The endocytosis of AMPA receptors is thought to be important in the expression of long-term depression (LTD) triggered by NMDA receptor activation. Although signaling pathways necessary for LTD induction have been identified, those responsible for the regulated internalization of AMPA receptors are unknown. Here we show that activation of NMDA receptors alone can trigger AMPA receptor endocytosis through calcium influx and activation of the calcium-dependent protein phosphatase calcineurin. A distinct signaling mechanism mediates the AMPA receptor endocytosis stimulated by insulin. These results demonstrate that although multiple signaling pathways can induce AMPA receptor internalization, NMDA receptor activation enhances AMPA receptor endocytosis via a signaling mechanism required for the induction of LTD.

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

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Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-Daspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.F ast excitatory synaptic transmission in the mammalian central nervous system is mediated primarily by ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type and Nmethyl-D-aspartate (NMDA)-type ionotropic glutamate receptors, which are coexpressed at many synapses and subserve distinct physiological functions in synaptic transmission (1-3). Although the vast majority of excitatory synapses in the hippocampus expresses functional NMDA receptors (NMDARs), electrophysiological and anatomical data suggest that the number of AMPA receptors (AMPARs) expressed at individual synapses on CA1 pyramidal cells is highly variable (4-8). Furthermore, recent evidence suggests that the surface expression of AMPARs at individual synapses is not fixed but is dynamically regulated by neuronal activity (2, 9-12). This activity-dependent regulation of the synaptic expression of AMPARs may contribute to the changes in synaptic strength that occur during NMDAR-dependent long-term potentiation and long-term depression (11, 12). Surprisingly, little is known about the detailed molecular mechanisms that regulate the number of AMPARs at excitatory synapses. Previously, we showed a pronounced redistribution of AMPARs away from synaptic sites within minutes after the triggering of long-term depression (12) or pharmacological activation of AMPARs (13). Herein, we show that this process is mediated by dynamin-dependent endocytosis and identify a role of NMDAR activation in promoting AMPAR endocytosis under physiological conditions. Materials and MethodsCell Culture and Immunocytochemistry. Hippocampal cultures were prepared as described (12, 13) and were used for experimentation at 2-3 weeks after plating. Surface AMPARs were stained with an antibody recognizing an extracellular epitope (amino acids 271-285) of the rat GluR1 subunit (Oncogene Research). Before treatment, antibody (5 g͞ml) was applied to live cells for 15 min at 37°C in conditioned cell medium. Cells were then briefly...

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