cAMP-Dependent Protein Kinase Mediates Activity-Regulated Synaptic Targeting of NMDA Receptors

Abstract

Chronic activity blockade increases synaptic levels of NMDA receptor immunoreactivity in hippocampal neurons. We show here that blockade-induced synaptic NMDA receptors are functional and mediate enhanced excitotoxicity in response to synaptically released glutamate. Activity blockade increased the cell surface association of NMDA receptors. Blockade-induced synaptic targeting of NMDA receptors did not require protein synthesis but required phosphorylation and specifically cAMPdependent protein kinase (PKA). Furthermore, activation of PKA was sufficient to induce synaptic targeting of NMDA receptors regardless of receptor activity status. These results implicate PKA activity downstream of receptor blockade as a mediator of enhanced synaptic transport or stabilization of NMDA receptors. Synaptic clustering of NR1-green fluorescent protein was observed in living neurons in response to NMDA receptor and cAMP phosphodiesterase antagonists and occurred gradually over the course of a day. This pathway represents a cellular mechanism for synaptic homeostasis and is likely to function in metaplasticity, long-term regulation of the ability of a synapse to undergo potentiation or depression. Key words: NMDA receptor; synaptogenesis; activity; synaptic clustering; excitotoxicity; subcellular localization; hippocampus; NR1-GFPThe NMDA-type glutamate receptor plays a central role in circuit development, memory formation, and many forms of synaptic plasticity in the mammalian brain. The NMDA receptor is composed of the essential NR1 subunit and one or more of the modulatory NR2A-D and NR3 subunits (Nakanishi, 1992;Seeburg, 1993;Mori and Mishina, 1995). NMDA receptor channel opening requires ligand binding (by presynaptic glutamate release) and removal of Mg 2ϩ block (by postsynaptic depolarization), thus conferring on the NMDA receptor the ability to function as a molecular coincidence detector (Mayer et al., 1984). Through its Ca 2ϩ permeability, NMDA receptor function is linked with many downstream signal transducing pathways in the neuron. The magnitude and kinetics of calcium elevation at the synapse are thought to be major determinants of long-term effects on synaptic efficacy (Lisman, 1989;Abraham and Bear, 1996).The level of NMDA receptor function at the synapse critically regulates brain function and cell survival. Mice expressing 5% of normal levels of NR1 exhibit increased motor activity, stereotypy, and deficits in social and sexual interactions, behaviors associated with schizophrenia (Mohn et al., 1999). Deletion of NR1 targeted postnatally selectively to CA1 of the hippocampus results in mice that are viable but deficient in spatial learning and formation of temporal memory (Tsien et al., 1996;Huerta et al., 2000). In contrast, overactivation of NMDA receptors contributes substantially to neuronal death during epilepsy, stroke, trauma, and neurodegenerative disorders (McDonald and Johnston, 1990;Choi, 1994;Rothman and Olney, 1995;During et al., 2000).NMDA receptor function is regulated during development and by ...

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