Adult Single-nucleus Neuronal Transcriptomes of Insulin Signaling Mutants Reveal Regulators of Behavior and Learning

The insulin/insulin-like signaling (IIS) pathway regulates many of C. elegans’ adult functions, including learning and memory ¹ . While whole-worm and tissue-specific transcriptomic analyses have identified IIS targets ^2,3 , a higher-resolution single-cell approach is required to identify changes that confer neuron-specific improvements in the long-lived insulin receptor mutant, daf-2 . To understand how behaviors that are controlled by a small number of neurons change in daf-2 mutants, we used the deep resolution of single-nucleus RNA sequencing to define each neuron type’s transcriptome in adult wild-type and daf-2 mutants. First, we found surprising differences between wild-type L4 larval neurons and young adult neurons in chemoreceptor expression, synaptic genes, and learning and memory genes. These Day 1 adult neuron transcriptomes allowed us to identify adult AWC-specific regulators of chemosensory function and to predict neuron-to-neuron peptide/receptor pairs. We then identified gene expression changes that correlate with daf-2’s improved cognitive functions, particularly in the AWC sensory neuron that controls learning and associative memory ⁴ , and used behavioral assays to test their roles in cognitive function. Combining deep single-neuron transcriptomics, genetic manipulation, and behavioral analyses enabled us to identify genes that may function in a single adult neuron to control behavior, including conserved genes that function in learning and memory.