Identifying regulators of associative learning using a protein-labelling approach in C. elegans

The ability to learn and form memories is critical for animals to make choices that promote their survival. The biological processes underlying learning and memory are mediated by a variety of genes in the nervous system, acting at specific times during memory encoding, consolidation, and retrieval. Many studies have utilised candidate gene approaches or random mutagenesis screens in model animals to explore the key molecular drivers for learning and memory. We propose a complementary approach to identify this network of learning regulators: the proximity-labelling tool TurboID, which promiscuously biotinylates neighbouring proteins, to snapshot the proteomic profile of neurons during learning. To do this, we expressed the TurboID enzyme in the entire nervous system of C. elegans and exposed animals to biotin only during the training step of a gustatory associative learning paradigm. Our approach revealed hundreds of proteins specific to ‘trained’ worms, including components of molecular pathways previously implicated in learning and memory formation in multiple species. We validated several novel regulators of learning involved in neurotransmission, including cholinergic receptors (ACC-1, ACC-3, GAR-1, LGC-46) and the putative glutaminase GLNA-3. These previously uncharacterised learning regulators show a clear impact on appetitive gustatory memory, but do not appear to have a generalised role in learning. In summary, we have shown that our approach to use proximity labelling to profile the brain of a small animal during training is a feasible and effective method to advance our knowledge on the biology of learning. ### Competing Interest Statement The authors have declared no competing interest.