NMDA receptors play a critical role in synaptic transmission, plasticity, and excitotoxicity. These ligand-gated ion channels are unique due to their voltage-dependent magnesium block, requiring both glutamate binding and membrane depolarization for activation. NMDA receptors are implicated in learning and memory and are a key target in neurological disorders such as Alzheimer’s disease, schizophrenia, and epilepsy.
This assay selectively isolates NMDA-mediated fEPSP by blocking AMPA receptors and reducing extracellular magnesium, ensuring that the recorded signals originate purely from NMDA receptor activation.
The pure NMDA component of the fESPS is validated using D-AP5, a selective NMDA receptor antagonist, that dose-dependently inhibits NMDA-mediated fEPSPs, achieving complete blockade at 10 µM. in parallel Perampanel, an AMPA receptor antagonist, does not affect NMDA responses, confirming the “purity” of the NMDA-mediated fESPS.
Ketamine and memantine exhibit dose-dependent inhibitions, while D-serine, an NMDA receptor co-agonist, enhances fEPSPs.
In addition, this assay enables to discriminate between different NMDA subunits. Developmental changes in NMDA receptor subunit composition were observed using specific allosteric modulators. In young rats, approximately 40% of NMDA currents are mediated by NR2B-containing receptors, while 60% are mediated by NR2A-containing receptors, as animals mature, the NR2A/NR2B ratio shifts to approximately 85/15%.
MEA approach provides a reliable, mid-throughput protocol for evaluating NMDA receptors function
This provides the unique opportunity to test NMDA modulators, more specifically allosteric modulators, and to investigate the role of NMDA receptor dysfunction in neurological and psychiatric disorders.
Synaptic activity is assessed by recording field excitatory post-synaptic potentials (fEPSPs) elicited in the CA1 region through electrical stimulation of the Schaffer collateral pathway. Stimulation is applied at the CA3/CA1 border of the Schaffer collaterals, with an intensity set to 40% of the maximum input intensity (Imax), as determined from the input-output (I-O) curve.
To record NMDA receptor–mediated synaptic responses, low Mg2+ACSF (0.1 mM) was used to facilitate an activation of NMDA receptor–gated Ca2+channels, 10 µM NBQX is added to the aCSF to block AMPA-Kainate mediated components.
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