PROJECT SUMMARY/ABSTRACT NMDA-type glutamate receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the central nervous system (CNS). Seven NMDA receptor subunits have been cloned (GluN1, GluN2A-D, and GluN3A-B) that assemble into tetrameric receptors. Most NMDA receptors in the CNS are composed of two GluN1 and two GluN2 subunits, which are intensely investigated and well understood. By contrast, many properties of GluN3-containing NMDA receptors remain unresolved. In recent years, GluN3-containing NMDA receptors have been implicated in synapse maturation and synaptic plasticity, and linked to several CNS disorders. Selective ligands for GluN3-containing receptors are missing, thereby hampering studies of their roles in neuronal signaling. Structure and function of neuronal GluN3-containing receptors are poorly understood. Physiological conditions of prolonged exposure to glycine result in accumulation of GluN3- containing receptors in a strongly desensitized state. The overarching goal of this project is to mitigate these barriers and guide future studies on neuronal signaling by GluN3-containing NMDA receptors composed of two GluN1 and two GluN3A subunits. In Aim 1, we will investigate ion channel properties of native GluN3A- containing receptors. Functional and pharmacological properties of the recently discovered native GluN1/3A receptors are unknown. We will use electrophysiological recordings to investigate function and pharmacology of native GluN1/3A receptors in brain slices and recombinant GluN1/3A receptors expressed in HEK cells. This aim will also investigate physiologically relevant conditions that can enable activation of current responses from native GluN1/3A receptors. In Aim 2, we develop the molecular pharmacology of GluN3A-containing NMDA receptors. The discovery of neuronal GluN1/3A receptors strengthens the rationale to develop GluN3A- selective ligands. We will evaluate novel orthosteric ligands and allosteric modulators at GluN1/3A receptors in order to develop more potent and selective GluN3 ligands. The goal of this aim is to provide lead compounds and structure-activity relationships that can guide the development of novel GluN3-selective pharmacological tools. In Aim 3, we will define structural determinants of physiological roles for neuronal GluN3A-containing receptors. Neuronal GluN3A expression prevents synapse maturation, and neurons in GluN3A-deficient mice display increased dendritic spine density compared to wild type mice. We will identify the structural (e.g. receptor domains) and functional features (e.g. agonist binding) of GluN3A-containing NMDA receptors that mediate changes in spine density and evaluate effects of GluN3A-selective ligands on dendritic spine density.