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.

Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development, during the integration of postembryonic-born GABAergic neurons into the circuit, produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, probably through distinct mechanisms in the developing and mature nervous system.

Formation of the nervous system requires a complex series of events including proper extension and guidance of neuronal axons and dendrites. Here we investigate the requirement for integrins, a class of transmembrane cell adhesion receptors, in regulating these processes across classes of C. elegans motor neurons. We show α integrin/ina-1 is expressed by both GABAergic and cholinergic motor neurons. Despite this, our analysis of hypomorphic ina-1(gm144) mutants indicates preferential involvement of α integrin/ina-1 in GABAergic commissural development, without obvious involvement in cholinergic commissural development. The defects in GABAergic commissures of ina-1(gm144) mutants included both premature termination and guidance errors and were reversed by expression of wild type ina-1 under control of the native ina-1 promoter. Our results also show that α integrin/ina-1 is important for proper outgrowth and guidance of commissures from both embryonic and post-embryonic born GABAergic motor neurons, indicating an ongoing requirement for integrin through two phases of GABAergic neuron development. Our findings provide insights into neuron-specific roles for integrin that would not be predicted based solely upon expression analysis.

The development of the nervous system requires precise outgrowth, extension, and wiring of both axons and dendrites to generate properly functioning neural circuits. The molecular mechanisms that shape neurite development, in particular dendritic development, remain incompletely understood. Dendrites are often highly branched and coated with actin-filled, thorny protrusions, called dendritic spines, that allow for increased numbers of synaptic contacts with neighboring neurons. Disruptions in dendritic spine development have been implicated in many neurological disorders such as autism, schizophrenia, and Alzheimer’s disease. Although the development of dendritic spines is vital for cognitive function, understanding the mechanisms driving their outgrowth and stabilization in vivo remains a challenge. Our recent work identifies the presence of dendritic spine-like structures in the nematode Caenorhabditis elegans and provides initial insights into mechanisms promoting spine outgrowth in this system. Specifically, we show that neurexin/nrx-1 is a critical molecular component in directing the development of synaptic connections and promoting spine outgrowth. Our investigation provides important insights into the molecular machinery that sculpt synaptic connectivity, and continuing efforts in this system offer the potential for identifying new mechanisms governing both synaptic partner selection and dendritic spine outgrowth.

A high degree of cell and circuit-specific regulation has complicated efforts to precisely define roles for synaptic adhesion proteins in establishing circuit connectivity. Here, we take advantage of the strengths of C. elegans for cell-specific analyses to investigate molecular coordination of pre- and postsynaptic development. We show that developing dendritic spines emerge from the dendrites of wild type GABAergic motor neurons following the localization of active zone proteins and the formation of immature synaptic vesicle assemblies in presynaptic terminals. Similarly, clusters of postsynaptic receptors and F-actin are visible in GABAergic dendrites prior to spine outgrowth. Surprisingly, these developmental processes occur without a requirement for synaptic activity. Likewise, the initial stages of spine outgrowth and receptor clustering are not altered by deletion of the C. elegans ortholog of the transsynaptic adhesion protein, neurexin/NRX-1. Over time, however, dendritic spines and postsynaptic receptor clusters are destabilized in the absence of presynaptic NRX-1/neurexin and collapse prior to adulthood. The kinesin-3 family member, UNC-104, delivers NRX-1 to presynaptic terminals and ongoing UNC-104 delivery is required into adulthood for the maintenance of postsynaptic structure. Our findings provide novel insights into the temporal order of synapse formation events in vivo and demonstrate a requirement for transsynaptic adhesion in stabilizing mature circuit connectivity.