Computational Molecular Characterization of the Interaction of Acetylcholine and the NMDA Receptor to Explain the Direct Glycine-Competitive Potentiation of NMDA-Mediated Neuronal Currents.

The activation of N-methyl-d-aspartate receptor (NMDAR) is triggered by the closure of bilobed (D1 and D2) clamshell-like clefts upon binding glycine (Gly) and glutamate. There is evidence that cholinergic compounds modulate NMDAR-mediated currents via direct receptor-ligand interactions; however, molecular bases are unknown. Here, we first propose a mechanistic structure-based explanation for the observed ACh-induced submaximal potentiation of NMDA-elicited currents in striatal neurons by predicting competitive inhibition with Gly. Then, the model was validated, in principle, by confirming that the coapplication of Gly and ACh significantly reduces these neuronal currents. Finally, we delineate the interplay of ACh with the NMDAR by a combination of computational strategies. Crystallographic ACh-bound complexes were studied, revealing a similar ACh binding environment on the GluN1 subunit of the NMDAR. We illustrate how ACh can occupy X-ray monomeric open, dimeric "semiopen" cleft conformations obtained by molecular dynamics and a full-active cryo-EM NMDAR structure, explaining the suboptimal NMDAR electrophysiological activity under the "Venus Flytrap model". At an evolutionary biology level, the binding mode of ACh coincides with that of the homologous ornithine-bound periplasmic LAO binding protein complex. Our computed results indicate an analogous mechanism of action, inasmuch as ACh may stabilize the GluN1 subunit "semiclosed" conformations by inducing direct and indirect D1-to-D2 interdomain bonds. Additionally, an alternative binding site was detected, shared by the known NMDAR allosteric modulators. Experimental and computed results strongly suggest that ACh acts as a Gly-competitive, submaximal potentiating agent of the NMDAR, possibly constituting a novel chemotype for multitarget-directed drug development, e.g., to treat Alzheimer's, and it may lead to a new understanding of glutamatergic neurotransmission.

Plasticity of GluN1 at Ventral Hippocampal Synapses in the Infralimbic Cortex.

Although the infralimbic cortex (IL) is not thought to play a role in fear acquisition, recent experiments found evidence that synaptic plasticity is occurring at ventral hippocampal (vHPC) synapses in IL during auditory fear acquisition as measured by changes in the N-methyl-D-aspartate (NMDA) receptor-mediated currents in male rats. These electrophysiological data suggest that fear conditioning changes the expression of NMDA receptors on vHPC-to-IL synapses. To further evaluate the plasticity of NMDA receptors at this specific synapse, we injected AAV particles expressing channelrhodopsin-EYFP into the vHPC of male and female rats to label vHPC projections with EYFP. To test for NMDA receptor changes in vHPC-to-IL synapses after fear learning, we used fluorescence-activated cell sorting (FACS) to quantify synaptosomes isolated from IL tissue punches that were positive for EYFP and the obligatory GluN1 subunit. More EYFP+/GluN1+ synaptosomes with greater average expression of GluN1 were isolated from male rats exposed to auditory fear conditioning (AFC) than those exposed to context and tones only or to contextual fear conditioning (CFC), suggesting that AFC increased NMDA receptor expression in males. In a second experiment, we found that pairing the tones and shocks was required to induce the molecular changes and that fear extinction did not reverse the changes. In contrast, females showed similar levels of EYFP+/GluN1+ synaptosomes in all behavioral groups. These findings suggest that AFC induces synaptic plasticity of NMDA receptors in the vHPC-to-IL projection in males, while female rats rely on different synaptic mechanisms.

GluN1 and GluN2A NMDA Receptor Subunits Increase in the Hippocampus during Memory Consolidation in the Rat.

It is widely accepted that NMDA receptors (NMDAR) are required for learning and memory formation, and for synaptic plasticity induction. We have previously shown that hippocampal GluN1 and GluN2A NMDAR subunits significantly increased following habituation of rats to an open field (OF), while GluN2B remained unchanged. Similar results were obtained after CA1-long-term potentiation (LTP) induction in rat hippocampal slices. Other studies have also shown NMDAR up regulation at earlier and later time points after LTP induction or learning acquisition. In this work, we have studied NMDAR subunits levels in the hippocampus and prefrontal cortex (PFC) after OF habituation and after object recognition (OR), to find out whether rising of NMDAR subunits is a general and structure-specific feature during memory formation. In 1, 2 and 3 month old rats there was an increase in hippocampal GluN1 and GluN2A, but not in GluN2B levels 70 min after OF habituation. This rise overlaps with early phase of memory consolidation, suggesting a putative relationship between them. The increases fell down to control levels 90 min after training. Similar results were obtained in the hippocampus of adult rats 70 min after OR training, without changes in PFC. Following OF test or OR discrimination phase, NMDAR subunits remained unchanged. Hence, rising of hippocampal GluN1 and GluN2A appears to be a general feature after novel "spatial/discrimination" memory acquisition. To start investigating the dynamics and possible mechanisms of these changes, we have studied hippocampal neuron cultures stimulated by KCl to induce plasticity. GluN1 and GluN2A increased both in dendrites and neuronal bodies, reaching a maximum 75 min later and returning to control levels at 90 min. Translation and/or transcription and mobilization differentially contribute to this rise in subunits in bodies and dendrites. Our results showed that the NMDAR subunits increase follows a similar time course both in vitro and in vivo. These changes happen in the hippocampus where a spatial representation of the environment is being formed making possible short term and long term memories (STM and LTM); appear to be structure-specific; are preserved along life; and could be related to synaptic tagging and/or to memory consolidation of new spatial/discrimination information.