Neurexin directs partner-specific synaptic connectivity in C. elegans

In neural circuits, individual neurons often make projections onto multiple postsynaptic partners. Here, we investigate molecular mechanisms by which these divergent connections are generated, using dyadic synapses in C. elegans as a model. We report that C. elegans nrx-1/neurexin directs divergent connectivity through differential actions at synapses with partnering neurons and muscles. We show that cholinergic outputs onto neurons are, unexpectedly, located at previously undefined spine-like protrusions from GABAergic dendrites. Both these spine-like features and cholinergic receptor clustering are strikingly disrupted in the absence of nrx-1. Excitatory transmission onto GABAergic neurons, but not neuromuscular transmission, is also disrupted. Our data indicate that NRX-1 located at presynaptic sites specifically directs postsynaptic development in GABAergic neurons. Our findings provide evidence that individual neurons can direct differential patterns of connectivity with their post-synaptic partners through partner-specific utilization of synaptic organizers, offering a novel view into molecular control of divergent connectivity.


Introduction
Neurons are typically wired into discrete circuits through stereotyped patterns of synaptic connections geared to perform specific functions. Individual neurons within circuits may receive convergent synaptic inputs from multiple classes of presynaptic partnering neurons, and likewise, make divergent synaptic outputs onto distinct postsynaptic targets. We have gained an understanding of some of the core mechanisms that sculpt convergent connectivity through studies of developmental processes such as activity-dependent synapse elimination (Brown et al., 1976;Campbell and Shatz, 1992;Okawa et al., 2014a;Sanes and Lichtman, 1999;Shatz and Kirkwood, 1984;Walsh and Lichtman, 2003). In contrast, the molecular processes controlling the establishment of divergent synaptic connections (between a single presynaptic partner and multiple postsynaptic target cells) are not clearly defined (Okawa et al., 2014b). Neural circuit models often represent divergent connections as a means for enabling the same signal from an individual presynaptic neuron to reach many different postsynaptic target cells. However, the strength of connections with postsynaptic partners can vary widely, strongly suggesting that presynaptic neurons have the capacity to establish and regulate connections with each postsynaptic target independently. While molecular guidance cues directing axon outgrowth have been well-studied, an understanding of the molecular mechanisms responsible for directing target-specific connectivity has remained elusive.
A primary mechanism for establishing nascent synapses is through the actions of synaptic adhesion molecules, also known as synaptic organizers (de Wit and Ghosh, 2016;Missler et al., 2012). These organizers are often anchored to the pre-and post-synaptic membranes (e.g. neurexins, neuroligins, leucine-rich repeat transmembrane proteins/LRRTMs) and promote synapse formation through trans-synaptic adhesion and signaling. The importance of these processes in establishing proper neural circuit connectivity is highlighted by the links between mutations in genes encoding these synaptic adhesion/organizing molecules and neuropsychiatric and neurodevelopmental disorders, such as autism spectrum disorder and schizophrenia (Kim et al., 2008;Reichelt et al., 2012;Rujescu et al., 2009). Intriguingly, synaptic organizers are capable of acting in a cell-specific manner to promote synapse formation (Chen et al., 2017;Siddiqui et al., 2013;Zhang et al., 2015). Thus, an exciting possibility is that individual neurons could encode connections with alternate synaptic partners through differential deployment of synaptic organizers.
We have investigated this possibility in the motor circuit of the nematode Caenorhabditis elegans where individual excitatory cholinergic motor neurons form synapses with both body wall muscles and GABAergic motor neurons. Through a screen for genes that govern the formation of these divergent synaptic connections, we demonstrate that the synaptic organizer nrx-1/neurexin directs the outgrowth of previously uncharacterized dendritic spine-like structures and the formation of synaptic connections with GABAergic neurons, but is not required for synaptic connectivity with muscles. Conversely, genes previously shown to be required for cholinergic connectivity with muscles (Francis et al., 2005;Gally et al., 2004;Pinan-Lucarré et al., 2014) are not required for the formation of synapses onto GABAergic neurons. Our findings demonstrate that cholinergic neurons utilize distinct molecular signals to establish synapses with GABAergic motor neurons versus body wall muscles, thus revealing that a single presynaptic neuron establishes divergent connections by employing parallel molecular strategies for the formation of connections with each postsynaptic partner. eLife digest Nervous systems are complex networks of interconnected cells called neurons.
These networks vary in size from a few hundred cells in worms, to tens of billions in the human brain. Within these networks, each individual neuron forms connections -called synapses -with many others. But these partner neurons are not necessarily alike. In fact, they may be different cell types. How neurons form distinct connections with different partner cells remains unclear.
Part of the answer may lie in specialized proteins called cell adhesion molecules. These proteins occur on the cell surface and enable neurons to recognize one another. This helps ensure that the cells form appropriate connections via synapses. Cell adhesion molecules are therefore also known as synaptic organizers. Philbrook et al. have now examined the role of synaptic organizers in wiring up the nervous system of the nematode worm and model organism Caenorhabditis elegans.
Motor neurons form connections with two types of partner cell: muscle cells and neurons. Philbrook et al. screened C. elegans that have mutations in genes encoding various synaptic organizers. This revealed that a protein called neurexin must be present for motor neurons to form synapses with other neurons. By contrast, neurexin is not required for the same neurons to establish synapses with muscles. Philbrook et al. found that neuron-to-neuron synapses arise at specialized finger-like projections. These resemble the dendritic spines at which synapses form in the brains of mammals, and had not been previously identified in C. elegans. In worms that lack neurexin, these spine-like structures do not form correctly, disrupting the formation of neuron-to-neuron connections.
Previous work has implicated neurexin in synapse formation in the mammalian brain. But this is the first study to reveal a role for neurexin in establishing partner-specific synaptic connections. Mutations in synaptic organizers, including neurexin, contribute to disorders of brain development. These include schizophrenia and autism spectrum disorders. Learning more about how neurexin helps establish specific synaptic connections may help us understand how these disorders arise.

Results
Clusters of the GFP-tagged acetylcholine receptor subunit ACR-12 are localized to spine-like dendritic protrusions on DD GABAergic neurons To establish a system to investigate mechanisms instructing synaptic connectivity, we labeled postsynaptic specializations on dorsally directed GABAergic DD neurons using cell-specific expression (flp-13 promoter) of the GFP-tagged acetylcholine receptor subunit ACR-12. Prior work showed that ACR-12 receptors in GABAergic motor neurons are clustered opposite cholinergic terminals and GABAergic expression of ACR-12::GFP rescues acr-12 mutant phenotypes (Barbagallo et al., 2017;Petrash et al., 2013). Moreover, postsynaptic ACR-12::GFP clusters relocate appropriately during developmental synaptic remodeling of the DD neurons, suggesting these clusters faithfully report synaptic inputs (He et al., 2015;Howell et al., 2015).
The morphology of DD neurons is highly polarized, facilitating clear visualization of the axonal and dendritic neuronal compartments. In the present work, we focus much of our analysis on the spatially isolated neurites of the DD1 neuron ( Figure 1A-C). In adults, the anterior DD1 process extends from the soma to enter the ventral nerve cord fascicle (the dendritic compartment), where prior EM studies show that approximately 26 synaptic inputs from cholinergic neurons are concentrated (the synaptic region, Figure 1C,D) (White et al., 1978(White et al., , 1976. The process then crosses the longitudinal midline of the worm via a commissural connection and enters the dorsal nerve cord where it forms en passant synaptic outputs onto body wall muscles (the axonal compartment) ( Figure 1A,B) (White et al., 1976). We find that ACR-12::GFP receptor clusters in DD1 are confined to the synaptic region of the ventral dendritic process in the mature animal ( Figure 1C). As C. elegans synapses are formed en passant, pre-and post-synaptic specializations typically appear, at the light level, to be localized along the main shafts of neuronal processes. Surprisingly, we noted that the majority of ACR-12::GFP clusters do not appear localized to the shaft of the primary DD1 dendritic process, instead appearing to protrude from the primary DD1 dendrite shaft ( Figure 1C). To investigate this finding in more detail, we examined morphological features in the synaptic region of the DD1 dendrite ( Figure 1D). Intriguingly, we noted finger-like structures (~0.3-1 mm in length) projecting outward from the DD1 dendrite in this region ( Figure 1D, Figure 1-figure supplement 1A). In contrast, these structures are not present in the asynaptic region of the process immediately adjacent to the cell soma ( Figure 1D). These protrusions are obscured by the processes of other ventral cord neurons when using promoters that provide for more broad expression (e.g. unc-47), and are therefore most clearly identifiable with specific labeling of DD neurons ( although the density of VD processes may complicate their detection. The dendritic protrusions concentrate clusters of ACR-12 receptors at their tips ( Figure 1E), and over 60% of ACR-12::GFP clusters appear localized to protrusions ( Figure 1F). The dendritic protrusions increase in abundance through larval development, and this increase correlates well with a similar developmental increase in ACR-12::GFP receptor clusters (Figure 1-figure supplement 2). Together, our results provide evidence that cholinergic receptors cluster at morphologically distinct finger-like structures present on DD neuron dendrites, raising the interesting possibility that these structures serve similar roles to dendritic spines in the mammalian nervous system.
Heteromeric ACR-12-containing AChRs are located on the cell surface opposite cholinergic release sites To explore the above possibility further, we evaluated the spatial relationship between ACR-12 clusters located on these spine-like structures and cholinergic release sites. We found that dendritic protrusions and ACR-12 receptor clusters are both located opposite clusters of cholinergic synaptic vesicles (Figure 2A,B), indicating that these likely represent mature synapses. We therefore next investigated whether these clusters indicate post-synaptic receptors residing at the cell surface. To address this question, we inserted an HA epitope tag into the extracellular C-terminus of ACR-12:: GFP (diagram in Figure 2C) (Gottschalk and Schafer, 2006). Injection of Alexa594 conjugated anti-HA antibody into live transgenic animals expressing this construct produces specific labeling in the DD1 synaptic region, and is also evident in coelomocytes (scavenger cells that take up excess antibody), confirming successful injection ( Figure 2C). The anti-HA signal colocalizes with ACR-12::GFP clusters in the synaptic region of DD1, but is not evident in the cell soma. In contrast, the intracellular GFP moiety produces fluorescence that is evident in both the soma and the synaptic region of the dendrite, representing both synaptic and internal receptor pools ( Figure 2C). Injection of anti-GFP antibody did not produce specific labeling, confirming that the intracellularly positioned GFP is not accessible to antibody (Figure 2-figure supplement 1A). Our analysis of ACR-12 localization in DD1 indicates that ACR-12 is incorporated into mature receptor complexes that are specifically targeted for transport to DD neuron dendrites, and reside on the cell surface at post-synaptic sites. We next sought to gain an understanding of the subunit composition of ACR-12 receptors in GABAergic neurons. Most acetylcholine receptors are formed as heteromeric combinations of five subunits. Prior work has demonstrated that partially or improperly assembled acetylcholine receptor intermediates are not transported out of the ER and are instead targeted for degradation (Blount and Merlie, 1990;Merlie and Lindstrom, 1983). We therefore reasoned that genetic ablation of obligate ACR-12 partnering subunits, by interfering with assembly, transport, and synaptic targeting of ACR-12 receptor complexes, could provide an efficient strategy for identifying subunit partners. We found that single mutations in the acetylcholine receptor subunit genes unc-38, unc-63, lev-1 or unc-29 strongly decrease ACR-12::GFP clustering. In wild type, we observe approximately 15 receptor clusters within the DD1 synaptic region. These clusters are eliminated almost completely with mutation of these subunit genes (Figure 2-figure supplement 1B,C). In contrast, mutations in other nAChR subunit genes with previously reported neuronal expression, such as acr-9 and acr-14 (Cinar et al., 2005;Fox et al., 2005), do not disrupt synaptic ACR-12 clustering (Figure 2-figure supplement 1B). Mutations in genes important for AChR assembly and trafficking (unc-50, unc-74, and ric-3) (Boulin et al., 2008;Eimer et al., 2007;Halevi et al., 2002;Haugstetter et al., 2005) also abolish synaptic clusters of ACR-12::GFP (Figure 2-figure supplement 1B,C). GABA neuronspecific expression of wild type cDNAs encoding individual AChR subunits (UNC-63 or UNC-38) or accessory proteins in the respective mutants is sufficient to restore ACR-12 clustering to wild type levels, providing support that both gene classes act cell-autonomously in GABA neurons to promote receptor assembly, maturation and synaptic delivery (Figure 2-figure supplement 1B,C). Cell-specific expression of either UNC-29::GFP or UNC-63::GFP in DD neurons produces punctate labeling in the DD1 synaptic region that closely resembles ACR-12::GFP clustering ( Figure 2D). Mutation of acr-12 in these transgenic animals significantly reduces UNC-29::GFP or UNC-63::GFP receptor clusters and fluorescence signal in the DD1 synaptic region ( Figure 2D, Figure 2-figure supplement 1D,E), providing further evidence that they coassemble with ACR-12. Together, our results indicate UNC-38, UNC-63, LEV-1, UNC-29 and ACR-12 subunits coassemble into a pentameric acetylcholine receptor in GABAergic neurons.

Neurexin directs cholinergic connectivity with GABAergic neurons
To decipher molecular mechanisms by which these newly defined post-synaptic structures develop, we examined ACR-12::GFP labeling in DD neurons of 28 strains carrying mutations in 36 candidate genes. These candidates predominantly encode scaffold and cell-cell interaction proteins previously   (Cinar et al., 2005) (Figure 3-figure supplement 1). Mutations in most genes tested (75%) produce no significant disruption in ACR-12 receptor clustering ( Figure 3A, light blue). A second group, comprising 8 of the 36 genes analyzed ( Figure 3A, green), produces mild to moderate (26-39%) decreases in ACR-12 clustering. Many of the mutants in this second group identify genes (e.g. lev-10, madd-4) that perform previously characterized functions in neuromuscular synapse development and muscle AChR clustering (Gally et al., 2004;Pinan-Lucarré et al., 2014)  One of the 36 mutations tested is clearly distinguishable by a striking decrease in ACR-12 clustering. Mutation of the nrx-1 gene (orange, Figure 3A) does not significantly affect transgene expression in GABAergic neurons (Figure 3-figure supplement 2C,D), but produces a~70% reduction in ACR-12 receptor clustering (nrx-1(ok1649), p<0.0001) ( Figure 3A,B). nrx-1 encodes the sole C. elegans ortholog of the synaptic organizer neurexin. Neurexin has been well documented to play roles in mammalian synapse formation and function (Chen et al., 2017;Dean et al., 2003;Graf et al., 2004;Missler et al., 2003). Roles for NRX-1 in C. elegans synapse formation remain, by comparison, less well defined. Moreover, roles for neurexin in establishing divergent connectivity have not been previously addressed in any system. Importantly, we do not observe appreciable alterations in the clustering of muscle AChRs in nrx-1 mutants ( Figure 3C), similar to previously reported findings (Hu et al., 2012). The profound alterations in ACR-12 localization described above, coupled with the lack of effect on muscle AChRs, therefore warranted an in-depth analysis of cholinergic synapses with GABAergic neurons in nrx-1 mutants.
We first sought to distinguish whether nrx-1 performs a specific role in synapse formation or serves more generalized functions in the developmental maturation of DD neurons. We studied the developmental remodeling of DD neurons, characterized by the dorsoventral repositioning of synaptic markers, which occurs at the L1/L2 transition in wild type animals (Jin and Qi, 2018;White et al., 1978). In particular, we examined the repositioning of pre-and post-synaptic markers expressed specifically in DD motor neurons (Figure 3-figure supplement 3A). Presynaptic remodeling, as measured using the synaptic vesicle marker mCherry::RAB-3, proceeds normally in nrx-1 mutants, indicating that this process does not require neurexin expression. The clustering of ACR-12 receptors, however, is impaired both prior to and following remodeling in nrx-1 mutants, suggesting that NRX-1 is required for receptor clustering in each of these developmental stages.
As is the case for mammals, the C. elegans nrx-1 locus encodes both long (nrx-1 L ) and short (nrx-1 S ) neurexin isoforms (Figure 3-figure supplement 3B). The long isoform encodes a single pass transmembrane protein harboring intracellular PDZ binding and interleaved extracellular LNS (laminin-neurexin-sex hormone-binding globulin) and EGF-like domains ( Figure 3D). The ok1649 allele generates an in-frame deletion, eliminating 861 bp predicted to encode an extracellular LNS domain, raising the possibility that partial NRX-1 function may still be present in this strain. We therefore expanded our analysis to include additional nrx-1 deletions, three of which (ds1, tm1961, and nu485) are predicted to primarily impact the long isoform, while another (wy778) removes the transmembrane and cytoplasmic domains shared by all NRX-1 isoforms (Calahorro and Ruiz-Rubio, 2013;Maro et al., 2015;Tong et al., 2015). All of the deletions tested disrupt ACR-12 receptor clustering in DD GABAergic neurons, with the most severe disruptions occurring in nrx-1(wy778) and  nrx-1(nu485) mutants ( Figure 3E). Strikingly, we also observe significant disruption of ACR-12 receptor clustering in the VD GABAergic neurons ( Figure 3F,G) of nrx-1 mutants, indicating a requirement for nrx-1 at synapses onto both GABAergic neuron classes.
In some instances (14 of 34 animals for nrx-1(wy778)), we noted that a few ACR-12 receptor clusters remain detectable. We investigated whether these residual ACR-12 receptors are localized to the cell surface by injecting Alexa594 conjugated anti-HA antibody into nrx-1 mutants expressing ACR-12::GFP::3xHA. Antibody fluorescent signal clearly colocalizes with ACR-12::GFP fluorescence, providing evidence that the few remaining receptor clusters in nrx-1 mutants are present at the cell surface (Figure 3-figure supplement 3C). We interpret this result to indicate that neurexin is not essential for membrane insertion, although we can't rule out that this process may occur less efficiently in nrx-1 mutants.
We next examined whether the localization of putative ACR-12 partnering subunits is also disrupted by mutation of nrx-1. As noted above, ACR-12 receptors are formed as heteromeric complexes in GABAergic neurons that likely incorporate the UNC-29 and UNC-63 AChR subunits. Neurexin deletion (wy778) reduces UNC-29::GFP and UNC-63::GFP clusters in DD1 by 60%, consistent with a requirement for neurexin in the proper localization of mature, heteromeric receptor complexes ( Figure 3-figure supplement 3D,E). In contrast to our findings for GABAergic neurons, nrx-1 deletion does not appreciably disrupt the clustering of AMPA-type glutamate receptors in interneurons (Figure 3-figure supplement 4A), consistent with the idea that neurexin is not globally required for the establishment of synaptic connectivity in worms. Additionally, we do not observe an appreciable decrease in cholinergic synaptic vesicle clusters (acr-2::SNB-1::GFP), although this analysis does not rule out all potential presynaptic defects (Figure 3-figure supplement 4B).
To elucidate mechanisms by which nrx-1 may instruct the formation of synapses between cholinergic and GABAergic motor neurons, we evaluated mutations in the nlg-1 gene. nlg-1 encodes the sole C. elegans ortholog of neuroligin, a well characterized binding partner of neurexin Boucard et al., 2005;Hu et al., 2012;Ichtchenko et al., 1995, Ichtchenko et al., 1996. We find that mutation of nlg-1 produces no appreciable defects in ACR-12 receptor clustering ( Figure 3A,B), indicating, surprisingly, that NRX-1 operates independently of NLG-1 to direct post-synaptic development in GABAergic neurons.

Post-synaptic morphological development requires NRX-1
We next investigated whether neurexin is required for the outgrowth or stabilization of the spinelike processes we observe in DD dendrites. Wild type animals (at the L4 stage) have an average of 7-8 of these spiny protrusions within the synaptic region of the anterior DD1 dendrite ( Figure 4A,B). In contrast, nrx-1 mutants have strikingly reduced numbers of spiny protrusions. For example, only two spiny protrusions are visible on average in DD1 dendrites of nrx-1(ok1649), and nrx-1(wy778) mutants show a near complete absence of spines ( Figure 4A,B). nrx-1 deletion disrupts both ACR-12 receptor clustering and spine outgrowth in posterior DD neurons to a similar extent as observed for DD1 ( Figure 4C, Figure 4-figure supplement 1A), indicating that the requirement for nrx-1 is shared across DD neurons. Roughly 15% of the receptor clusters remaining in nrx-1(ok1649) mutants are associated with the remaining spines (compared with 67% in wild type). In contrast, the number of dendritic receptor clusters is not appreciably altered by nrx-1 deletion (p=0.67, student's t test), suggesting NRX-1 may preferentially regulate spine-associated receptor clusters in DD neurons. nrx-1 deletion also significantly reduces spiny protrusions in L2 animals (the earliest stage at which they are visible) (Figure 4-figure supplement 1B), suggesting that neurexin is required for initial spine outgrowth, although an additional role in maintenance is also possible. In accordance with our finding that nlg-1 is not required for ACR-12::GFP localization, mutation of nlg-1 does not alter spine number ( Figure 4A,B), arguing against an essential role for neuroligin in the formation of cholinergic synapses with GABAergic neurons. Likewise, spine number is not appreciably altered in either acr-12 or unc-63 mutants ( Figure 4A,B), indicating that spine outgrowth proceeds normally in the absence of functional ACR-12 receptors.

nrx-1 is expressed and functionally required in cholinergic motor neurons
To understand how NRX-1 regulates the formation of cholinergic synapses with GABAergic neurons, we sought to define the requirements for nrx-1 expression. We first examined expression of a nrx-1 L ::GFP transcriptional reporter incorporating~4.8 kb of sequence upstream of the nrx-1 L start site, and found that this reporter is strongly expressed in cholinergic motor neurons ( Figure 5A). We next asked whether specific nrx-1 expression in cholinergic neurons is sufficient to rescue the post-synaptic defects of nrx-1 mutants. We found that cholinergic nrx-1 L expression reverses the ACR-12 clustering defects of nrx-1(wy778) mutants, while expression in either GABAergic neurons or muscles fails to rescue ( Figure 5B-D). Notably, cholinergic expression of nrx-1 L also restores spine-like protrusions in nrx-1 mutants (Figure 5-figure supplement 1A,B). Thus, our results suggest that presynaptic NRX-1 acts non-autonomously to direct post-synaptic assembly in GABAergic neurons. To investigate this possibility in more detail, we examined the subcellular localization of NRX-1 by expressing GFP-tagged NRX-1 L (NRX-1 L ::GFP) (Maro et al., 2015) in a subset of cholinergic neurons (DA/DB). Expression of unc-129::NRX-1 L ::GFP produces discrete puncta along the dorsal nerve cord where the synaptic outputs of DA/DB neurons are located ( Figure 5E). NRX-1 L ::GFP clusters colocalize with clusters of mCherry::RAB-3 fluorescence, providing evidence that NRX-1 L is preferentially localized to cholinergic presynaptic sites ( Figure 5E,F). Intriguingly, cholinergic expression of a rescuing transgene lacking the PDZ binding motif located at the intracellular NRX-1 C-terminus (nrx-1 L DPDZ) in nrx-1 mutants can also rescue ACR-12 clustering defects (   The COE-type transcription factor unc-3 directly controls neurexin expression We have previously demonstrated that mutation of the COE-type (Collier/Olf/Ebf) transcription factor unc-3 disrupts ACR-12 clustering in VD GABAergic neurons (Barbagallo et al., 2017). In light of our findings here that nrx-1 deletion similarly disrupts ACR-12 clustering and spine-like protrusion outgrowth in DD neurons ( Figure 6A,B), we next investigated the role of UNC-3 transcriptional regulation in the development of cholinergic connectivity with GABAergic DD neurons. Prior work has shown that activity of UNC-3 is essential for the specification of cholinergic neurotransmitter identity (Kratsios et al., 2015(Kratsios et al., , 2011. To investigate the requirement for unc-3 in ACR-12 clustering, we first evaluated whether cholinergic transmission itself is critical for the development of post-synaptic specializations on DD neurons. We found that mutations in the unc-17 cholinergic vesicular ACh transporter produces no appreciable changes in spine-like protrusion number or ACR-12 clusters ( Figure 6B), arguing against a strong requirement for cholinergic transmission in the formation of these structures. We next asked whether UNC-3 transcriptional regulation of nrx-1 expression is critical for the development of cholinergic connectivity with GABAergic neurons. To address this question, we first tested whether unc-3 is required for expression of the nrx-1 L ::GFP transcriptional reporter described above. We found that mutation of unc-3 significantly reduces nrx-1 L ::GFP fluorescence in motor neuron cell bodies of the ventral nerve cord, as well as the majority of fluorescence in ventral cord processes ( Figure 6C,D). The remaining ventral cord GFP fluorescence is associated with the processes of head neurons that project into the nerve cord, which are presumably not subject to unc-3 regulation. We next used fluorescent in situ hybridization (FISH) to determine the effects of unc-3 mutation on nrx-1 mRNA abundance. Fluorescent signals indicating nrx-1 mRNA are clearly associated with cholinergic motor neuron cell bodies (co-labeled with unc-17::GFP) in wild type animals ( Figure 6E), consistent with our prior analysis using the nrx-1 L ::GFP transcriptional reporter. The smFISH signals are strongly diminished in nrx-1(nu485) deletion mutants ( Figure 6E,F), confirming they accurately report nrx-1 mRNA abundance. Labeled nrx-1 mRNA signals in cholinergic motor neurons are strikingly reduced by mutation of unc-3, consistent with the possibility that nrx-1 is a transcriptional target of unc-3 ( Figure 6E,F).
We noted that a second nrx-1 L transcriptional reporter incorporating only~2 kb of nrx-1 regulatory sequence did not produce strong fluorescence in ventral cord motor neurons ( Figure 6C,D). We reasoned that regulatory elements required for nrx-1 expression in these neurons may be present in the sequence that differs across these two transcriptional reporters (2 versus 4.8 kb) ( Figure 6C,D). As both mammalian COE transcription factors and UNC-3 bind a conserved COE binding motif (TCCCNN G / A G / A G / A G / A ) to regulate transcription of target genes (Kim et al., 2005;Kratsios et al., 2011;Wang et al., 2015Wang et al., , 1993, we searched for COE binding motifs within this region. We identified a potential COE motif (TCCCAAAGGG) located approximately 20 bp from the 5' end of the 4.8 kb nrx-1 L ::GFP transcriptional reporter. Mutation of this site  Figure 6C,D), offering evidence that UNC-3 directly regulates nrx-1 transcription in ventral cord neurons. We reasoned that forced expression of nrx-1 L in cholinergic neurons using a promoter not subject to unc-3 regulation may allow NRX-1 to coordinate synapse development independently of UNC-3 transcriptional regulation. We expressed the nrx-1 L isoform using a regulatory region of the unc-3 gene that drives expression in ventral cord cholinergic neurons (Barbagallo et al., 2017). We found that cholinergic-specific expression of the nrx-1 L isoform significantly restored receptor clusters in the dendritic region of unc-3 animals ( Figure 6G), indicating that the lack of ACR-12 receptor clusters in unc-3 mutants is largely driven by the absence of nrx-1 expression, although additional phenotypes associated with mutation of unc-3 may contribute (e.g. variable nerve cord defasciculation) (Barbagallo et al., 2017). These findings define the gene regulatory mechanisms controlling neurexin expression in presynaptic neurons and illustrate their involvement in the establishment of synaptic connectivity.

nrx-1 deletion impairs cholinergic neurotransmission onto GABAergic neurons
To investigate how nrx-1 deletion impacts the spatial arrangement of pre-and post-synaptic specializations, we examined strains coexpressing ACR-12::GFP in GABAergic neurons with the synaptic vesicle marker mCherry::RAB-3 in cholinergic neurons. In the wild type, ACR-12 receptor clusters at the tips of spiny protrusions are submerged within the presynaptic domains of cholinergic axons, where synaptic contacts are presumably located ( Figure 7A, left). In nrx-1 mutants, however, we noted a gap between the neurites of the pre-and post-synaptic neurons ( Figure 7A, right), suggesting that nrx-1 coordinates the extension of receptor-bearing spiny protrusions to presynaptic domains of cholinergic axons. These results, in combination with the receptor clustering defects in VD GABAergic neurons and the lack of an appreciable effect on muscle synapses described above, predict that nrx-1 deletion would impair cholinergic synaptic activation of GABAergic neurons, while cholinergic transmission onto muscles would remain unaffected.
Cholinergic depolarization (5 s) evokes robust stimulus-coupled Ca 2+ transients in both GABAergic neurons (67% of stimuli) and muscles (88% of stimuli) that occur within 250 ms of stimulus onset (average response latency: 0.22 ± 0.06 s in motor neurons and 0.25 ± 0.02 s in muscles). These transients are not observed in the absence of cholinergic Chrimson expression or in the absence of retinal (Figure 7-figure supplement 1B,C), consistent with a requirement for presynaptic Chrimson-mediated depolarization. In both cell types, evoked Ca 2+ transients rise rapidly following stimulation (t rise : 0.48 ± 0.08 s in GABA neurons; 0.74 ± 0.06 s in muscles), and persist throughout the duration of stimuli before decaying to baseline. Motor neuron transients are typically shorter in duration (mean duration: 7.7 ± 1 s in GABA neurons; 10.8 ± 0.5 s in muscles) and decay more rapidly (t decay : 1.1 ± 0.4 s in GABA neurons; 4.6 ± 0.6 s in muscles) compared with muscle transients, likely reflecting differences in both synaptic connectivity and physiology across the two cell types.
For both GABA neurons and muscles, evoked Ca 2+ responses are eliminated almost completely by mutations that impair post-synaptic AChR function in the respective cell types (acr-12 or unc-29; acr-16, respectively). Specifically, acr-12 deletion reduces the mean peak amplitude of GABA neuron calcium responses to cholinergic stimulation by 60% ( Figure 7C-D, Figure 7-figure supplement 1D) and increases the failure rate (no response to stimulation) by 53% compared to wild type, consistent with prior electrophysiology studies (Petrash et al., 2013). Similarly, for muscles, the mean peak amplitude of calcium responses to cholinergic stimulation is reduced by 95% in unc-29;acr-16 double mutants ( Figure 7F-G, Figure 7-figure supplement 1E), and the failure rate is increased roughly 15-fold to 77%, consistent with prior electrophysiology studies of evoked synaptic responses in these double mutants (Francis et al., 2005). nrx-1 deletion reduces the mean peak amplitude of GABA neuron calcium responses to cholinergic stimulation by roughly 71% (Figure 7C-D, Figure 7figure supplement 1D), and increases the failure rate for GABA neuron recordings by 47%. By comparison, nrx-1 deletion does not produce a significant decrease in either mean peak fluorescence ( Figure 7F-G, Figure 7-figure supplement 1E) or the failure rate in recordings of evoked muscle activity. Together, these findings support a specific requirement for nrx-1 in cholinergic transmission  onto GABA neurons, while nrx-1 appears dispensable for transmission onto muscles under our recording conditions. Notably, both the failure rate and mean peak amplitude of evoked GABA neuron Ca 2+ responses are restored to wild type levels with expression of a rescuing nrx-1 L transgene in nrx-1 mutants using a cholinergic neuron-specific promoter ( Figure 7C-D, Figure 7-figure supplement 1D).
Consistent with a requirement for NRX-1 in cholinergic synaptic connectivity with GABAergic motor neurons, automated worm track analysis showed that nrx-1 mutants display defects in the amplitude of dorsoventral bending, a feature of worm movement previously associated with GABAergic function (McIntire et al., 1993;Petrash et al., 2013). These effects are rescued with cell-specific expression of nrx-1 L in cholinergic neurons (Figure 7-figure supplement 1F). Thus, presynaptic nrx-1 expression in cholinergic neurons is required for synaptic connectivity between cholinergic and GABAergic motor neurons, and deficits in these connections alter motor performance. Together, our data indicate that NRX-1 located in presynaptic cholinergic neurons is required for establishing synaptic connectivity with partnering GABAergic neurons, but not muscle cells. NRX-1 signaling promotes both receptor clustering and the outgrowth of post-synaptic spinelike morphological features in GABAergic dendrites. Our findings support a model where distinct synaptic organizers, acting on specific post-synaptic targets, are coordinately regulated with neuronal identity, perhaps offering a mechanism for independent developmental regulation of synaptic outputs across alternate partners (Figure 8).

Discussion
Neurons often make divergent synaptic connections onto multiple postsynaptic partners, and these connections are critical for proper neural circuit performance in the brain. In this study, we use dyadic C. elegans synapses between cholinergic motor neurons and their muscle and GABAergic motor neuron postsynaptic partners as a model to define novel molecular mechanisms controlling divergent connectivity. First, we identify spine-like dendritic specializations on GABAergic DD neurons. We find that heteromeric post-synaptic AChR complexes composed of the ACR-12, UNC-63, UNC-38, UNC-29 and LEV-1 subunits are localized to these structures. Second, we identify a novel neurexin signaling pathway required both for the formation of post-synaptic specializations and for AChR clustering. In contrast, nrx-1/neurexin is not required for the development of cholinergic synapses onto muscles, which instead require molecularly distinct pathways that have been described previously (Francis et al., 2005;Gally et al., 2004;Gendrel et al., 2009;Jensen et al., 2012;Pinan-Lucarré et al., 2014;Rapti et al., 2011). Third, we find that presynaptic nrx-1 expression in cholinergic neurons is required for the establishment of synapses with GABAergic neurons, and transcriptional regulation of nrx-1 is achieved through actions of the COE transcription factor unc-3. These findings suggest a model where cholinergic expression of nrx-1 initiates synapse formation with GABAergic neurons via NRX-1 mediated trans-synaptic signaling. Finally, the ACh-GABA connectivity defects that we observe in nrx-1 mutants are paralleled by significant impairment of evoked cholinergic transmission onto GABAergic neurons, and altered sinusoidal movement. In contrast, functional connectivity with muscles is unaffected. Together, our findings provide evidence that distinct molecular signaling pathways act in parallel to establish divergent connections at dyadic synapses in the motor circuit, raising the interesting possibility that differential use of synaptic organizers may be similarly utilized in the brain to provide a molecular code directing divergent connectivity (Figure 8).
Heteromeric ACR-12 receptor complexes cluster in specialized postsynaptic domains of GABAergic DD dendrites Using candidate deletion analysis and cell-specific rescue, we identified AChR subunits and accessory proteins required for the assembly and localization of ACR-12 receptors in GABAergic neurons, defining the subunit composition of this neuronal receptor. Our findings implicate four additional receptor subunits (UNC-38, UNC-63, UNC-29, LEV-1) that co-assemble with ACR-12 to form pentameric receptor complexes in GABAergic neurons, and demonstrate that three accessory proteins (UNC-74, UNC-50, RIC-3) with more generalized roles in AChR assembly and maturation (Boulin et al., 2008;Halevi et al., 2002;Jospin et al., 2009) are also required. These findings provide evidence that ACR-12 receptors in GABA neurons are similar in subunit composition to muscle L-AChRs, differing only in the inclusion of the ACR-12 subunit in GABA neurons, whereas muscle L-AChRs incorporate the LEV-8 subunit (Boulin et al., 2008;Towers et al., 2005). Consistent with our analysis, a prior study showed that ACR-12 can be co-purified with the UNC-29 or LEV-1 subunits (Gottschalk et al., 2005). We have previously shown that unc-29 is expressed in GABAergic motor neurons, and UNC-29::GFP localizes similarly to ACR-12::GFP in DD neurons, both in the mature animal and during developmental remodeling of these neurons (He et al., 2015).
We show that ACR-12 receptor complexes are concentrated at the tips of spine-like dendritic protrusions in GABAergic DD neurons. Spiny processes associated with D-type GABAergic neurons had been noted in prior electron microscopy studies (White et al., 1976;1986), but, to our knowledge, were not characterized further. We find that these AChR-containing dendritic protrusions are apposed by presynaptic clusters of cholinergic vesicles and increase in number during the course of larval development, perhaps representing new synaptic connections formed with post-embryonic born cholinergic neurons that are integrated into the circuit following the L1/L2 transition (White et al., 1978). While further investigation of these structures will undoubtedly reveal additional insights, the characteristics we define here raise the interesting possibility that these dendritic . Distinct molecular scaffolds direct partner-specific connectivity. Distinct molecular scaffolds coordinate post-synaptic development in GABAergic neurons vs muscle. NRX-1/neurexin located at sites of presynaptic cholinergic release acts to coordinate ACR-12 receptor localization (green) and spine outgrowth in GABA neurons. A complex of proteins direct receptor clustering (brown) at the neuromuscular junction (Gally et al., 2004;Gendrel et al., 2009;Pinan-Lucarré et al., 2014;Rapti et al., 2011). NRX-1 and MADD-4 (Kratsios et al., 2015) expression in cholinergic neurons is transcriptionally co-regulated with neurotransmitter identity by the COE transcription factor UNC-3. DOI: https://doi.org/10. 7554/eLife.35692.035 protrusions are structural specializations for housing neurotransmitter receptors and other proteins required for post-synaptic signaling, perhaps representing an evolutionary precursor to mammalian dendritic spines.
The synaptic organizer neurexin directs post-synaptic development in a partner-specific manner nrx-1 deletion impairs both AChR localization and spiny outgrowths in DD neurons, creating a gap between the pre-and post-synaptic neurons. Similarly, in Drosophila, mutation of the single neurexin gene dnrx causes disorganization of synaptic structure at the neuromuscular junction (Li et al., 2007). Knockout of two out of the three mouse alpha neurexins reduces dendrite length and total spine number in the cortex (Dudanova et al., 2007), though significant numbers of dendritic spines remain detectable. In our studies, we find that nrx-1 is required for ACR-12 receptor localization in GABAergic DD and VD neurons, although we observe spine-like protrusions only in DD neurons. These findings may point toward the idea that NRX-1 is not solely involved in directing spine development, but serves an additional role in receptor clustering. Further, we show that dendritic protrusions form independently of a requirement for AChRs containing either ACR-12 or UNC-63, offering additional support that spine outgrowth and receptor clustering may be independently regulated by NRX-1. Similarly, dendritic spines on mouse CA1 pyramidal neurons form normally in the absence of functional glutamate receptors (Lu et al., 2013).
Although GABA neuron ACR-12 AChRs and muscle L-AChRs share very similar subunit composition (Boulin et al., 2008;Lewis et al., 1980), we find that genes required for proper localization of muscle L-AChRs (e.g. madd-4, lev-10) play comparatively minor roles at synapses onto GABA neurons. Conversely, loss of nrx-1 function specifically affects GABAergic, but not muscle, AChR clustering, and cholinergic transmission onto muscles appears largely unaffected. Our cell-specific rescue experiments indicate nrx-1 acts in cholinergic neurons to coordinate post-synaptic development in GABAergic neurons via trans-synaptic signaling.
Numerous studies support neuroligin as a primary trans-synaptic binding partner with neurexin (Boucard et al., 2005;Comoletti et al., 2006;Ichtchenko et al., 1995, Ichtchenko et al., 1996Nguyen and Südhof, 1997). Indeed, C. elegans NRX-1/neurexin function has been characterized almost exclusively in the context of its partnership with NLG-1/neuroligin. A retrograde neurexinneuroligin signaling pathway that regulates neurotransmitter release has been described, involving signaling through the Ca 2+ channel auxiliary subunit UNC-36/a2d (Hu et al., 2012;Tong et al., 2017). Consistent with this representing a distinct mechanism from that described in our work, mutation of unc-36 has no appreciable effect on post-synaptic development in our experiments. Postsynaptic expression of NLG-1/neuroligin is required at GABAergic synapses. However, MADD-4/Punctin likely acts as a major presynaptic partner for NLG-1 in this case, with NRX-1 playing a comparatively minor role (Maro et al., 2015;Tong et al., 2015;Tu et al., 2015). Finally, recent work also implicates neurexin-neuroligin signaling in sexually dimorphic neurite plasticity (Hart and Hobert, 2018). In contrast to these studies, we find that NRX-1 operates independently of NLG-1 to direct the formation of cholinergic synapses with GABAergic neurons. How therefore might presynaptic neurexin direct postsynaptic maturation? Our analysis shows that presynaptic NRX-1 localizes properly in the absence of acr-12, arguing against a requirement for direct binding of neurexin to the postsynaptic receptor. Prior studies offer strong evidence for alternate neurexin binding partners that support trans-synaptic signaling (Boucard et al., 2012;de Wit et al., 2009;Ko et al., 2009;Missler et al., 1998;Petrenko et al., 1996;Pettem et al., 2013;Sugita et al., 2001;Uemura et al., 2010). For several of these gene families (e.g. neurexophilins, cerebellins), clear C. elegans orthologs are not present. Others are included in our candidate analysis (e.g. casy-1/calsyntenin, lat-2/latrophilin), but single gene mutations do not produce post-synaptic defects comparable to mutation of nrx-1, suggesting either the possibility of a novel post-synaptic nrx-1 binding partner or redundant post-synaptic mechanisms. Additional genetic or proteomic studies will be required to distinguish between these possibilities and address this important question.

Transcriptional control of partner-specific synaptic connectivity
Transcriptional regulators of neurexin expression are only beginning to be elucidated (Runkel et al., 2013). Previously, we found that mutation of the COE-type transcription factor unc-3 disrupts AChR clustering in GABAergic dendrites (Barbagallo et al., 2017). These disrupted clusters are unlikely to reflect a requirement for acetylcholine release in AChR clustering, as neither tetanus toxin expression nor mutation of unc-17/vAChT produces appreciable defects in ACR-12 clustering (Barbagallo et al., 2017) (this study). Here, we find that neurexin is a transcriptional target of UNC-3, and we propose that UNC-3 regulation of nrx-1 expression directs development of postsynaptic specializations in GABAergic neurons. Prior work indicates that UNC-3 transcriptional regulation of MADD-4/Punctin is essential for proper development of cholinergic synapses with muscles (Kratsios et al., 2015). However, madd-4 is dispensable for cholinergic receptor clustering in neighboring GABAergic neurons (Barbagallo et al., 2017) (this study). Thus, cholinergic connectivity with distinct synaptic targets-muscles and GABAergic neurons-is coordinately regulated with cholinergic neuronal identity by unc-3 transcriptional control of alternate synaptic organizers.
Our work here defines a novel yet essential role for NRX-1 during synapse formation. Neurexin acts in a target-specific manner to coordinate postsynaptic development. Presynaptic neurexin instructs receptor localization and the development of spine-like processes in postsynaptic GABAergic neurons, and nrx-1 expression is critical for cholinergic transmission onto GABAergic neurons, while not required for signaling onto neighboring muscle. Our work suggests synaptic target-specific utilization of organizers such as neurexin may specify divergent connectivity, and provide a molecular mechanism for target-specific regulation of synapse development.

Materials and methods
Strains C. elegans strains were maintained at room temperature (22-24˚C) on nematode growth media plates (NGM) seeded with the Escherichia coli strain OP50. All strains are derivatives of the N2 Bristol strain (wild type). Transgenic strains were obtained by microinjection to achieve germline transformation (Mello et al., 1991) and identified with co-injection markers as previously (Barbagallo et al., 2017). Integrated lines were produced by X-ray irradiation and outcrossed to wild type. A complete list of all strains used in this work is included in Supplementary file 1.

Molecular biology
Plasmids were constructed using the two-slot Gateway Cloning system (Invitrogen) as described previously (Bhattacharya et al., 2014) and confirmed by restriction digest and/or sequencing as appropriate. All plasmids and primers used in the study are described in Supplementary files 2 and 3 respectively.

Confocal microscopy
For all imaging, nematodes were immobilized with sodium azide (0.3 M) on a 2 or 5% agarose pad. Each n represents analysis of the nerve cord from an independent animal. Images were obtained using either a 3i (Intelligent Imaging Innovations) Everest spinning-disk confocal microscope or Olympus BX51WI spinning disk confocal equipped with a 63x objective. All DD1 confocal images were obtained by imaging L4 hermaphrodites of similar size in the region near the pharynx using identical image and laser settings for each marker, and receptor clusters were quantified in a region from the DD1 cell body to connecting commissure (i.e. the synaptic region).
Analysis of synapse number/fluorescence intensity was conducted using either Volocity 6.3 or ImageJ software (open source) using defined intensity threshold values acquired from control experiments for each fluorescent marker. Specifically, the 'find objects' function in Volocity was used, excluding objects > 10 mm 2 and <0.2 mm 2 . Alternatively, the 'analyze particles' function of ImageJ was used. For ImageJ analyses, background fluorescence was first subtracted by calculating the average intensity of each image in a region devoid of puncta. In some cases, fluorescence intensity within a region of interest was also measured and normalized to wild type control as indicated. Confocal montages of the nerve cord were assembled by using the 'straighten to line' function in ImageJ. Only images where the DD1 neuron was clearly distinguishable from neighboring cells were included in analyses.
For measurements of dendritic morphology, post-synaptic protrusions in the ventral dendritic region anterior to the DD1 soma (i.e. the synaptic region) were quantified in L4 hermaphrodites. For posterior DD neurons, post-synaptic protrusions were quantified anterior to the DD3 soma (25 mm region). All protrusions ! 0.3 mm were analyzed, measuring from the base of the main dendritic process to the tip of the protrusion.
Staging and timecourse of receptors/protrusion growth during development Spine and receptor cluster number in the DD1 synaptic region were analyzed in synchronized animals using strains IZ1458 and IZ1464 as described previously (He et al., 2015). Briefly, embryos for each strain were picked to separate 60 mm unseeded plates and allowed to hatch for 40 min. Newly hatched L1 larvae were moved to freshly seeded plates, and the midpoint of the 40 min in which the embryos hatched was considered t = 0. Plates were incubated at 25˚C for 28, 34, 46, and 52 hr. Developing protrusions ! 0.2 mm in the synaptic region were analyzed, and ACR-12 receptor clusters were quantified as above.
Single molecule RNA fluorescent in situ hybridization (FISH) and imaging Custom Stellaris FISH probes against nrx-1 mRNA were obtained from Biosearch Technologies as a mix of 48 probes conjugated to CAL Fluor Red 590 Dye. Experiments were performed using wild type, unc-3(e151) mutants, and nrx-1(nu485) mutants expressing either unc-47::GFP (oxIs12), unc-4:: GFP (wdIs5), or unc-17::GFP (vsIs48) markers to label populations of GABA and ACh motor neurons. Synchronized populations of L3-L4 larval animals were fixed and hybridized as described previously (Ji and van Oudenaarden, 2012;Raj et al., 2008). Images were obtained using spinning disk microscopy as above. Z-projections were analyzed in ImageJ using the 'analyze particles' function. Following background subtraction, the total number of nrx-1 mRNA molecules was calculated for a 45 x 5.5 mm straightened region of the anterior ventral nerve cord using a defined intensity threshold across all images.

Injection of fluorescent antibodies for in vivo labeling of nAChRs
For staining of ACR-12 receptors at the cell surface, mouse monoclonal a-HA antibodies (16B12) coupled to Alexa594 were diluted in injection buffer (20 mM K 3 PO 4 , 3 mM K citrate, 2% PEG 6000, pH 7.5). Antibody was injected into the pseudocoelom of early L4 stage wild type or nrx-1(wy778) animals as described previously (Gottschalk and Schafer, 2006). Animals were allowed to recover for six hours on seeded NGM plates. Only animals in which fluorescence was observed in coelomocytes (indicating uptake of excess antibody and successful injection) were included in the analysis. Injections of anti-GFP Alexa594 antibody followed the same protocol.
Imaging was carried out on a Yokogawa CSU-X1-A1N spinning disk confocal system equipped with EM-CCD camera (Hammamatsu, C9100-50) and 40X C-Apochromat 1.2 NA water immersion objective. Optogenetic stimulation experiments employed a 625 nm (40 W) LED (Mightex Systems). Optical output through the objective was 0.3 mW/mm 2 at the focal plane of the specimen. Simultaneous GCaMP excitation (488 nm) and emission (525 nm) acquisition and Chrimson activation were achieved using a 556 nm edge BrightLine single-edge short-pass dichroic beam splitter positioned in the light path (Semrock).
Data were acquired using Volocity software. Images were binned at 4 Â 4 during acquisition and sampled at 10 Hz. GABA motor neuron and muscle ROIs in respective experiments were identified by mCherry fluorescence. Recordings from motor neuron cell bodies were obtained systematically, beginning at the anterior end of the ventral nerve cord and moving in a posterior direction. Each field typically contained 1-5 GABA motor neurons. Only recordings of neurons located anterior to the vulva were included in the analysis. Muscle recordings were obtained either directly anterior or posterior to the vulva.
Photobleaching correction was carried out by fitting an exponential function to the data (Correct-Bleach plugin, ImageJ). A linear fit (Igor Pro, Wavemetrics) of the background fluorescence was subtracted from the cell body fluorescence across all time points. Pre-stimulus baseline fluorescence (F 0 ) was calculated as the average of the corrected background-subtracted data points in the first 4 s of the recording and the corrected fluorescence data was normalized to prestimulus baseline as DF/F 0 , where DF = F -F 0 . Peak DF/F 0 was determined by fitting a Gaussian function to the DF/F 0 time sequence using Multi peak 2.0 (Igor Pro, WaveMetrics). All data collected were analyzed, including failures (no response to stimulation). Peak DF/F 0 values were calculated from recordings of >10 animals per genotype. Mean peaks ± SEM were calculated from all peak DF/F 0 data values and normalized to the wild type mean. Latency was calculated as the time required from stimulus onset for fluorescence (DF/F 0 ) to reach two times the pre-stimulus baseline standard deviation (Larsch et al., 2015). Duration was measured as the time between the onset of the transient and the completion of the decay back to the baseline. Rise and decay time constants were determined from the time constant of exponential fits between the baseline and peak fluorescence as appropriate.
with behavioral experiments, data collection and analysis; Mark J Alkema, Data availability All data generated or analysed during this study are included in the manuscript and supporting files.