NMDA-type ionotropic glutamate receptors mediate excitatory neurotransmission in the central nervous system (CNS) and play critical roles in brain functions. Aberrant NMDA receptor signaling is implicated in many CNS diseases, and NMDA receptors are receiving widespread interest as therapeutic targets. Although it has proven difficult to translate preclinical findings into clinical efficacy, the many NMDA receptor ligands from these studies have created a valuable pharmacological toolbox that continues to support neuroscience research. Most NMDA receptors are composed of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. There are four different GluN2 subunits (GluN2A-D) that endow NMDA receptors with distinct functional properties and different developmental and regional expression in the CNS. Selective modulation of NMDA receptors that contain a specific GluN2 subunit can therefore target a subset of receptor subtypes expressed in disease-relevant neuronal populations. NMDA receptors require simultaneous binding of glycine (or D-serine) to GluN1 and glutamate to GluN2 for activation, but mainly rely on synaptic release of glutamate for activation in the CNS, since extracellular glycine (or D-serine) is continuously present. Thus, glutamate binding to GluN2 primarily mediates phasic activation of synaptic NMDA receptors, while agonist occupancy at GluN1 can modulate response amplitude. We will investigate two distinct modes of NMDA receptor modulation, both of which display GluN2 subunit-selectivity and affect the GluN1 agonist binding site to modulate NMDA receptor responses. In Aim 1, we will define binding contacts and mechanism of action for a new class of GluN2A-selective negative allosteric modulators that bind the subunit interface between GluN1 and GluN2A agonist binding domains (ABDs) to negatively modulate agonist binding to GluN1. In Aim 2, we will determine the structural basis for GluN2 subunit-specific activity of novel glycine site agonists with remarkably high potency and unprecedented variation in agonist efficacy among NMDA receptor subtypes. By replacing endogenous glycine or D-serine, these GluN2-dependent agonists can modulate NMDA receptors when activated by synaptic glutamate release. In Aim 3, we will modulate neuronal NMDA receptors in acute rodent brain slices using novel GluN2A-selective NAMs and GluN1 agonists. NMDA receptor-positive neurons in the adult CNS express at least two different GluN2 subunits and many NMDA receptors are assembled with two different GluN2 subunits (i.e. triheteromeric receptors). We will evaluate the activity of the novel modulators at recombinant triheteromeric NMDA receptor subtypes (GluN1/2A/2B, GluN1/2B/2D, and GluN1/2A/2C) and at native NMDA receptors in distinct neuronal populations with defined expression of these GluN2 subunit combinations. These investigations will uncover previously unrecognized features of NMDA receptor modulation and provide new avenues for basic...