Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses

Abstract

At the synapse, presynaptic membranes specialized for vesicular traffic are linked to postsynaptic membranes specialized for signal transduction. The mechanisms that connect pre- and postsynaptic membranes into synaptic junctions are unknown. Neuroligins and beta-neurexins are neuronal cell-surface proteins that bind to each other and form asymmetric intercellular junctions. To test whether the neuroligin/beta-neurexin junction is related to synapses, we generated and characterized monoclonal antibodies to neuroligin 1. With these antibodies, we show that neuroligin 1 is synaptic. The neuronal localization, subcellular distribution, and developmental expression of neuroligin 1 are similar to those of the postsynaptic marker proteins PSD-95 and NMDA-R1 receptor. Quantitative immunogold electron microscopy demonstrated that neuroligin 1 is clustered in synaptic clefts and postsynaptic densities. Double immunofluorescence labeling revealed that neuroligin 1 colocalizes with glutamatergic but not gamma-aminobutyric acid (GABA)ergic synapses. Thus neuroligin 1 is a synaptic cell-adhesion molecule that is enriched in postsynaptic densities where it may recruit receptors, channels, and signal-transduction molecules to synaptic sites of cell adhesion. In addition, the neuroligin/beta-neurexin junction may be involved in the specification of excitatory synapses.

Figure 1

Specificity of mAbs to neuroligin. (A) Immunoblots of rat brain homogenates and COS cells expressing neuroligins 1, 2, and 3 (10 μg per lane) probed with mAbs 4C12, 5G10, and 4F9. (B) Immunoblots of mouse brain homogenates from homozygous (−/−) and heterozygous (+/−) neuroligin 1-mutant mice and wild-type control mice (+/+).

Figure 2

Light-microscopic localization of neuroligin 1 in rat brain. Shown are sections of the overall neocortex (A) or of layers 4 and 5 of the neocortex (B). Numbers indicate cortical layers. Low-magnification view of the entire hippocampal formation (C) or high-magnification view of the apical dendrites of pyramidal neurons from the CA1 region (D). DG, dentate gyrus; ml, molecular layer; pl, pyramidal cell layer; and sr, stratum radiatum. All sections were stained with antibody 4C12 and developed by using horseradish peroxidase-labeled secondary antibodies. [Bar = 100 μm (A, B, and D) or 250 μm (C).]

Figure 3

Expression of neuroligin 1 in development. Brain homogenates from rats of the indicated age were analyzed by immunoblotting with antibodies to neuroligins, PSD-95, NMDA-receptor type 1 (NMDAR1), and msec7. Expression levels were quantified with iodinated secondary antibodies at the following ages: E12 and E16, embryonic days 12 and 16; 0, day of birth; P1–P60, postnatal days 1–60.

Figure 4

Quantitative immunogold electron microscopy of neuroligin 1 and synaptophysin 1. (A) Representative ultrathin sections from rat neocortex stained with antibodies to neuroligin 1 (a–f) and synaptophysin (g). Neuroligin 1 immunoreactivity decorates synaptic clefts (a–e) and postsynaptic densities (f). Arrowheads indicate postsynaptic densities. (Bars = 0.1 μm.) (B) Quantitation of gold particles in randomly chosen sections labeled with antibodies to neuroligin 1 or synaptophysin. Total number of gold particles identified in the indicated subcellular compartments are given. Gold particles at locations that are not recognizable were designated nonassignable.

Figure 5

Confocal micrographs of brain sections double labeled for neuroligin 1 and GluR2/3 glutamate receptors (A–I) or for GABA_Aβ receptors and glutamate receptors (J–R). Note the colocalization of GluR2/3 receptors (a marker for excitatory synapses) with neuroligin 1 in somata and dendrites of pyramidal neurons in neocortex and hippocampus and in Purkinje cells of the cerebellum (A–I). GABA_Aβ receptors, in contrast, are segregated (J–R). (Bar = 100 μm.)