Input-specific localization of NMDA receptor GluN2 subunits in thalamocortical neurons

ABSTRACT Molecular and functional diversity among synapses is generated, in part, by differential expression of neurotransmitter receptors and their associated protein complexes. N -methyl- D -aspartate receptors (NMDARs) are tetrameric ionotropic glutamate receptors that most often comprise two GluN1 and two GluN2 subunits. NMDARs generate functionally diverse synapses across neuron populations through cell-type-specific expression patterns of GluN2 subunits (GluN2A – 2D), which have vastly different functional properties and distinct downstream signaling. Diverse NMDAR function has also been observed at anatomically distinct inputs to a single neuron population. However, the mechanisms that generate input-specific NMDAR function remain unknown as few studies have investigated subcellular GluN2 subunit localization in native brain tissue. We investigated NMDAR synaptic localization in thalamocortical (TC) neurons expressing all four GluN2 subunits. Utilizing super resolution imaging and knockout-validated antibodies, we revealed subtype- and input-specific GluN2 localization at corticothalamic (CT) versus sensory inputs to TC neurons in 4-week-old male and female C57Bl/6J mice. GluN2B was the most abundant postsynaptic subunit across all glutamatergic synapses followed by GluN2A and GluN2C, and GluN2D was localized to the fewest synapses. GluN2B was preferentially localized to CT synapses over sensory synapses, while GluN2A and GluN2C were more abundant at sensory inputs compared to CT inputs. Furthermore, postsynaptic scaffolding proteins PSD95 and SAP102 were preferentially localized with specific GluN2 subunits, and SAP102 was more abundant at sensory synapses than PSD95. This work indicates that TC neurons exhibit subtype- and input-specific localization of diverse NMDARs and associated scaffolding proteins that likely contribute to functional differences between CT and sensory synapses. HIGHLIGHTS NMDAR subtypes and synaptic scaffolding proteins show preferential localization at specific inputs to thalamocortical neurons. GluN2B was preferentially localized to corticothalamic synapses, while GluN2A and GluN2C were more abundant at sensory inputs to thalamocortical neurons. Colocalization between synaptic scaffolding proteins with NMDARs was GluN2 subtype-dependent. NMDAR subsynaptic organization in thalamocortical neurons is input- and GluN2-subtype specific.