Genome-wide analysis reveals pathways important for the development and maturation of excitatory synaptic connections to GABAergic neurons

A high degree of cell and circuit-specific regulation has presented challenges for efforts to precisely define molecular mechanisms controlling synapse formation and maturation. Here, we pursue an unbiased forward genetic approach to identify C. elegans genes involved in the formation and maturation of cholinergic synaptic connections with GABAergic motor neurons as indicated by the distribution of GFP-tagged postsynaptic AChRs in GABAergic dendrites. We identified mutations in 3 genes that identify key processes in synapse/circuit maturation: postsynaptic receptor assembly, cargo trafficking, and synapse structural organization. Mutation of the RUN domain (RPIP8, UNC-14, and NESCA) cargo adaptor unc-14 dramatically impacted both dendritic spines and overall GABAergic neuron morphology. In contrast, mutation of the nicotinic acetylcholine alpha subunit unc-63 caused a failure in AChR assembly in GABAergic neurons but did not significantly alter dendritic spine structure or abundance. Notably, specific expression of wild type unc-14 cDNA in either GABAergic neurons or presynaptic cholinergic neurons was not sufficient to rescue the unc-14 mutant phenotype while pan-neuronal expression provided significant rescue, indicating that disruptions in GABAergic neuron morphology arise from compound effects. Finally, we obtained a mutation in the Liprin-α synaptic scaffold syd-2 that produces a stop codon in a C-terminal SAM domain and has severe effects on dendritic spines and AChR localization. Our unbiased strategy identified key genes that implicate three distinct cellular processes important for synapse/circuit development and maturation. The identification of these genes from our screen highlights how mechanisms for receptor assembly, cargo trafficking and synapse structural organization each contribute to circuit connectivity.