan at contacts with dendrites.
(B and C) Fibroblasts expressing neurexin-1β-CFP cocultured with hippocampal neurons expressing YFP-neuroligin-1 (B) or YFP-neuroligin-2 (C) induced clusters of YFP-neuroligin lacking immunoreactivity for synapsin in contacting dendrites. The transfected neuron is indicated by *.
(D) Fibroblasts expressing neurexin-1β-CFP cocultured with hippocampal neurons expressing YFP-neuroligin-1 induced overlapping clusters of recombinant YFP-neuroligin-1 and endogenous neuroligin-2 in contacting dendrites. Smaller clusters of gephyrin and PSD-95 were apparent within the neuroligin-positive dendrite regions.
(E) Fibroblasts expressing neurexin-1β-CFP cocultured with hippocampal neurons induced clusters of neuroligin-2, gephyrin, and PSD-95 in contacting dendrites. Within the neurexin contact region as assayed by confocal microscopy, gephyrin clusters were nearly all associated with high concentrations of neuroligin-2, whereas the association of PSD-95 clusters with neuroligin-2-immunopositive regions was more variable.
Scale bars (A)-(C) 10 μm, (D) and (E) 5 μm.

Neurexin Clusters GABA and NMDA Glutamate Receptors
(A and B) Fibroblasts expressing neurexin-1β-CFP cocultured with hippocampal neurons induced clusters of GABA_ARγ2 (A) and NR1 (B) in contacting dendrites. Induced clusters (arrowhead) lacked immunoreactivity for synapsin, unlike endogenous synaptic clusters (arrow).
(C) Neurexin-1β-CFP did not induce clustering of GluR1; only endogenous clusters colocalizing with VGlut1 were observed (arrows). Unlike the other receptors, GluR1 levels are relatively high in all dendrite shafts regardless of fibroblast contact.
(D and E) The number and total integrated intensity of nonsynaptic clusters of GABA_ARγ2 and NR1, but not GluR1, was greater for neuronal processes within areas contacted by fibroblasts expressing neurexin-1β-CFP than mCFP (* p < 0.05, n = 20 cells each).
Scale bar 10 μm.

Mislocalized Expression of Neuroligin Disrupts Postsynaptic Protein Localization and Synaptic Transmission
(A) High level extrasynaptic expression of HA-neuroligin-2 (left panels) but not HA-CD8 control (right panels) in hippocampal neurons disrupts dendritic clustering of gephyrin, GABA receptor, PSD-95, and NMDA receptor. Most of the clusters seen in the left panels are from untransfected dendrites.
(B–D) The number, total area, and total integrated intensity of clusters per dendrite length of gephyrin, GABA receptor, PSD-95, and NMDA receptor are reduced in neurons expressing mislocalized HA-neuroligin-2 compared with HA-CD8 control (* p < 0.001, n = 20 cells each).
(E and F) Representative recordings show reduction in mIPSCs (E) and mEPSCs (F) in neurons expressing mislocalized HA-neuroligin-2 compared to HA-CD8 control.
(G and H) The frequency (G) and amplitude (H) of both mIPSCs and mEPSCs is reduced in neurons expressing high levels of HA-neuroligin-2 compared to HA-CD8 control (* p < 0.01, ** p < 0.05, Kolmogorov-Smirnov test, n = 11–13 cells each; only the neurons exhibiting PSCs were included in the amplitude comparison).
Scale bar 10 μm.

Neuroligin Clusters Glutamate and GABA Synaptic Vesicles; Neurexin Clusters Glutamate and GABA Postsynaptic Scaffolding Proteins
(A and B) Fibroblasts (F) expressing HA-neuroligin-2 cocultured with hippocampal neurons (N) induced clusters of VGlut1 (A) and GAD (B) in contacting glutamatergic and GABAergic axons. The induced clusters of VGlut1 or GAD lacked postsynaptic PSD-95 or gephyrin immunoreactivity (arrowheads) in contrast to endogenous synaptic clusters (arrows).
(C) Fibroblasts (F) expressing neurexin-1β-CFP cocultured with hippocampal neurons (N) induced clusters of gephyrin and PSD-95 in contacting dendrites. Induced clusters (arrowheads) lacked immunoreactivity for the presynaptic antigen synapsin, unlike endogenous synaptic clusters (arrows). As shown in the magnified inset, induced clusters of gephyrin and PSD-95 were partially overlapping but largely distinct.
(D) Coculture of fibroblasts (F) expressing CFP with neurons resulted in only endogenous synaptic clusters (arrows) but no induced clusters of gephyrin or PSD-95 at contacts with dendrites.
(E and F) Number and total integrated intensity of nonsynaptic clusters of gephyrin and PSD-95 was greater for neuronal processes within areas contacted by fibroblasts expressing neurexin-1β-CFP than mCFP (* p < 0.001, n = 40 cells each).
(G) The neurexin-1β LNS domain was essential for synaptogenic activity, but the β-specific region and glycosylated region were not. Numbers indicate the percentage of fibroblasts expressing the indicated construct (extracellular domain shown) associated with nonsynaptic clusters of gephyrin or PSD-95 in a neuron coculture assay (n = 100 cells counted each). The neurexin LNS domain could not be functionally replaced with the agrin LNS domain. Full-length agrin, N-cadherin, or NgCAM had no synaptogenic activity in this assay.
Scale bars 10 μm.