Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the NMDA receptor-associated scaffolding complex, GKAP and DLC2, using a novel variation of Fluorescence Fluctuation Microscopy called two-photon scanning Number and Brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which in turn potentiated GKAP self-association. In dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of 2 DLC2 and 2 GKAP monomers, while in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission via ordering of GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2–GKAP interactions in glutamergic signalling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.