A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity

C Peter Bengtson,Hilmar Bading,Oliver Dick

doi:10.1186/1471-2202-9-11

Abstract

BackgroundExtrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic) may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death.ResultsHere we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours) periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6%) reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting.ConclusionThe slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity-dependent neuroprotection. Other factors, in particular calcium signaling to the nucleus and the induction of survival promoting genes are more likely to mediate acquired neuroprotection.

Highlights

Extrasynaptic NMDA receptors couple to a cAMP response element binding protein (CREB) shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival
MK-801 treatment in bursting networks effectively isolates the extrasynaptic pool of NMDA receptors To establish the necessary parameters to isolate extrasynaptic NMDA receptors in hippocampal cultures, we first examined the blockade of synaptic NMDA receptor evoked EPSCs by MK-801 during bicuculline-induced bursts of action potential (AP)
NMDA and AMPA components of EPSCs can be differentiated from each other, the continuous barrage of EPSCs in AP bursting cultures necessitated the blockade of AMPA receptors

Summary

Introduction

Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The differential role of NMDA receptors provides a new concept explaining how the same receptor, dependent on its location, can couple to both survival and death This concept represents an alternative to the "Ca2+ load" hypothesis, which attempts to assign a toxic threshold to Ca2+ influx associated with NMDA receptor activation [8,9]. The neuroprotective effects of preconditioning neurons with low concentrations of NMDA are mediated, at least in cultured hippocampal networks, via AP-induced stimulation of synaptic NMDA receptors [20]. The molecules responsible for synaptic NMDA receptorinduced survival represent potential clinical targets to reduce neuron loss associated with pathological conditions including stroke and neurodegenerative diseases in which NMDA receptor-mediated excitotoxicity has been implicated [21,22,23,24,25,26,27]

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