Nanoscale rules governing the organization of glutamate receptors in spine synapses are subunit specific.

Heterotetrameric glutamate receptors are essential for the development, function, and plasticity of spine synapses but how they are organized to achieve this is not known. Here we show that the nanoscale organization of glutamate receptors containing specific subunits define distinct subsynaptic features. Glutamate receptors containing GluA2 or GluN1 subunits establish nanomodular elements precisely positioned relative to Synaptotagmin-1 positive presynaptic release sites that scale with spine size. Glutamate receptors containing GluA1 or GluN2B specify features that exhibit flexibility: GluA1-subunit containing AMPARs are found in larger spines, while GluN2B-subunit containing NMDARs are enriched in the smallest spines with neither following a strict modular organization. Given that the precise positioning of distinct classes of glutamate receptors is linked to diverse events including cell death and synaptic plasticity, this unexpectedly robust synaptic nanoarchitecture provides a resilient system, where nanopositioned glutamate receptor heterotetramers define specific subsynaptic regions of individual spine synapses.