Investigating Atypical Signaling of N-Methyl-D-Aspartate Receptors

N-methyl-D-aspartate (NMDA) receptors are ionotropic ligand-gated channels that require ligand binding jointly with membrane depolarization to become active. This unique characteristic of the NMDA receptor coupled with previously published data suggests they may signal in a metabotropic fashion i.e., ligand binding triggers downstream conformational changes and signaling events independent of ion influx. It has been shown that long-term depression (LTD) can still be maintained in hippocampal slices treated with the open channel blocker MK-801 while the ligand binding site antagonist D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) blocks LTD. However, conflicting data was published suggesting MK-801 does not block LTD warranting further study. My work aimed to answer the following question: can NMDA receptors signal in a metabotropic fashion? First, live cell imaging in primary hippocampal cultures infected with GCaMP6s or GCaMP6s fused to postsynaptic density protein 95 (PSD95-GCaMP6s) were used to measure NMDA receptor dependent calcium signaling events. Next, phosphorylation levels of proteins that had been implicated in a proposed metabotropic signaling cascade were measured to determine the plausibility of this model and subsequent metabotropic NMDA receptor signaling. Lastly, extracellular field recordings were conducted to replicate and advance previously published data that originally suggested NMDA receptors signal in a metabotropic fashion. D-AP5 and MK-801 reduced NMDA receptor dependent calcium signaling events similarly in both GCaMP6s and PSD95-GCaMP6s infected cultures indicating ionotropic NMDA receptor signaling. The biochemical screen was unable to substantiate the previously published model of metabotropic signaling but did further support my calcium imaging data whereby MK-801 can decrease postsynaptic calcium influx within 20 minutes. In our system, metabotropic NMDA receptor signaling is merely an artifact from the confounding effects of picrotoxin and a higher concertation of MK-801. By using a more physiologically relevant experimental design I showed MK-801 is a less effective blocker of LTD and requires pretreatment while D-AP5 can block this form of plasticity in as little as 15-minutes. The data from my project helps advance our understanding of downstream postsynaptic excitatory signaling and could assist with the development of therapeutics that target the NMDA receptor.