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.

Presynaptic boutons pass through several stages of structural and functional development before they form a mature presynaptic compartment (1). Mature boutons contain an electron-dense matrix of scaffolding proteins called the cytomatrix of active zones (CAZ), which is assembled exclusively at sites of neurotransmitter release and thought to control exocytotic neurotransmitter release from SVs. Key CAZ-proteins are Munc13s, RIMs, CAST/ERC, Piccolo/Aczonin, and Bassoon (2). Immature presynaptic boutons differ from mature ones by several characteristics. For example, immature but not mature boutons of cultured hippocampal neurons require F-actin to prevent dispersal of their constituents (3). Moreover, the number of SVs per bouton that undergo exocytosis, the rate of exocytosis, and the responsiveness of the exocytotic machinery to stimulation are increased in mature compared with immature neurons (4).

It is largely unknown how these presynaptic maturation steps are controlled. However, transsynaptic signaling is likely to be involved. Members of the Neuroligin (Nlgn) family of postsynaptic transmembrane proteins are thought to bind presynaptic transmembrane receptors of the Neurexin family to mediate transsynaptic interactions (5). Expression of Nlgns in non-neuronal cells leads to assembly of the presynaptic machinery in cocultured neurons (6). Overexpression of Nlgns in neurons increases the number of presynaptic boutons contacting the dendrites of transfected neurons (5, 7–9). This effect of Nlgns requires neurotransmission (10). Moreover, synapse numbers are normal, but synapse function is impaired in mutant mice lacking 1 (10) or 3 (11) Nlgn genes, suggesting that Nlgns are critically involved in steps following synapse formation. Neuroligin mutations have been linked to cases of autism (9), and silencing of Neuroligin1 (Nlgn1), an isoform specific for excitatory synapses (10, 12), interferes with long-term potentiation and associative fear memory (13), highlighting the importance of Neuroligins for brain function and behavior. Mechanistically, an essential role for Nlgn1 in regulating postsynaptic NMDA receptor function has emerged (10, 13). Here, we report that Nlgn1 also critically controls key steps of presynaptic bouton maturation.

Results

Nlgn1 Induces F-Actin Independence of CAZ Proteins.

One way of assaying the maturation of presynaptic boutons is to monitor the sensitivity of presynaptic markers to F-actin disrupting drugs. On day 5 in vitro (DIV5), presynaptic accumulations of the SV-marker Synaptophysin and the CAZ-marker Bassoon are dispersed upon treatment with the F-actin disrupting drug Latrunculin A (LatA). After DIV12, these markers are essentially resistant to LatA-treatment. Acquisition of F-actin independence can thus serve as a readout for bouton maturation (3).

We transfected cultured hippocampal neurons with a GFP-tagged version of Nlgn1 (Nlgn1GFP) or with GFP alone on DIV2, treated the cultures with LatA on DIV5, and immunostained for Bassoon. Transfection of Nlgn1GFP caused a 2.3-fold increase of the number of Bassoon puncta on transfected neurons (Fig. 1), consistent with the well-known effect of Nlgn overexpression on bouton number in older cultures. As expected (3), F-actin depolymerization resulted in the loss of nearly all detectable Bassoon puncta in pEGFP-expressing cells (Fig. 1 and Table 1). By contrast, 79% of Bassoon puncta on Nlgn1GFP expressing neurons were resistant to LatA-treatment (Fig. 1E and Table 1). Similarly, 85% of Bassoon puncta were resistant to LatA-treatment in untransfected DIV18 cultures (Table 1). These results indicate that presynaptic boutons formed in immature cultures on Nlgn1 overexpressing neurons have features of mature boutons with respect to the LatA-resistance of Bassoon. In addition, overexpression of Nlgn1 rendered presynaptic Piccolo and RIM1 LatA-resistant, but not Synaptophysin, VAMP2, VGlut2, and VGAT (Table 1), suggesting that this effect of Nlgn1 is specific for the CAZ.

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).

Table 1.

Effects of actin depolymerization on CAZ and SV proteins

	LatA	Puncta/30-μm dendrite
		Bassoon	Piccolo	RIM1	VAMP2	Syphysin	VGlut2	VGat
DIV5	−	5.1 ± 2.0	6.1 ± 2.3	5.85 ± 1.51	6.7 ± 2.1	7.8 ± 1.5	4.08 ± 2.1	7.3 ± 2.4
	+	0.83 ± 0.7***	1.1 ± 0.91**	1.2 ± 0.79**	2.1 ± 1.2**	2.0 ± 0.5*	0.88 ± 0.3*	0.17 ± 0.13*
	LatA R	16%	18%	21%	31%	26%	22%	23%
DIV18	−	20.8 ± 5			13.8 ± 1.9	21.2 ± 2.9	12.2 ± 1.7	9.6 ± 2.4
	+	17.6 ± 3.6ns			7.5 ± 1.3***	20.1 ± 2.2ns	12.3 ± 1.5ns	10.3 ± 1.5ns
	LatA R	85%			54%	95%	101%	107%
DIV5 Nlgn1GFP	−	6.90 ± 1.2	6.33 ± 0.47	6.08 ± 1.8	4.9 ± 1.3	6.2 ± 2.0	3.8 ± 0.23	7.5 ± 1.2
	+	5.44 ± 1.1ns	4.7 ± 0.83ns	4.94 ± 1.6ns	1.7 ± 0.4**	0.9 ± 0.7***	1.0 ± 0.32*	2.1 ± 0.93*
	LatA R	79%	74%	81%	35%	15%	26%	28%
DIV5 pEGFP-F	−	2.96 ± 0.55	3.1 ± 1.15	2.96 ± 1.2	2.3 ± 1.1	2.72 ± 1.2		5.5 ± 1.2
	+	0.7 ± 0.75**	0.9 ± 1.1*	0.98 ± 0.95*	0.7 ± 0.89*	0.8 ± 1.7*		0.86 ± 0.9*
	LatA R	24%	29%	33%	30%	29%		16%

Syphysin, synaptophysin; LatA R, LatA resistance. Numbers are means ± SD, 80–250 puncta (7–15 cells); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; paired t test.

The Intracellular Domain of Nlgn1 Is Necessary for Early CAZ Maturation.

It is well established that Nlgn1 alone, for example, bound to beads or expressed in non-neuronal cells, induces the assembly of a functional presynaptic release apparatus in axons (9). We tested the presynaptic F-actin dependence of such hemisynapses by coculturing Nlgn1GFP-expressing HEK293 cells with DIV5 neurons. LatA-treatment led to a dramatic reduction in the number of Bassoon clusters in Nlgn1-induced hemisynapses (Fig. S1), suggesting that to promote F-actin independence in presynaptic terminals Nlgn1 requires neuron-specific, postsynaptic interactions. To test this notion, we transfected hippocampal neurons with truncated versions of Nlgn1 on DIV2 followed by LatA-treatment on DIV5. The constructs NlgnB through NlgnG contain deletions in the intracellular domain of increasing size (Fig. 2A), but all constructs are properly targeted to the plasma membrane (14). Overexpression of all constructs increased the number of incoming terminals (Fig. 2B). However, only Nlgn1GFP, NlgnA and NlgnB induced F-actin independence of Bassoon, while NlgnC, -F, and- G did not (Fig. 2B). As NlgnG lacks the intracellular domain, it appears that the extracellular Neurexin-binding domain is sufficient to induce presynaptic terminal assembly, while intracellular portions are required for promoting CAZ-maturation.

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).

Nlgn1GFP Promotes the Functional Maturation of Presynaptic Boutons.

Is the Nlgn1-induced acquisition of F-actin independence accompanied by advanced functional maturation? In mature cultures, a larger fraction of boutons responds to a given stimulus than in immature cultures. We stimulated DIV5 neurons using high K⁺-depolarization and identified responsive boutons by measuring the uptake of an antibody directed against the luminal domain of the SV-protein Synaptotagmin1. This antibody is only taken up where SVs undergo cycles of exo-and endocytosis (15–17). The total number of boutons was identified by staining for Piccolo. The fraction of boutons undergoing depolarization induced SV recycling was significantly increased by postsynaptic expression of Nlgn1GFP, but not of NlgnG (Fig. 3).

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.

We next tested whether overexpression of Nlgn1 affected synaptic transmission. We first looked at the responsiveness of spontaneous transmission events (“minis”) to the phorbol ester PMA, which depends on the maturational stage of presynaptic boutons. In particular, mini frequency is increased by PMA application in DIV15, but not in DIV5 cultures (18). As expected, the frequency of minis recorded from untransfected or GFP expressing cells on DIV5 did not increase in response to PMA application. In contrast, PMA application did increase mini frequency in Nlgn1 ovexpressing DIV5 cells (Fig. 4 A and B), indicating advanced functional maturation of presynaptic boutons.

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.

To look at the effect of Nlgn1 overexpression on evoked synaptic transmission, we estimated the probability of neurotransmitter release (P_r) using the open channel blocker MK801, which produces a progressive decrease in postsynaptic currents whose time course depends on P_r (19). We found that NMDA receptor currents could only be recorded reliably in neurons on DIV6 or older. In contrast, after overexpressing Nlgn1 we could reliably record synaptic NMDA currents in DIV5 cultures, suggesting that Nlgn1 overexpression accelerates synaptic maturation. When stimulating synapses at 0.1 Hz in the presence of MK801, the EPSC amplitudes decreased progressively (Fig. 4 C and D). In untransfected DIV6–8 cultures, the response to the second stimulus was more strongly reduced than the subsequent responses, possibly reflecting a transiently high initial P_r, which has been reported for immature synapses in autaptic cultures (20). The remaining part of the decay was monoexponential with a τ of 6.16 ± 0.47 stimuli (n = 7). DIV5 cells overexpressing Nlgn1 had an entirely monoexponential decay curve with τ = 2.85 ± 0.54 stimuli (n = 6; Fig. 4 C–E). Thus, overexpression of Nlgn1 in immature cultures has 2 effects: First, cells become capable of evoked NMDA receptor dependent synaptic transmission. This might involve enhanced pre- or postsynaptic maturation or both. Second, an overall increase in P_r with an apparent loss of an initial high P_r component is caused, indicating a presynaptic change.

To investigate the roles of Nlgn1 in more detail, we looked at parameters of SV recycling. It has been shown using styryl dyes that both the number of recycling SVs per bouton (the “recycling SV pool size”) and the rate of dye release (reflecting the rate of exocytosis) increase with the developmental stage of the culture, in particular after DIV5 (4). We therefore tested these parameters (Fig. 4F and Fig. S2). Overexpression of Nlgn1GFP, but not of NlgnG, increased the recycling SV pool size to levels of DIV14 boutons (Fig. 4F). Similarly, Nlgn1GFP enhanced the rate of dye release, while NlgnG had only a mild effect on release kinetics (Fig. 4G). Thus, Nlgn1GFP increases the number of presynaptic boutons, and induces both structural maturation of the CAZ and the functional maturation of the neurotransmitter release machinery, while an intracellularly truncated Nlgn-construct increases bouton number, but does not influence maturation.

Overexpression of Nlgn1 also affected the localization of synaptic proteins on DIV5 as analyzed by immunofluorescence (Fig. S3). Virtually all accumulations of Bassoon or Piccolo, that is, presumably active zones, were positive for Synapsin 1 and Synaptophysin with and without overexpression of Nlgn1. However, Nlgn1 significantly enhanced the immunofluorescence intensity of Synapsin1 and Synaptophysin clusters at these sites, indicating increased recruitment of these presynaptic proteins or of SVs to synapses. Conversely, overexpression of Nlgn1 did not affect the synaptic immunofluorescence intensity of the postsynaptic proteins PSD95 and GluR1, but enhanced the fraction of Bassoon or Piccolo clusters positive for these postsynaptic proteins (Fig. S3).

Nlgn1 Is Necessary for Distinct Aspects of Presynaptic Maturation.

To test whether Nlgn1 is necessary for presynaptic maturation, we tested the stage of structural and functional maturation of presynaptic boutons from Nlgn1 knock out (KO) mice (11). In DIV13 cultures of Nlgn1 KO mice, P_r was indistinguishable from that determined in wild-type litter mates (τ = 5.59 ± 0.69 stimuli, n = 6; s = 7.18 ± 1.02 stimuli, n = 5, respectively), consistent with results from slice cultures where suppression of the Nlgn1 isoform alone did not affect P_r (21). Likewise, FM4–64 destaining kinetics were not affected (Fig. S4). By contrast, both the LatA-resistance of Bassoon and the recycling SV pool size were reduced to levels of DIV5 neurons in DIV18 Nlgn1 KO cultures (Fig. 5), indicating specific defects in presynaptic maturation in Nlgn1-deficient neurons.

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.

Interestingly, chronic application of the NMDA receptor blocker AP5 from DIV3-DIV18 to standard rat cultures prevented the developmental acquisition of LatA-resistance of Bassoon, i.e., had the same effect as knockout of Nlgn1. AP5 also blocked the aquisition of LatA-resistance of Bassoon in boutons induced by Nlgn1-overexpression between DIV2 and DIV5 (Fig. S5). Thus, this form of structural, presynaptic maturation appears to be regulated by Nlgn1 and NMDA-receptor activity.

Discussion

Nlgns have been implicated in synapse formation, maturation, and function, as well as in cases of autism, but the mechanisms of their action have remained controversial. On the one hand, Nlgns may have a role in synapse formation, as overexpression of Nlgns increases and knock-down of Nlgns decreases the number of synapses in neuronal cultures (9). On the other hand, mice lacking Nlgns1–3 have a normal number of synapses and Nlgns do have a role in synapse function, as evident from the impairment of synaptic transmission in KO animals (10, 11). Here we specifically tested the hypothesis that Nlgns signal to presynaptic boutons to promote defined steps of presynaptic bouton maturation. We predicted (a) that overexpression of Nlgns in immature cultures would enhance the number of functional presynaptic boutons—as well-established for advanced cultures stages—and (b) that the developmental characteristics of these boutons would resemble those of mature cultures. To set a well-defined time window, we specifically tested presynaptic parameters that are known to remain at an immature level until DIV5 and aquire mature features after DIV10 (3, 4, 18). A key finding of our study is that presynaptic boutons formed on Nlgn-overexpressing neurons in immature cultures have features of mature boutons.

Control of Presynaptic Maturation by Nlgn1.

Overexpression of Nlgn1 on DIV5 promoted presynaptic maturation in 5 assays: First, LatA-resistance of CAZ-markers was induced. Second, the size of the recycling SV pool was increased to mature levels. Third, the rate of stimulation-induced dye-release from SVs was enhanced. Fourth, the fraction of boutons responding to high K⁺ stimulation was increased. Fifth, spontaneous synaptic transmission became responsive to PMA-augmentation. These 5 parameters have been shown to develop only after DIV5 in untreated cultures and are considered hallmarks of presynaptic maturation (3, 4, 18). Moreover, on DIV5, NMDA-receptor responses could only be evoked in Nlgn1-overexpressing, but not in control neurons. This effect of Nlgn1 could arise from the recruitment of functional NMDA-receptors to synaptic sites, from induction of presynaptic release competence, or both. The Nlgn1-induced increase in release probability, measured in the MK801 assay, and the increased percentage of functional active zones, determined in the synaptotagmin-uptake assay, are consistent with a presynaptic contribution to this effect.

Release probability was enhanced in the overexpression paradigm, but unaffected in KO cultures. This is consistent with results from acute perturbation experiments on brain slices, which showed that presynaptic release probability is only decreased upon simultaneous perturbation of 3 Nlgn isoforms, suggesting redundant control of release probability by Neuroligins (21). In contrast, the LatA-resistance of Bassoon and the recycling SV-pool size were enhanced to mature levels upon overexpression of Nlgn1 on DIV5, and remained at immature levels in Nlgn1 KO cultures on DIV18, indicating an essential role of Nlgn1 for these 2 aspects of maturation. This adds an essential function of Nlgn1 in presynaptic maturation to its role in maintaining or regulating postsynaptic NMDA receptor function (10, 13, 22, 23).

Effects of Nlgn1 on Active Zones and SVs.

We show that Nlgn1 is required for the LatA-resistant, that is, F-actin independent anchoring of Bassoon, Piccolo, and Rim1. The molecular mechanisms underlying the developmental acquisition of presynaptic LatA-resistance are not known. LatA-resistance could be a corollary of increasing linkage between pre- and postsynaptic scaffolds. In this scenario, Nlgn1 would be essential to establish this level of pre- and postsynaptic anchoring. Alternatively, LatA-resistance could reflect specific interactions within the CAZ induced by Nlgn1 that might have inherent functional consequences. Overexpression of Nlgn1 did not speed up the acquisition of LatA-resistance of synaptic vesicle markers, although these markers develop F-actin independence in parallel with Bassoon during normal culture development (3). This indicates that during the development of neuronal cultures 2 separate pathways lead to the acquisition of F-actin independence of SVs and the CAZ, respectively, with CAZ-related maturation being under the control of Nlgn1.

Do we have evidence for functional consequences of this effect of Nlgn1 on the architecture of the CAZ? Nlgn1 induced the responsiveness of presynaptic boutons to PMA-augmentation. PMA is an analogue of diacylglycerol (DAG) which specifically enhances neurotransmitter release from a set of SVs called the readily releasable pool, and this is primarily mediated by PMA/DAG induced recruitment of the CAZ-protein Munc13 to the plasmamembrane (24). This allows for 2 conclusions: first, the readily releasable pool of SVs is one of the targets of Nlgn1. Second, Munc13, being the primary mediator of the PMA/DAG effect, is a likely effector of the action of Nlgn1. We propose that the molecular mechanisms that underlie Nlgn1-induced LatA-resistance of the CAZ might anchor Munc13 in a way that allows its PMA/DAG induced action on SVs to take place.

In addition, Nlgn1 appears to regulate other SV pools. Using the high K⁺ maximal stimulation protocol for FM-dye uptake and release we found that the total recycling pool size was reduced by 50% in Nlgn KO cultures. Moreover, Nlgn1 overexpression leads to increased fluorescence of the SV markers Synapsin and Synaptophysin, consistent with increased recruitment of SVs. A developmental increase in the number of reserve pool vesicles has been reported to be a step in presynaptic maturation (25), and our data argue for an essential role of Nlgn1 in promoting this event.

Distinct Effects of Nlgn1 on Presynaptic Assembly and Maturation.

Nlgn1 expressed in non-neuronal cells induces the formation of functional presynaptic boutons in axons contacting transfected cells (9). We find here that in such a co-culture assay Nlgn1 does not promote CAZ-maturation, indicating that enhancing bouton maturation requires neuron-specific postsynaptic interaction partners. Using purely neuronal cultures we found that the extracellular domain of Nlgn1 together with its transmembrane region is sufficient to enhance the number of boutons, but intracellular portions are required to induce structural and functional maturation in these boutons. Thus, the positive effects of recombinant Nlgn1 on presynaptic bouton number and bouton maturation can be assigned to distinct domains of Nlgn1 and, presumably, distinct molecular mechanisms.

Our study is consistent with work performed on cholinergic chick ciliary ganglion neurons, where the Neurexin-binding domain of Nlgn was sufficient to induce recruitment of presynaptic proteins to nascent boutons, while the 14 membrane-proximal amino acids of the intracellular domain were needed to align the boutons with receptor clusters and therefore allow for synaptic transmission (26). Our study adds 2 important aspects to this notion. First, the extracellular domain of Nlgn1 with its transmembrane anchor is sufficient to generate functional presynaptic boutons. Second, an intracellular portion of Nlgn1 significantly longer than 14 amino acids was necessary to promote the structural and functional maturation of these boutons. In our assay, NlgnB (amino acids 1–828) was able to, and NlgnC (amino acids 1–809) was not able to promote bouton maturation. A candidate binding partner for the maturation-promoting regions could be S-SCAM, a beta-Catenin binding protein which binds amino acids 793–828 of Nlgn (27). Interestingly, N-Cadherin may contribute to the aquisition of LatA-resistance (28), raising the possibility of a functional interaction between Neurexin-Nlgn-based and N-Cadherin-based cell adhesion in presynaptic maturation. Taking together the data from the different model systems, we hypothesize that in neuronal cultures Neurexin-binding of Nlgn1 drives the assembly of functional yet immature boutons, a membrane-proximal intracellular region of Nlgn1 aligns pre- and postsynaptic terminals, and a more distal region of the intracellular domain of Nlgn1 identified here induces structural and functional bouton maturation.

We also find that the developmental acquisition of F-actin independence of Bassoon is activity-dependent, as it is blocked by chronic application of the NMDAR-blocker AP5. This highlights that the CAZ undergoes maturation events and that these events respond to activity. AP5 also blocked the action of Nlgn1-overexpression in promoting F-actin independence of Bassoon in young neurons. Moreover, chronic NMDA receptor blockade and knockout of Nlgn1 each prevented the developmental acquisition of F-actin independence of Bassoon to a similar, large degree. This raises the possibility that Nlgn1 and NMDA receptor activity might act in the same pathway of CAZ-maturation.

Presynaptic boutons are generated—and must be expected to undergo subsequent maturation—during pivotal stages of brain development, including initial wiring of the brain, and after adult neurogenesis, as well as during remodeling of presynaptic input in regions undergoing structural plasticity (29). Therefore, it is important to understand the molecular mechanisms and regulation of bouton maturation. In addition, isoforms of Nlgn have been implicated in the pathogenesis of autism. Our study pinpoints molecular mechanisms of bouton maturation and Nlgn-based signaling that might contribute to such disorders.

Methods

Briefly, dissociated hippocampal cultures were prepared from E19 Wistar rats or newborn mice as described (30) and were cultured at a density of 60,000 cells/cm² on poly-l-lysine-coated coverslips in neurobasal medium supplemented with B27 and glutamine (Invitrogen). Transfection was performed on DIV2 using calcium phosphate (30) and 5 types of experiments were carried out at DIV5: 1) LatrunculinA treatment and immunocytochemistry, 2) Vesicle labeling with Synaptotagmin1 antibodies, 3) Fluorescent imaging with FM dyes, 4) Estimation of P_r using MK801, and 5) whole cell recordings in the presence of PMA. All protocols are described thoroughly in SI Methods.