In a study published in Cell on Jan. 23, a research team led by ZHU Shujia from the Center for Excellence in Brain Science and Intelligence Technology of the Chinese Academy of Sciences (CAS), along with LI Yang from the Shanghai Institute of Materia Medica of CAS, dissected the assembly and architecture of endogenous N-methyl-ᴅ-aspartate receptors (eNMDARs) in adult mammalian cerebral cortex and hippocampus. 

Learning and memory are fundamental brain functions that underlie human cognition and perception of the world, which rely on development- and activity-dependent synaptic plasticity. NMDA receptors, members of excitatory ionotropic glutamate receptor family, are essential to these processes. They regulate strength of synaptic connections, playing a critical role in advanced brain functions. In higher brain structures involved in cognition, such as the cerebral cortex and hippocampus, they are especially vital for cognitive function. 

NMDA receptors are heteromeric tetramers composed of two obligatory GluN1 and two alternative subunits, either GluN2 (N2A to N2D) or GluN3 (N3A and N3B). Over the past decade, molecular understanding of NMDA receptors has been limited to studies conducted in recombinant over-expression systems. This is largely due to the low abundance of eNMDARs in brain and the lack of effective purification methods, which have hindered physiological investigations of these receptors and their subtype diversity.

In this study, researchers first enriched eNMDARs from brain tissue of adult wild-type rats using a high-affinity antibody labelled with an affinity tag. During cryo-electron microscopy data processing, they took advantage of a convolutional network-based model to separate eNMDARs from the heterogeneous pool of endogenous proteins. By combining biochemical and algorithmic purification techniques, they finally resolved the native receptors mediating physiological synaptic plasticity in the brain at near-atomic resolution.

Researchers identified three major eNMDAR subtypes: GluN1-N2A-N2B tri-heteromeric, GluN1-N2B and GluN1-N2A di-heteromeric receptors, accounting for 45%, 35% and 20% of NMDA receptors in cortex and hippocampus, respectively. GluN1-N2A-N2B tri-heteromeric tetramer highlighted the functional integration of GluN2A and GluN2B subunits in vivo. Its structure showed a distinct assembly and asymmetric architecture. Conformational variations were identified in GluN2B subunit between GluN1-N2A-N2B tri-heteromeric and GluN1-N2B di-heteromeric receptors. These structural discrepancies of the subunit across different receptor subtypes provided insight into functional diversities of eNMDARs. 

These findings uncovered the molecular basis by which eNMDARs precisely tune excitatory synaptic transmission and synaptic plasticity in adult mammals. Notably, subunit composition of eNMDARs undergoes alterations across different developmental stages and brain regions. Besides, researchers established a paradigm for mapping spatiotemporal atlas of eNMDARs throughout brain, which will enhance the understanding of learning and memory, such as how synaptic plasticity differs across ages. 

This study paved the way for exploring pathological changes in eNMDARs under different disease models.