REGIONAL AND CELLULAR LOCALIZATION OF NR3 SUBUNITS OF THE NMDA RECEPTOR AND REGULATION OF THEIR INTRACELLULAR DISTRIBUTION IN RESPONSE TO DOPAMINE RECEPTOR AND NMDA RECEPTOR AGONISTS

Abstract

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity, neurodevelopment and learning and memory. NR3 subunits (NR3A and NR3B) are relatively new players in a well-established field of NMDARs, previously involving only NR1 and NR2 subunits. Their incorporation into conventional NMDARs forms glutamate-activated NR1/NR2/NR3 triheteromeric receptors, while the omission of glutamate-binding NR2 results in excitatory glycine-activated NR1/NR3 diheteromeric receptors in heterologous cells. Both types of NR3-containing NMDARs exhibit several differences in receptor properties when compared to conventional NR1/NR2 receptors. However, knowledge of biophysical and pharmacological properties alone is inadequate unless complemented with spatiotemporal mapping efforts and an understanding of how localization of specific receptor configurations is regulated. Therefore, in this study, we focused on a wide scale mapping of NR3B protein by examining its regional, cellular and subcellular spatial distribution in a rat model. We also monitored the developmental expression of NR3B in crude synaptosomal and purified synaptic fractions. These studies were performed alongside other NMDAR subunits for comparison. It is important to take into account the expression profile of other NMDAR subunits since any impact on receptor function will depend on the relative availability of multiple subunits in spatially-defined subcellular environments at specific developmental time points. In order to study the synaptic distribution of NR3B more closely, we have adopted two strategies: (i) biochemical fractionation and enrichment, and (ii) immunolabeling of cultured postnatal neurons. Through fractionation experiments, we have extracted unique differences in the synaptic expression profile of NR3B versus other NMDAR subunits. Through culturing of low density postnatal neurons, we have established a model for NR3B expression and localization studies that is suitable for imaging after drug treatment. In actual fact, the search for a suitable model of NR3B endogenous expression is a challenging undertaking due to the late postnatal expression of NR3B, and low viability of neurons obtained from older postnatal animals. To study the effect of different configurations of NR3B-containing NMDARs on surface expression, we utilized a heterologous overexpression system. In particular, we tested a hypothetical masking mechanism by a NR3B region present near the distal carboxyl terminal end. In relation to expression at synaptic and extrasynaptic sites, we tested the relative strength of interaction of various diheteromeric and triheteromeric NR3B-containing configurations with a postsynaptic scaffolding protein, the postsynaptic density-95 (PSD-95). Our results demonstrated that certain configurations of NR3B-containing receptors interact more effectively with this scaffolding protein, thus suggesting that certain configurations might be better anchored at postsynaptic sites than others. Taken together, we have addressed NR3B spatiotemporal expression from the global- to subcellular level. We have also provided a developmental slant in examining synaptic distribution patterns of NMDAR subunits, which frames our understanding of how NMDAR subunits might be regulated in their expression at the synapse across development. We show that certain NMDAR subunits greatly enhance surface expression of NR3B. Additionally, we have identified a novel model of endogenous NR3B protein expression for localization studies. This study has filled various knowledge gaps and by doing so, we believe it provides a firmer foundation for many future NMDAR-related studies.