Postsynaptic Neuroligin1 regulates presynaptic maturation

Abstract

Presynaptic nerve terminals pass through distinct stages of maturation after their initial assembly. Here we show that the postsynaptic cell adhesion molecule Neuroligin1 regulates key steps of presynaptic maturation. Presynaptic terminals from Neuroligin1-knockout mice remain structurally and functionally immature with respect to active zone stability and synaptic vesicle pool size, as analyzed in cultured hippocampal neurons. Conversely, overexpression of Neuroligin1 in immature neurons, that is within the first 5 days after plating, induced the formation of presynaptic boutons that had hallmarks of mature boutons. In particular, Neuroligin1 enhanced the size of the pool of recycling synaptic vesicles, the rate of synaptic vesicle exocytosis, the fraction of boutons responding to depolarization, as well as the responsiveness of the presynaptic release machinery to phorbol ester stimulation. Moreover, Neuroligin1 induced the formation of active zones that remained stable in the absence of F-actin, another hallmark of advanced maturation. Acquisition of F-actin independence of the active zone marker Bassoon during culture development or induced via overexpression of Neuroligin1 was activity-dependent. The extracellular domain of Neuroligin1 was sufficient to induce assembly of functional presynaptic terminals, while the intracellular domain was required for terminal maturation. These data show that induction of presynaptic terminal assembly and maturation involve mechanistically distinct actions of Neuroligins, and that Neuroligin1 is essential for presynaptic terminal maturation.

Fig. 1.

Overexpression of Nlgn1 induces F-actin independence of Bassoon in young neurons. Cultured hippocampal neurons were transfected with Nlgn1GFP or pEGFP on DIV2, treated with Latrunculin A (LatA) on DIV5, and immunostained for Bassoon. Presynaptic clusters of Bassoon are lost upon treatment with LatA (A and B). Overexpression of Nlgn1GFP increases the number of Bassoon puncta along dendrites. These puncta do not disassemble upon LatA treatment (C and D). (Scale bar, 15 μm.) (E) Quantification of the number of Bassoon puncta per 30-μm dendrite. Mean ± SEM; n = number of exp., 8–10 cells per exp.; **, P < 0.01, paired t test. Increase of Bassoon puncta induced by Nlgn1GFP overexpression was verified with 1-way ANOVA (***, P < 0.0001).

Fig. 2.

The extracellular domain of Nlgn1-GFP is sufficient to induce presynaptic accumulation of Bassoon, but intracellular regions are required for presynaptic maturation. (A) Schematic representation of Nlgn1 constructs. TM, Transmembrane domain; Black bar, extracellular domain. (B) Number of Bassoon puncta on transfected neurons with and without LatA-treatment on DIV5. All constructs increase synapse numbers, but NlgnC, -F, and -G fail to induce CAZ maturation. Mean ± SEM; n = number of exp., 8–10 cells per exp.; ***, P < 0.001, **, P < 0.01, *, P < 0.05, paired t test (verified by 1-way ANOVA test).

Fig. 3.

Overexpression of Nlgn1GFP, but not of NlgnG or GFP, on DIV5 increases the percentage of active zones that recycle SVs upon depolarization. Vesicle recycling was assayed by uptake of an antibody against the luminal domain of Synaptotagmin1 (red). Presynaptic terminals were immunostained for the active zone marker Piccolo (blue). (Scale bars, 5 μm.) (A–C) Image of a GFP expressing neuron (green in C). The majority of Piccolo puncta show no detectable staining (arrows point to examples) or weak staining (i.e., close to background staining; arrowhead) for Synaptotagmin-antibody uptake. (D–F) In contrast, virtually all Piccolo puncta on a Nlgn1GFP expressing neuron colocalize with sites of antibody uptake. (G) Quantification of the effects of Nlgn1GFP and NlgnG compared to controls. Mean ± SEM; n = the number of puncta (15–17 cells); *, P < 0.05, paired t test.

Fig. 4.

Overexpression of Nlgn1 in immature neurons induces functional bouton maturation. (A and B) Overexpression of Nlgn1 makes presynaptic terminals responsive to PMA on DIV5. (A) Sample traces showing miniature EPSCs before and after addition of PMA. (B) Frequency but not amplitude of miniature EPSCs was increased by Nlgn1GFP; n = number of cells, **, P < 0.01 t test. (C) Overexpression of Nlgn1 in immature neurons results in an increase in release probability. Sample traces of pharmacologically isolated NMDAR-EPSCs recorded in the presence of MK801 (10 μM) from immature (DIV 5–8) cultures under control conditions and overexpression of Nlgn1. Key indicates stimulus number. (D) Mean amplitudes of NMDAR-EPSCs across multiple cells (n = 6–7), normalized to the first EPSC obtained in the presence of MK801 (Note: For better illustration we depicted the relative amplitudes of stimulus 2 with a slight offset). (E) Decay time constants of single exponential fits calculated for each cell. In case of immature untreated neurons only the slow component starting from stimulus 3 was fitted (**, P < 0.01 unpaired t test). (F and G) Overexpression of Nlgn1GFP but not GFP or NlgnG, enhances the recycling SV pool size and the rate of FM dye release on DIV5 significantly. (F) Quantification of the recycling SV pool size; means ± SEM; n = number of puncta (8–10 cells); **, P < 0.01, paired t test. (G) Time course of FM4–64 unloading; means ± SEM.

Fig. 5.

Neuroligin 1 is required for distinct aspects of structural and functional maturation of presynpatic boutons. (A) Nlgn1 KO has no effect on release probability in mature cultures. Left: Sample traces of pharmacologically isolated NMDAR-EPSCs recorded in the presence of MK801 (10 μM) from cultures derived from Nlgn1 KO mice and wild type litter mates (DIV 13). Key indicates stimulus number. Middle: Mean amplitudes of NMDAR-EPSCs across multiple cells (n = 5–6), normalized to the first EPSC obtained in the presence of MK801. Right: Decay time constants of single exponential fits calculated for each cell (n.s. = P > 0.05 unpaired t test). (B) Immunofluorescence images showing loss of synapses after LatA-treatment of DIV18 neurons from Nlgn1 KO mice, but not from WT mice (Scale bar, 20 μm). (C) Quantification showing a reduction in the number of F-actin independent Bassoon puncta in DIV18 cultures from Nlgn1 KO mice. Mean ± SEM; n = number of exp., 10 cells per exp., *, P < 0.05, paired t test. (D) The size of the pool of actively recycling synaptic vesicles is reduced in boutons of Nlgn1 KO cultures. Mean ± SEM; n = number of puncta (10 cells); *, P < 0.05, paired t test.