Optochemical profiling of NMDAR molecular diversity at synaptic and extrasynaptic sites

Neurotransmitter receptors are critical for neuronal communication. They often form large multimeric complexes that differ in their subunit composition, distribution, and signaling properties. N-methyl-D-aspartate receptors (NMDARs), a class of glutamate-gated ion channels with essential roles in brain development and plasticity, co-exist as multiple subtypes, with GluN2A diheteromers, GluN2B diheteromers, and GluN2A/GluN2B triheteromers prevailing in the adult forebrain. Studying individual subtypes in native tissues with subunit stoichiometry resolution remains challenging, and the relative abundance and subcellular distribution of these subtypes remain controversial. Here we develop and use the photochemical tool Opto2B for specific and reversible modulation of GluN2B diheteromers, while leaving other receptor subtypes (in particular GluN2A/GluN2B triheteromers) unaffected. Using Opto2B, we characterize the differential contribution of GluN2B diheteromers to synaptic and extrasynaptic NMDAR pools during mouse development. Our results suggest that GluN2A receptors predominate in both pools in adult hippocampal CA1 pyramidal cells, with no preferential contribution of GluN2B diheteromers to extrasynaptic currents, challenging the common view that GluN2A and GluN2B NMDARs segregate in synaptic and extrasynaptic compartments, respectively. Our study addresses long-standing questions on extrasynaptic NMDARs and paves the way for interrogating NMDAR signaling diversity with unprecedented molecular and spatio-temporal resolution.