Dissecting the genetic landscape of GPCR signaling through phenotypic profiling in C. elegans.

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans. These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts.

Developmental history modulates adult olfactory behavioral preferences via regulation of chemoreceptor expression in Caenorhabditiselegans.

Developmental experiences play critical roles in shaping adult physiology and behavior. We and others previously showed that adult Caenorhabditiselegans which transiently experienced dauer arrest during development (postdauer) exhibit distinct gene expression profiles as compared to control adults which bypassed the dauer stage. In particular, the expression patterns of subsets of chemoreceptor genes are markedly altered in postdauer adults. Whether altered chemoreceptor levels drive behavioral plasticity in postdauer adults is unknown. Here, we show that postdauer adults exhibit enhanced attraction to a panel of food-related attractive volatile odorants including the bacterially produced chemical diacetyl. Diacetyl-evoked responses in the AWA olfactory neuron pair are increased in both dauer larvae and postdauer adults, and we find that these increased responses are correlated with upregulation of the diacetyl receptor ODR-10 in AWA likely via both transcriptional and posttranscriptional mechanisms. We show that transcriptional upregulation of odr-10 expression in dauer larvae is in part mediated by the DAF-16 FOXO transcription factor. Via transcriptional profiling of sorted populations of AWA neurons from control and postdauer animals, we further show that the expression of a subset of additional chemoreceptor genes in AWA is regulated similarly to odr-10 in postdauer animals. Our results suggest that developmental experiences may be encoded at the level of olfactory receptor regulation, and provide a simple mechanism by which C. elegans is able to precisely modulate its behavioral preferences as a function of its current and past experiences.

Correction: Kinesin-3 mediated axonal delivery of presynaptic neurexin stabilizes dendritic spines and postsynaptic components.

[This corrects the article DOI: 10.1371/journal.pgen.1010016.].

Kinesin-3 mediated axonal delivery of presynaptic neurexin stabilizes dendritic spines and postsynaptic components.

The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein neurexin/NRX-1 is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that NRX-1 delivery to presynaptic terminals is dependent on kinesin-3/UNC-104 and show that ongoing UNC-104 function is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation and maintenance events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion.

The Caenorhabditis elegans Tubby homolog dynamically modulates olfactory cilia membrane morphogenesis and phospholipid composition.

Plasticity in sensory signaling is partly mediated via regulated trafficking of signaling molecules to and from primary cilia. Tubby-related proteins regulate ciliary protein transport; however, their roles in remodeling cilia properties are not fully understood. We find that the C. elegans TUB-1 Tubby homolog regulates membrane morphogenesis and signaling protein transport in specialized sensory cilia. In particular, TUB-1 is essential for sensory signaling-dependent reshaping of olfactory cilia morphology. We show that compromised sensory signaling alters cilia membrane phosphoinositide composition via TUB-1-dependent trafficking of a PIP5 kinase. TUB-1 regulates localization of this lipid kinase at the cilia base in part via localization of the AP-2 adaptor complex subunit DPY-23. Our results describe new functions for Tubby proteins in the dynamic regulation of cilia membrane lipid composition, morphology, and signaling protein content, and suggest that this conserved family of proteins plays a critical role in mediating cilia structural and functional plasticity.

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.

Excitatory neurons sculpt GABAergic neuronal connectivity in the C. elegans motor circuit.

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 post-embryonic-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.

Transcriptional Control of Synaptic Remodeling through Regulated Expression of an Immunoglobulin Superfamily Protein.

Neural circuits are actively remodeled during brain development, but the molecular mechanisms that trigger circuit refinement are poorly understood. Here, we describe a transcriptional program in C. elegans that regulates expression of an Ig domain protein, OIG-1, to control the timing of synaptic remodeling. DD GABAergic neurons reverse polarity during larval development by exchanging the locations of pre- and postsynaptic components. In newly born larvae, DDs receive cholinergic inputs in the dorsal nerve cord. These inputs are switched to the ventral side by the end of the first larval (L1) stage. VD class GABAergic neurons are generated in the late L1 and are postsynaptic to cholinergic neurons in the dorsal nerve cord but do not remodel. We investigated remodeling of the postsynaptic apparatus in DD and VD neurons using targeted expression of the acetylcholine receptor (AChR) subunit, ACR-12::GFP. We determined that OIG-1 antagonizes the relocation of ACR-12 from the dorsal side in L1 DD neurons. During the L1/L2 transition, OIG-1 is downregulated in DD neurons by the transcription factor IRX-1/Iroquois, allowing the repositioning of synaptic inputs to the ventral side. In VD class neurons, which normally do not remodel, the transcription factor UNC-55/COUP-TF turns off IRX-1, thus maintaining high levels of OIG-1 to block the removal of dorsally located ACR-12 receptors. OIG-1 is secreted from GABA neurons, but its anti-plasticity function is cell autonomous and may not require secretion. Our study provides a novel mechanism by which synaptic remodeling is set in motion through regulated expression of an Ig domain protein.

ACR-12 ionotropic acetylcholine receptor complexes regulate inhibitory motor neuron activity in Caenorhabditis elegans.

Heterogeneity in the composition of neurotransmitter receptors is thought to provide functional diversity that may be important in patterning neural activity and shaping behavior (Dani and Bertrand, 2007; Sassoè-Pognetto, 2011). However, this idea has remained difficult to evaluate directly because of the complexity of neuronal connectivity patterns and uncertainty about the molecular composition of specific receptor types in vivo. Here we dissect how molecular diversity across receptor types contributes to the coordinated activity of excitatory and inhibitory motor neurons in the nematode Caenorhabditis elegans. We show that excitatory and inhibitory motor neurons express distinct populations of ionotropic acetylcholine receptors (iAChRs) requiring the ACR-12 subunit. The activity level of excitatory motor neurons is influenced through activation of nonsynaptic iAChRs (Jospin et al., 2009; Barbagallo et al., 2010). In contrast, synaptic coupling of excitatory and inhibitory motor neurons is achieved through a second population of iAChRs specifically localized at postsynaptic sites on inhibitory motor neurons. Loss of ACR-12 iAChRs from inhibitory motor neurons leads to reduced synaptic drive, decreased inhibitory neuromuscular signaling, and variability in the sinusoidal motor pattern. Our results provide new insights into mechanisms that establish appropriately balanced excitation and inhibition in the generation of a rhythmic motor behavior and reveal functionally diverse roles for iAChR-mediated signaling in this process.

A tale of two receptors: Dual roles for ionotropic acetylcholine receptors in regulating motor neuron excitation and inhibition.

Nicotinic or ionotropic acetylcholine receptors (iAChRs) mediate excitatory signaling throughout the nervous system, and the heterogeneity of these receptors contributes to their multifaceted roles. Our recent work has characterized a single iAChR subunit, ACR-12, which contributes to two distinct iAChR subtypes within the C. elegans motor circuit. These two receptor subtypes regulate the coordinated activity of excitatory (cholinergic) and inhibitory (GABAergic) motor neurons. We have shown that the iAChR subunit ACR-12 is differentially expressed in both cholinergic and GABAergic motor neurons within the motor circuit. In cholinergic motor neurons, ACR-12 is incorporated into the previously characterized ACR-2 heteromeric receptor, which shows non-synaptic localization patterns and plays a modulatory role in controlling circuit function.(1) In contrast, a second population of ACR-12-containing receptors in GABAergic motor neurons, ACR-12GABA, shows synaptic expression and regulates inhibitory signaling.(2) Here, we discuss the two ACR-12-containing receptor subtypes, their distinct expression patterns, and functional roles in the C. elegans motor circuit. We anticipate our continuing studies of iAChRs in the C. elegans motor circuit will lead to novel insights into iAChR function in the nervous system as well as mechanisms for their regulation.