Ketamine induces synaptic and monoaminergic remodeling in human iPSC-derived neurons

Major depressive disorder (MDD) is a leading cause of global disability, and a substantial proportion of patients develop treatment-resistant depression (TRD), highlighting the urgent need for mechanistically novel and fast-acting antidepressants. Ketamine has emerged as a paradigm-shifting therapy due to its rapid and robust antidepressant effects, yet its molecular mechanisms in human neurons remain incompletely understood. Here, we investigated the cellular and transcriptomic effects of ketamine in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons. Neuronal cultures were treated with ketamine (1 µM) for 72 hours and analyzed using immunocytochemistry, western blotting, and RNA sequencing. Ketamine treatment did not alter gross neuronal morphology or presynaptic organization but significantly increased synaptic localization of the postsynaptic scaffolding protein PSD-95, suggesting rapid functional strengthening of existing excitatory synapses. At the signaling level, ketamine selectively modulated the Akt pathway without inducing changes in BDNF protein levels or MAPK activation, and without broadly altering excitatory or inhibitory synaptic protein expression. Transcriptomic profiling revealed 604 differentially expressed genes, with significant enrichment of pathways related to serotonergic and dopaminergic neurotransmission. Notably, ketamine upregulated genes involved in monoamine synthesis and signaling, including DDC and DRD1 , while downregulating genes associated with GPCR ligand binding and neurotransmitter transport. SynGO analysis further identified selective regulation of synapse-associated genes, predominantly affecting presynaptic and neurotransmitter uptake mechanisms. Together, these findings indicate that ketamine exerts rapid antidepressant-relevant effects in human neurons by enhancing excitatory synaptic organization and engaging downstream transcriptional programs linked to monoaminergic signaling. This hiPSC-based model provides a mechanistic framework for understanding ketamine’s rapid action and a foundation for future studies on differential responses in TRD patients.