Activation of NMDA-type glutamate receptors (NMDARs) drives signaling and neuronal plasticity that mediates
brain circuit wiring and learning. However, NMDAR overactivation can trigger neuronal cell death and is linked
to neurodegeneration, and hypofunction of NMDARs is a leading model of the etiology of schizophrenia and
other cognitive disorders. Further, the subsynaptic organization of NMDARs influences the probability of their
activation during synaptic transmission, and extrasynaptic receptors are thought to play critical roles in cell death
and gene expression. Thus, neurons utilize a variety of mechanisms to control the abundance of NMDARs on
the cell surface and particularly in synapses. These mechanisms are complex in part because NMDARs are
tetramers, formed from two obligatory GluN1 subunits and two subunits typically from the GluN2 family. Notably,
NMDARs with different GluN2 compositions display different biophysical characteristics, and the GluN2 subunits
also guide different protein interactions and signaling. Accordingly, different subtypes of NMDARs drive vastly
different forms of physiological plasticity and are linked to different disorders. Thus, identifying how neurons
control abundance of specific NMDAR subtypes in synapses has been a longstanding goal in neuroscience.
Unfortunately, our understanding of these mechanisms has been crucially restricted by inability to visualize one
of the key classes of NMDARs in neurons, the triheteromeric receptors, which contain GluN1 and two different
GluN2 subunits (most commonly GluN2A+GluN2B). Triheteromeric receptors are thought to be the majority of
NMDARs in adult brain, but there are as yet no tools to distinguish them from other NMDAR subtypes in neurons.
Thus, their distribution with neurons remains mysterious, and the mechanisms controlling their subcellular
trafficking remain almost totally unknown. To overcome this, we here introduce a new tool to visualize
triheteromeric NMDARs in neurons. Our strategy is based on bimolecular complementation, and we tagged
GluN2A and GluN2B with two parts of a modified fluorescent protein that complement to produce fluorescence
only when an NMDAR is assembled containing both the GluN2A and GluN2B subunits (split-tagged NMDARs).
Preliminary data demonstrate that split-tagged receptors traffic normally within neurons and accumulate strongly
within synapses, and whole-cell recordings demonstrate that their activation by glutamate is unaltered by the
presence of the tags. Proceeding from these results, we propose to develop and validate several versions of
split-tagged triheteromeric NMDARs useful for different experiments. We will use confocal and super-resolution
imaging to provide the first maps of triheteromeric NMDAR distribution in neurons. Finally, because the rate of
NMDAR turnover in synapses is critical for determining synaptic strength and plasticity, we will use FRAP and
single-molecule tracking to provide the first measures of synaptic exchange of triheteromeric NMDARs. This
work will fill longstanding gaps in our knowledge of NMDARs and lay necessary groundwork for investigation of other aspects of triheteromeric NMDAR trafficking in healthy neurons and disease models.