The soluble neurexin-1β ectodomain causes calcium influx and augments dendritic outgrowth and synaptic transmission

Classically, neurexins are thought to mediate synaptic connections through trans interactions with a number of different postsynaptic partners. Neurexins are cleaved by metalloproteases in an activity-dependent manner, releasing the soluble extracellular domain. Here, we report that in both immature (before synaptogenesis) and mature (after synaptogenesis) hippocampal neurons, the soluble neurexin-1β ectodomain triggers acute Ca2+-influx at the dendritic/postsynaptic side. In both cases, neuroligin-1 expression was required. In immature neurons, calcium influx required N-type calcium channels and stimulated dendritic outgrowth and neuronal survival. In mature glutamatergic neurons the neurexin-1β ectodomain stimulated calcium influx through NMDA-receptors, which increased presynaptic release probability. In contrast, prolonged exposure to the ectodomain led to inhibition of synaptic transmission. This secondary inhibition was activity- and neuroligin-1 dependent and caused by a reduction in the readily-releasable pool of vesicles. A synthetic peptide modeled after the neurexin-1β:neuroligin-1 interaction site reproduced the cellular effects of the neurexin-1β ectodomain. Collectively, our findings demonstrate that the soluble neurexin ectodomain stimulates growth of neurons and exerts acute and chronic effects on trans-synaptic signaling involved in setting synaptic strength.

In the neuronal survival assay, Neurexide displayed a significant survival effect on hippocampal neurons, whereas a noticeable (but statistically insignificant) trend in cerebellar granule neurons survival was seen (Supplementary Fig. S1e). As for NX-1βe, the stimulation of outgrowth by Neurexide in hippocampal neurons was inhibited by ω-conotoxin MVIIA ( Supplementary Fig. S2f), indicating that Neurexide promotes outgrowth in a Ca 2+ -dependent manner similar to that of NX-1βe. We finally tested whether Neurexide interacts with NL-1. Surface plasmon resonance analysis indeed showed that this interaction occurs with sub-μM affinity (Supplementary Fig. S2g, estimated K D = 480 nM). The lower affinity compared to full-length NX-1βe correlates with the higher concentration of Neurexide needed for optimal effect (Fig. 2d). The neuritogenic effect of NX-1βe was absent in rat neurons after knock down of NL-1 (KD; cells transfected with the shRNA containing p-GFP-V-RS vector were identified by their green fluorescence), or in NL-1 knockout (KO) mouse neurons, compared to wildtype (WT wildtype, Htz heterozygous; n = 4-7 cultures). (d) Fluorescence micrographs of an immature neuron (DIV 1) soma and a piece of neurite from the same cell before NX-1βe application (left), during (middle), and after wash-out (right). The cell was loaded with Fura-2AM and shown is the ratio of fluorescence after 340/380 nm excitation, which reports on [Ca 2+ ] i . Scale bar = 3 µm (soma), 1 µm (neurite). (e-g) Quantification of 340/380 fluorescence ratios in soma and neurites before, during, and after 20 s exposure to NX-1βe shows a reversible increase in cytoplasmic [Ca 2+ ]. (e) The omission of Ca 2+ from the solution (0 mM Ca 2+ ) abrogated the [Ca 2+ ] i increase, which was also seen upon addition of the Cd 2+ (f). (g) The N-type Ca 2+ channel blocker ω-conotoxin blocked most Ca 2+ -influx.
Scientific Reports | (2020) 10:18041 | https://doi.org/10.1038/s41598-020-75047-z www.nature.com/scientificreports/ In sum, the effect of the NX-1β ectodomain on neurite outgrowth and survival is mimicked by a minimal peptide modeled after the NX-1β:NL1 binding site, indicating that we have identified the minimal region of the ectodomain required for this effect.
The NX-1β ectodomain acutely enhances glutamatergic synaptic transmission. After having established that both the NX-1β ectodomain and the synthetic peptide Neurexide affect neuritogenesis, we set out to investigate whether the compounds affect synaptic transmission in already formed synapses. To this end, we used rat hippocampal glutamatergic neurons grown in autaptic culture for 10-14 days 53 . Since only one autoinnervating neuron is present per glial island, differences in neuronal survival will not affect the results in this preparation. In voltage clamp experiments individual action potentials (APs) are evoked upon brief depolarization, while spontaneous APs are effectively prevented when keeping the membrane potential at -70 mV during inter-stimulus intervals. Therefore, it is possible to assess both evoked postsynaptic currents and mini events in the same experiment without the use of tetrodotoxin (TTX). After establishing a whole-cell configuration, the neurons were stimulated by AP pairs (inter-stimulus interval 50 ms) every 20 s. First evoked EPSC (eEPSC) amplitude and Paired-Pulse Ratio (PPR) were quantified for each stimulus pair, and miniature EPSCs (mEPSCs) were analyzed in-between stimuli. We acutely exposed rat glutamatergic neurons to NX-1βe using a local superfusion multibarrel system during the recording (concentration ~ 55 pM, similar to the concentration causing maximal stimulation of neuritogenesis, Fig. 1a). Strikingly, local application of NX-1βe acutely enhanced synaptic transmission manifested as an increased frequency of mEPSC release (Fig. 3a1) and an increase in eEPSC amplitude (Fig. 3a3). This occurred concomitantly with a decrease in PPR (Fig. 3a4), indicative of an increase in synaptic release probability 54 . The mEPSC amplitude remained unchanged (Fig. 3a2). Importantly, the effects of NX-1βe were reversible upon wash ( Fig. 3a1-4, Supplementary Fig. S4), indicating that this type of synaptic enhancement requires continued presence of NX-1βe. The increase in mEPSC frequency and decrease of PPR indicate that the effect of NX-1βe is presynaptic. Note that the mEPSC frequency sharply increases after each paired stimulation due to increases in calcium concentrations, resulting in a sawtooth pattern. During NX-1βe application the activity-induced increase www.nature.com/scientificreports/ in mEPSC frequency is even more pronounced (higher peaks) and lasts longer (slower relaxation), indicating that NX-1βe affects presynaptic calcium homeostasis. Indeed, when glutamatergic neurons were incubated with BAPTA-AM to chelate Ca 2+ , the vast majority of mEPSCs were blocked, also during NX-1βe application (Supplementary Fig. S4). This shows that mEPSCs are Ca 2+ -dependent both in the presence and absence of NX-1βe.
Scientific Reports | (2020) 10:18041 | https://doi.org/10.1038/s41598-020-75047-z www.nature.com/scientificreports/ eEPSC amplitude and PPR, which establishes the effect of NX-1βe also in mouse neurons. In contrast, in neurons from NL-1 KO littermates the effects were largely abrogated ( Fig. 3b1-4), indicating that the effect mostly depends on NL-1 expression. Strikingly, application of our peptide Neurexide (10 μM, Supplementary Fig. S5) mimicked the effects of NX-1βe on mEPSC frequency, eEPSC amplitude and PPR, whereas again the mEPSC amplitude was unaffected. Overall, our data show that the NX-1β ectodomain acutely enhances synaptic function in glutamatergic neurons through an increase in release probability. This increase is mimicked by Neurexide emulating the NX1β:NL-1 interaction site and therefore it is not caused by the intracellular part of NX-1β (see "Discussion"). This shows that NX1βe can modify synaptic efficacy on a time scale of seconds in a NL1-dependent manner.
The NX-1β ectodomain causes Ca 2+ -influx via NMDA-receptors. To identify the intracellular events, which lead to increased presynaptic release upon exposure to NX-1βe, we loaded autaptic glutamatergic neurons with Fura-2 and combined electrophysiological measurements with fluorescence imaging. Application of NX-1βe rapidly led to an increase in intracellular [Ca 2+ ], measured as an increase in 340/380 nm fluorescent ratio in the cell body and in dendrites ( Fig. 4a3-4). Note, the increase in mEPSC frequency and eEPSC amplitude were verified in the same cells ( Fig. 4a1-2). Previous experiments show that NL-1 interacts with and stabilizes NMDA-receptors at the synapse 30,55 . Therefore, we asked if the source of the Ca 2+ increase might be influx through NMDA-receptors. Indeed, superfusion with the NMDA-blocker AP-5 abolished both the NX-1βe induced increase in intracellular [Ca 2+ ] ( Fig. 4b3-4), and the effect of NX-1βe on mEPSC frequency and eEPSC amplitude (Fig. 4b1,b2). Note that AP5 applied alone already reduced Ca 2+ influx during AP stimulation (Fig. 4b4), indicating that our experimental conditions do allow for NMDA-receptor stimulation. Thus Ca 2+ -influx through NMDA-receptors is a prerequisite for the stimulatory effect of NX-1βe on presynaptic release.
NMDA-receptors are most commonly found postsynaptically, where they interact with NL-1, but they can also be expressed presynaptically 56 . Given the finding that NX-1βe affects presynaptic release and N-type VGCC in developing neurons, we wanted to investigate the effect of NX-1βe on presynaptic [Ca 2+ ], which could be due to presynaptic NMDA-receptors 56 or VGCCs. An effect on Ca 2+ channels could be expected if the added NX-1βe interferes with presynaptic NMDA-receptors or Ca 2+ channels 24,57 . Fura-infusion into autaptic neurons does not allow for distinction between pre-and postsynaptic compartments, so in other experiments we expressed a genetically encoded Ca 2+ -sensor fused to synaptophysin, syGCaMP2, which is targeted to synaptic vesicles 58 . Patching the cells and stimulating with short AP trains (10 stimuli @ 40 Hz) before, during and after NX-1βe application revealed clear increases in fluorescence, as expected ( Supplementary Fig. S6). We found again NX-1βe increased eEPSC amplitude and concomitantly decreased PPR ( Supplementary Fig. S6d,f). However, NX-1βe application did not modify the activity dependent fluorescence increase (ΔF/F), indicating that AP-induced increases in [Ca 2+ ] i were unaffected. However, the basal fluorescence slightly increased upon application of NX-1βe, suggesting that resting presynaptic [Ca 2+ ] was mildly affected by NX-1βe ( Supplementary Fig. S6c). The signal-to-noise relationship of a genetically expressed Ca 2+ indicator is generally less than that of Fura-2, therefore small changes in presynaptic [Ca 2+ ] i might be underestimated. Together, these data support the notion that [Ca 2+ ] i is increased by NX-1βe, which leads to a potentiation of synaptic release (see "Discussion").
Long-term (hours) exposure to the NX-1β ectodomain homeostatically down regulates the readily-releasable pool of vesicles. Previous investigations making use of the NX-1βe added to neuronal cultures reported a decrease in mEPSC frequency 59 , which correlated with impaired NL-1-dependent synapse formation, presumably due to inhibition of the NX1:NL1 interaction 22,59 . In those experiments, the NX-1β ectodomain was added during the period of active synaptogenesis over longer time (2-3 days) and at higher concentrations than in the present study. The different findings prompted us to investigate whether the effect of the ectodomain might be time or activity dependent. Indeed, after exposure of autaptic neurons to the NX-1βe for 10 days during the period of synaptogenesis (from DIV 1 to 10), the mEPSC frequency and eEPSC amplitudes were severely reduced ( Supplementary Fig. S7a,d). In contrast, the mEPSC amplitude and decay time were unchanged ( Supplementary Fig. S7b,c), indicating a presynaptic effect. In parallel experiments, NX-1βe was applied for only 2 h to mature cultures to investigate synaptic adaptation independent of synapse development. Intriguingly, 2 h of NX-1βe exposure of mature neurons led to identical changes in synaptic features (DIV 10-14 days; Supplementary Fig. S7), while overnight exposure to NX-1βe did not affect synapse number or neuronal morphology (Supplemental Fig. S10). Taken together, the effects seen here are not due to NX-1βe induced differences in synaptogenesis (see also below).
The acute upregulation of synaptic strength by NX-1βe identified above might indirectly lead to down-regulation of vesicular release, in a homeostatic plasticity regulative manner which counteracts the global increase in synaptic strength 60,61 . To investigate this point, we added either TTX or a mixture of CNQX and AP5, to block ionotropic glutamate receptors, immediately before adding the NX-1β ectodomain and compared these groups to neurons that were treated only with NX-1βe for 2 h. Also in this independent experimental series 2 h of NX-1βe exposure-when added alone-reduced mEPSC frequency, and eEPSC amplitude (Fig. 5a,d). mEPSC amplitude and decay were unaffected (Fig. 5b,c). Strikingly, application of either TTX or CNQX/AP5 abrogated the effects of NX-1βe (Fig. 5a,d), indicating that neural activity and ionotropic glutamatergic neurotransmission are both prerequisites for long-term NX-1βe induced homeostatic down-regulation of synaptic strength.
Application of hypertonic sucrose solution can be used to probe the readily-releasable pool (RRP) of vesicles 62 . The Rosenmund and Stevens experiments showed that the RRP was depressed in neurons treated with long-term NX-1βe (Fig. 5e), while the vesicular release probability (eEPSC charge divided by the sucrose pool) remained unchanged ( Fig. 5f. The same was found in another independent set of experiments ( Supplementary Fig. S7e www.nature.com/scientificreports/ The reduction in RRP was complete within 2 h of exposure to NX-1βe and did not change, even when NX-1βe was present continuously for 10 days ( Supplementary Fig. S7e,f). This reduction of RRP size sufficiently explains the inhibition of spontaneous and evoked synaptic transmission caused by long-term NX-1βe exposure.
Overall, these results show that the calcium dependent increase in vesicular release probability induced by acute addition of NX-1βe leads to a secondary long-term activity-dependent down-regulation of the RRP.
Endogenous metalloproteases and α-secretases regulate the size of the readily-releasable pool. If shedding of ectodomains from endogenous proteins (including, but not limited to, NX-1β) is involved in setting the synaptic strength in neuronal cultures, then blocking metalloproteases to prevent shedding should induce compensation of the synaptic strength in the opposite direction. We therefore incubated mouse autaptic cultures (DIV 12-14 days) overnight with two broad-spectrum metalloprotease inhibitors, GM6001 (38 µM) and TAPI-1 (30 µM), which have been shown to prevent NX-1β (and NL-1) ectodomain shedding 35 . Other culture dishes from the same neuronal preparations were incubated only with NX-1βe, or left as controls. Strikingly, metalloprotease inhibitors caused an increase in mEPSC frequency, eEPSC amplitude and RRP size beyond control values (Fig. 6a-c), whereas NX-1βe again caused a decrease in those parameters (see Supplementary Fig. S8  . (a1-4) Autaptic glutamatergic neurons were subjected to patch clamp with (membrane impermeable) Fura-2 in the pipette. Electrophysiological and fluorescence measurements were performed simultaneously. NX-1βe was applied during the grey shaded time period, which led to increased mEPSC frequency (a1), increased eEPSC amplitude (a2), increased 340/380 fluorescence ratio in cell soma (a3), and in dendrites (a4), indicative of an increase in cytoplasmic [Ca 2+ ]. Note that the increase in fluorescence ratio was much smaller in these cells compared to immature neurons (Fig. 1). (b1-b4) Blocking NMDA receptors with AP5 during NX-1βe application blocked the increase in mEPSC frequency (b1), and eEPSC amplitude (b2), as well as the increase in 340/380 fluorescence amplitude in cell soma (b3) and dendrites (b4). These data demonstrate that Ca 2+ -influx via NMDA-receptors is a prerequisite for upregulating synaptic strength.
Scientific Reports | (2020) 10:18041 | https://doi.org/10.1038/s41598-020-75047-z www.nature.com/scientificreports/ for additional parameters from this experiment, and Supplementary Fig. S9 for similar findings in rat neurons). Immunostaining of neurons for synaptophysin and MAP2 did not show any significant differences in synaptic number or dendritic branching after overnight exposure to NX-1βe, or GM6001/TAPI-1 ( Supplementary  Fig. S10). These findings are consistent with the notion that regulated shedding of endogeneous ectodomains can be involved in bidirectional alterations of synaptic strength. We next investigated whether NL-1 is a prerequisite for the physiological effects of the NX-1βe and the effect of metalloprotease inhibitors. Notably, all effects were abolished in neurons cultured from NL-1 KO mice (Fig. 6a-c). These data show that NL-1 is an obligatory intermediary key player for metalloproteases to modulate presynaptic strength, both acutely and during long-term exposure. However, it does not rule out that other metalloprotease targets-of which there are many in the synapse 41 -could also be required.   Figure 5. Activity-dependent reduction in the Readily-Releasable Pool (RRP) of vesicles by long-term exposure to NX-1βe. (a-f) Exposure to NX-1βe for ~ 2 h resulted in a decrease in mEPSC frequency (a), a decrease in eEPSC amplitude (d), and a decrease in RRP size as estimated by sucrose application (e). The mEPSC amplitude (b), and decay time (c), as well as the vesicular release probability (P ves ) were unchanged (f). When NX-1βe exposure was combined with either Tetrodotoxin (TTX) to block neuronal activity or CNQX/AP5 to block ionotropic glutamatergic neurotransmission, the effect of long-term NX-1βe exposure was abrogated.

Discussion
This study delineates roles for the NX-1β ectodomain in neuronal development (dendrite formation and neuronal survival), and in acute and chronic modulation of synaptic transmission (temporarily increasing presynaptic efficacy, while concurrently triggering down-regulation of the RRP to rebalance synaptic strength). Neurite outgrowth is a complex process accompanying neuronal differentiation. This process involves different CAMs (e.g. NCAM, N-cadherin and L1) and neurotrophic factors (e.g. NGF and BDNF) and leads to intracellular events, including activation of receptor tyrosine kinases, Ca 2+ -influx, inhibition of actin-capping proteins and altered actin dynamics 63,64 . Our data obtained from immature neurons indicate that NX-1β and NL-1 expressed before synaptogenesis 45 can be recruited for neuritogenesis. Indeed, it has been shown that NL-1 with the B-insert induces neurite outgrowth through interaction with NX-1 65 . Reciprocally, we show here that the soluble NX-1β ectodomain without the SS4 insert strongly induced neuritogenesis in NL-1-expressing hippocampal neurons. Interestingly, both dose-response curves are bell-shaped (present study and 50,65 ), and thus the effect is not apparent at 1 nM NX-1βe (Fig. 1a) or 10 nM soluble NL-1 65 . Previous experiments did not report NX-1βe-induced neurite outgrowth 22,59 , consistent with the higher concentrations used in those studies. This result together with the fact that the employed NX-1βe is a dimer (a FC-chimera) and the finding that , while neurons exposed to NX-1βe showed the opposite effects-compared to ctrls. In NL-1 KO littermates, the inhibition of metalloproteases was without effect (a-c, right panels). All panels: top: representative traces; bottom: summarized and averaged data).
Scientific Reports | (2020) 10:18041 | https://doi.org/10.1038/s41598-020-75047-z www.nature.com/scientificreports/ tetrameric and dimeric Neurexide were more effective than monomeric peptide indicates that clustering of NL-1 (or NX-1β 65 ), is involved. Using Fura-imaging we show that NX-1βe induces Ca 2+ -influx via mechanisms that include N-type Ca 2+ channels, and blocking N-type Ca 2+ -channels abolishes the neuritogenic effect of NX-1βe. Ca 2+ -influx has repeatedly been implicated in supporting growth of neurites [66][67][68] , and thus, clustering of NL-1 might act as a gatekeeper mechanism to allow localized Ca 2+ -influx to stimulate formation of dendrites in the part of the neuron where NL-1 is ligated. The complementary data obtained with soluble NL-1 (NX-1-dependent outgrowth of axons 65 ) and NX-1βe/Neurexide [NL-1 dependent outgrowth of dendrites ( Supplementary Fig. S3)] is parallel to previous findings, that NL-1 binding to NX-1β induces presynaptic specializations 69 , and that binding of NXs to NLs induces post-synaptic specializations 21 . Our data thus indicate that even before synaptogenesis, NL-1 and NX-1β can stimulate neurite outgrowth. These data were obtained in vitro and it should be stressed that in vivo there are likely many redundant pathways for synaptogenesis and neurite outgrowth, some of which are independent of NXs and NLs. In developing cultured neurons, postsynaptic NL-1 enhanced the size of the RRP 27 , whereas cleaving of NL-1 led to a decrease in release probability (increase in PPR) within 30 min 40 . Notably, we show here that the acute application of NX-1βe or Neurexide to neurons that have already formed functional synapses increases synaptic release probability (decreases PPR) and increases mEPSC frequency within a few seconds in a NL-1 dependent manner. Two different NL-1 binding compounds (NX-1βe or Neurexide), which are produced using different protocols elicits identical results, which are abolished in NL-1 KO cells, and depends on NMDA-receptors. The two latter findings-and the known interaction between NL-1 and NMDA-receptors-make it likely that NX-1βe acts at least in part via binding to NL-1. However, it should be kept in mind that there are many other interaction partners of NX-1β (see "Introduction"), and they typically bind via sites that overlap with the NL-1 binding site. It is therefore possible that other NX binding partners are also required for the effect seen.
A generalized adaptation of both pre-and postsynaptic Ca 2+ -homeostasis seems to underlie the NX-1βedependent effects of synaptic release. We show that NX-1βe causes acute Ca 2+ -influx and that blocking NMDA receptors blocks the effect of NX-1βe on presynaptic release. Using presynaptic Ca 2+ -imaging, we found a small change in presynaptic Ca 2+ -homeostasis upon NX-1β application, suggesting that the mechanism could involve a direct (NL-1 dependent) effect on presynaptic boutons or involve a retrograde signal that travels from the post-to the presynapse to trigger changes in [Ca 2+ ] presynaptic . Since the effect of neurexin ectodomain or neurexide application was acute and appeared within a few seconds, it can be effectively ruled out that Ca 2+ or another signal could diffuse intracellularly from the postsynaptic to the presynaptic side to elicit the effect. The effects on presynaptic release could be blocked by inhibiting NMDA-receptors and might involve NMDA-receptors on either the postsynaptic or the presynaptic side 56 of the synapse; neurexin ectodomain might interact with either or both upon binding to NL-1 in the synapse. Interestingly, it has been found that dopamine acting on D1-receptors can stimulate Ca 2+ -influx through NMDA-receptors in D2R-neurons in a reaction that requires metalloproteinase activity in an intermediate step 70 . Furthermore, NX-1β has been shown to regulate presynaptic calcium channels via a retrograde synaptic endocannabinoid signaling pathway 32 . NX-1βe does not contain the C-terminal end of Neurexin, and Neurexide, which only contains the minimal NL-1 binding sequence, elicited a similar effect. Thus, the cytoplasmic tail of neurexin is not required for NX:NL-dependent strengthening of the presynapse, just as it is dispensable for synapse formation 71 . It is in principle possible that application of NX-1βe could break up existing NX:NL-dimers, leading to relaxation of a persistent presynaptic inhibition by the cytoplasmic neurexin-tail. Alternatively, or in addition, the acute increase in postsynaptic [Ca 2+ ] i upon NX-1βe application might represent a further augmentation of a signaling process already going on tonically 32,55,72,73 .
Whereas short-term exposure to NX-1βe increases the release probability, longer-term treatment results in a decrease in the RRP, which is complete within 2 h. A significant effect on mEPSC amplitude was seen in one experiment ( Supplementary Fig. S8), but not in others ( Supplementary Fig. S7, Fig. 5b). This indicates that the effects were predominantly presynaptic. The fact that the down-regulation is abolished in the NL-1 KO, by blocking ionotropic glutamatergic transmission, or by inhibiting activity with TTX, suggests that it is a downstream consequence of the initial synaptic strengthening induced by NX-1βe, rather than a separate phenomenon. The activity dependent reduction in RRP size is the hallmark of presynaptic homeostatic mechanisms 61,74,75 , which counteracts the short-term NX-1βe-induced increase in release probability. Previously, a role for α-NXs in synaptic homeostasis at the mouse neuromuscular junction has been described 76 . Our data imply the acute effects of NX-1βe on synaptic function can be similarly controlled using homeostatic mechanisms in central synapses.
This mechanism might explain the reduction in RRP size upon K + -depolarization in autaptic hippocampal neurons 77 , which indeed is also expected to lead to increased Ca 2+ influx, and activity-induced enhancement of metalloprotease activity 35,40,78 . Either K + or NX-1βe application will increase synaptic transmission and activity, which might stimulate metalloproteases to cleave NL-1 35 . This suggestion aligns with data indicating presynaptic down-regulation upon acute cleavage of NL-1 40 and also with the increase in eEPSC size and presynaptic vesicle pool seen upon NL-1 overexpression 26,27,30 . Consistent with this model we show that overnight incubation with a mixture of two metalloprotease inhibitors induces an increase in RRP and eEPSCs size. Metalloproteases are likely to cleave many different synaptic proteins; however, strikingly we found that they did not modulate synaptic function in NL-1 KO neurons, indicating that NL-1 is a necessary component-not necessarily the only one-for their action in cultured neurons.
Collectively, our findings provide evidence for the involvement of the NX-1β ectodomain in neurite formation and in acute and chronic regulation of synaptic transmission. Using a synthetic peptide, modeled based on the minimal NL-1 binding sequence, Neurexide, we show that this effect can be mimicked by pharmacological manipulation. This adds to mounting evidence that NXs are intricately involved in synaptic plasticity mechanisms. Heterozygous crosses were used to recover knockout animals (NL-1 KO). All animals used for experiments were genotyped using a PCR-protocol 79 . All animals were maintained in an AAALAC-accredited stable and all protocols were performed in accordance with institutional guidelines as overseen and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Copenhagen. Adult mice were sacrificed by cervical dislocation; embryos were sacrificed by decapitation.
Primary hippocampal and cerebellar granule neuron cultures for neurite outgrowth and neuronal survival. Hippocampal neurons were isolated from Wistar rats on embryonic day 19 or C57Bl6 mice on embryonic day 18 (Charles River, Sulzfeld, Germany) essentially as previously described 64,80 . Cerebellar granule neurons were isolated from Wistar rats on postnatal day 7, as described 80  Analysis of neuronal survival. Potassium deprivation. Cerebellar granule neurons were seeded at a density of 62,500 cells/cm 2 in eight-well poly-d-lysine (0.01 μg/cm 2 )-coated LabTek Permanox slides (Nunc) and grown for 7 days at 37 °C and 5% CO 2 82 . Apoptosis was induced by reducing the potassium levels in the medium from 40 to 5 mM and increasing concentrations of NX-1βe or Neurexide were added. Forty-eight hours later, the cells were fixated, stained and numbers of survived neurons was determined as previously described 82 .
Oxidative stress. Hippocampal neurons were grown as described for the potassium deprivation model. On DIV 7, NX-1βe or Neurexide were added. One hour later, H 2 O 2 (Sigma-Aldrich) was added to a final concentration of 60 µM, and the cultures were incubated for 24 h, fixated, stained, and analyzed similarly to the potassium deprivation model.
Surface plasmon resonance analysis. The analysis was performed with a Biacore 2000 machine (GE Healthcare, Hilleroed, Denmark). NL-1 (cat#4340-NL; R&D Systems) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare). Immobilization was performed at 5 µl/min, and the activation and deactivation time was 7 min. Injections of 45 μl NL-1 (0.033 μg/μl) in 10 mM sodium-acetate, pH 4.0, resulted in immobilization of ~ 2800 resonance units (RU). The analysis was performed at 25 °C using Ca 2+ -supplemented HBS-P (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005% (v/v) Surfactant P20, 3 mM CaCl 2 ) as running buffer for analysis of NX-1β or Neurexide binding to NL-1. The NX-1β Fc chimera (0.05-0. 8  . Cells were whole-cell voltage clamped at − 70 mV with a double EPC-10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) under control of Patchmaster v2x32 software (HEKA Elektronik). Currents were low-pass filtered at 3 kHz and stored at 20 kHz. Patch pipettes were pulled from borosilicate glass using a multi-step puller (P-897; Sutter Instruments). Pipette resistance ranged from 3 to 5 MΩ and was compensated to 85%. Only cells with series resistances < 15 MΩ were included in analysis. All recordings were made at room temperature. EPSCs were evoked by depolarizing the cell from − 70 to 0 mV for 2 ms. A fast local multi-barrel perfusion system (Warner SF-77B, Warner Instruments) was used to establish acute application of NX-1βe or Neurexide. For dendritic Ca 2+ -measurements in mature neurons Fura-2 (200 µM) was added to the internal medium and infused for ~ 20 min to allow optimal infusion. Neurons used for presynaptic Ca 2+ -measurements were transduced with syGCaMP2 58 expressing lentiviral particles on DIV1 and recorded on DIV 10-14. Experiments were conducted in regular external recording medium on an inverted Zeiss Axiovert 200 microscope equipped with an F-Fluar 40×/1,30 numerical aperture oil-immersion objective (Carl Zeiss Microscopy). Fluorophores were excited by a monochromator (Polychrome V, TILL Photonics) controlled by TILLVision, and images (1376 × 1040 pixels) were acquired with a cooled digital 12-bit CCD camera (SensiCam, PCO-Tech). A custom analysis procedure in Igor Pro (Wavemetrics Inc.) was used for offline analysis of evoked and sucrose responses. Spontaneous events were detected using Mini Analysis program (Synaptosoft). Fiji (ImageJ) was used for analysis of fluorescence data.
Ca 2+ -measurements in immature rat neurons. Rat neurons were prepared as described above (primary hippocampal and cerebellar granule neuron cultures for neurite outgrowth and neuronal survival). After 24 h the neuronal cultures were treated with 6 µM Fura-2AM (Sigma) for 30 min at 37 °C and 5% CO 2 (dark). After incubation with Fura-2AM the cells were washed 2 times with prewarmed supplemented neurobasal medium. After washing the treated cells were put back into the incubator for 10 min at 37 °C and 5% CO 2 (dark) to complete enzymatic removal of the acetoxymethyl (AM) group of internalized Fura-2AM. Imaging experiments were conducted on the experimental setup described above, but without patching the cells. Bath applications were performed using a local gravity-driven perfusion system.
Statistics and graphical presentation. For neuronal outgrowth data, n denotes the number of cultures; a minimum of 150 neurons per condition were included. For electrophysiological recordings, the results are shown as average ± SEM with n referring to the number of cells for each group unless otherwise stated. When comparing two groups, the variances were first compared using an F test. In case of homoscedastic data (F test insignificant), we tested differences between group means using a Student's t test. In case of heteroscedastic data (F test significant), we tested difference between group medians using a Mann-Whitney U test. Significance was assumed when p < 0.05. Graphical presentation and statistical testing was performed using SigmaPlot 12.3 (Systat Software Inc). In figures, the significance levels are indicated by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001).