Development and application of fluorescent use-dependent NMDA receptor ligands (360G-Wellcome-078972_Z_05_Z)

An important unresolved question in excitatory neurotransmission is the spatial extent of activation of NMDA receptors following presynaptic glutamate release: is NMDA receptor activation confined to local postsynaptic receptors, or does glutamate spread to and activate extrasynaptic NMDA receptors or even receptors within other synapses in the vicinity? This question has hitherto been addressed only tangentially by applying neuroanatomical and in vitro electrophysiological and pharmacological methods, or by performing simulations constrained by quantitative estimates of presynaptic glutamate release, uptake, diffusion and receptor properties. Fluorescence microscopy in acute brain slices potentially offers a complementary approach, but it has only been applied to probe the down-stream consequences of NMDA receptor activation for Ca2+ transients inside neurons, which are indirectly related to NMDA receptor binding. We propose a new method based on fluorescence microscopy of receptor ligands. We will attach a fluorescent moiety to molecules derived from the high-affinity use-dependent ligand MK-801 (dizocilpine), and screen them for biological activity in a recombinant expression system. We will then apply the most promising candidates to acute hippocampal slices and evoke glutamate release at a subset of synapses to label activated NMDA receptors. By imaging the spatial distribution of fluorescence we will obtain a read-out of the history of activation of NMDA receptors for comparison to electrophysiological signals.