A finely tuned balance between excitation and inhibition (E/I) is essential for proper brain function. Disruptions in the GABAergic system, which alter this equilibrium, are a common feature in various types of neurological disorders, including autism spectrum disorders (ASDs). Mutations in Phosphatase and Tensin Homolog (PTEN), the main negative regulator of the phosphatidylinositol 3-phosphate kinase/Akt pathway, are strongly associated with ASD. However, it is unclear whether PTEN deficiencies can differentially affect inhibitory and excitatory signaling. Using the Caenorhabditis elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) inputs regulate muscle activity, we found that daf-18/PTEN mutations impact GABAergic (but not cholinergic) neurodevelopment and function. This selective impact results in a deficiency in inhibitory signaling. The defects observed in the GABAergic system in daf-18/PTEN mutants are due to reduced activity of DAF-16/FOXO during development. Ketogenic diets (KGDs) have proven effective for disorders associated with E/I imbalances. However, the mechanisms underlying their action remain largely elusive. We found that a diet enriched with the ketone body β-hydroxybutyrate during early development induces DAF-16/FOXO activity, therefore improving GABAergic neurodevelopment and function in daf-18/PTEN mutants. Our study provides valuable insights into the link between PTEN mutations and neurodevelopmental defects and delves into the mechanisms underlying the potential therapeutic effects of KGDs.

Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the Caenorhabditis elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells, most notably in gonadal cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel sites of monoaminergic neurotransmitter uptake. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.