NMDA glutamate receptor is a ligand-gated ion channel that mediates a major component of excitatory neurotransmission in the central nervous system (CNS). NMDA receptors are activated by simultaneous binding of two different agonists, glutamate and glycine/ D-serine1. With aging, glutamate concentration gets altered, giving rise to glutamate toxicity that contributes to age-related pathologies like Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and dementia88,95. Some treatments for these conditions include NMDA receptor blockers like memantine130. However, when completely blocking the receptors, there is a restriction of the receptor's normal physiological function59. A different approach to regulate NMDAR receptors is thorough allosteric modulators that could allow cell type or circuit-specific modulation, due to widely distributed GluN2 expression, without global NMDAR overactivation59,65,122.

In one study, we hypothesized that the compound CNS4 selectively modulates NMDA diheteromeric receptors (GluN2A, GluN2B, GuN2C, and GluN2C) based on (three) different glutamate concentrations. Electrophysiological recordings carried out on recombinant NMDA receptors expressed in xenopus oocytes revealed that 30μM and 100μM of CNS4 potentiated ionic currents for the GluN2C and GluN2D subunits with 0.3μM Glu/100μM Gly. However, when using 300μM Glu/100μM Gly, CNS4 inhibited the relative response in the GluN2D subunit and had no effect on the remaining subunits. CNS4 reduced the response to glutamate alone for GluN2A but increased it for GluN2B and did not appear to replace glutamate. Another set of electrophysiological recordings measuring current-voltage relationship was made in order to understand ion flow across the channel in the presence of CNS4. 100μM CNS4 numerically increased the ionic inward current through the channel pore with more positive membrane potential, reflected by a significant difference in reversal potential values, in the GluN2C and GluN2D subunits. CNS4 also exhibited a non-voltage dependent activity and it did not appear to compete with magnesium which naturally blocks the receptor.

Finally, the effect of CNS4 on calcium uptake and cellular viability was study in neurons from primary rat brain culture. Cortial and striatal neurons were given excessive doses of synthetic agonist NMDA in order to hyperactivate native NMDAR. In the calcium assay, 100µM of CNS4 significantly increased calcium upatake when given with 300µM NMDA compared with NMDA alone in cortex and when given with 100µM and 300µM NMDA in striatum. In the MTS assay, CNS4 did not alter neuronal viability in either cortical or striatal neurons compared with NMDA alone. Also, when CNS4 was used in non treated neurons it did not alter neuronal viability. Findings from the primary brain culture let us conclude that CNS4 could facilitate calcium influx and possibly be non toxic for neurons.