Previous studies have demonstrated that synaptic AMPARs

Previous studies have demonstrated that synaptic AMPARs can differ greatly in their mobility; some rapidly and constitutively endothelin receptor antagonist in and out of the synaptic membrane, while others remain somewhat stable in the synaptic membrane (Luscher et al., 1999, Luthi et al., 1999). We find that mGluR- and NMDAR-activation differentially mediate the endocytosis of these two populations of AMPARs in hippocampal pyramidal neurons. NMDAR-activation drives the endocytosis of the subpopulation of GluA2 AMPARs which constitutively cycle rapidly into and out of the membrane surface while mGluR activation internalizes the less mobile surface AMPARs. Glutamate Receptor Interacting Proteins (GRIPs) which regulate GluA2 trafficking (Dong et al., 1997, Dong et al., 1999, Mao et al., 2010, Osten et al., 2000), appear to play a key role in defining these two populations of AMPARs. Knock down of GRIP by siRNA increases the population of cycling AMPARs and blocks mGluR, not NMDAR mediated receptor internalization. Finally activation of group I metabotropic glutamate receptors but not NMDARs, dissociates GluA2 from GRIP and removes these receptors from the membrane surface. Our results suggest that by inducing the internalization of different populations of AMPARs, NMDAR and mGluR activation couple to forms of synaptic depression that are likely to be functionally distinct.
Results
Discussion Our results demonstrate that NMDAR and mGluR activation induce the internalization of two distinct populations of AMPARs. NMDAR activation results in the loss of a population of rapidly cycling surface AMPARs not directly associated with GRIP1/2. In contrast, mGluR activation decreases GRIP1/2-GluA2 association and internalizes surface GluA2s previously bound by GRIP1/2. By demonstrating that NMDA and mGluR-activation couple to the endocytosis of different subpopulations of GluA2 AMPARs, these findings provide an explanation for previously proposed differences in expression mechanisms between NMDAR- and mGluR- induced LTD (Oliet et al., 1997). The hypothesis that these two forms of LTD induce endocytosis of different populations of surface GluA2 receptors is supported by the observed additivity of GluA2 endocytosis when both NMDAR and mGluR are activated simultaneously. While it is possible that the simultaneous activation of these two receptors could result in an interaction of the downstream signaling pathways that could impact AMPAR endocytosis, analysis of the link between rapidly cycling AMPARs and NMDAR- or mGluR-mediated AMPAR endocytosis further supports the internalization of different populations of receptors by NMDAR and mGluR activation. Blockade of the rapidly cycling population of AMPARs prevented the NMDAR-mediated changes in surface AMPARs, consistent with previous electrophysiological data. Additionally, constitutive GluA2 endocytosis was selectively reduced after NMDAR-induced AMPAR endocytosis consistent with NMDAR activation alone depleting the pool of rapidly cycling GluA2s. As these measurements are of endocytosis in isolation (i.e. not exocytosis as well), they are indirect measurements of constitutive AMPAR cycling. However, in most of our experiments we have measured endocytosis when surface AMPAR expression is expected to be stabilized, and therefore endocytosis is in balance with exocytosis, making it a reasonable indicator of receptor turnover (Carroll et al., 2001). The mechanistic link between NMDAR activation and cycling AMPARs is still not clear. One possibility is that constitutive endocytosis during rapid cycling delivers these receptors into endosomal compartments where they can then be sequestered through protein interactions, possibly PICK1 (Citri et al., 2010, Sossa et al., 2006) or GRIP1/2 (DeSouza et al., 2002). The inability of mGluR activation to alter constitutive AMPAR cycling or for mGluR-mediated AMPAR internalization to be affected by disrupting AMPAR cycling indicated that this receptor pathway is more closely linked to receptors which are maintained out of this highly mobile population.