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Key Event Relationship Overview

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Description of Relationship

Upstream Event Downstream Event/Outcome
NMDARs, Overactivation Intracellular Calcium overload, Increased

AOPs Referencing Relationship

AOP Name Type of Relationship Weight of Evidence Quantitative Understanding
Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment. Directly Leads to Moderate

Taxonomic Applicability

Name Scientific Name Evidence Links

How Does This Key Event Relationship Work

The NMDA receptor is distinct from the other glutamate receptors in two ways: first, it is both ligand-gated and voltage-dependent; second, it requires co-activation by two ligands: glutamate and either glycine or D-serine. Following membrane depolarization, the co-agonists, L-glutamate and glycine must bind to their respective sites on the receptor to open the channel. On activation, the NMDA receptor allows the influx of extracellular calcium ions into the postsynaptic neuron and neurotransmission occurs (reviewed in Higley and Sabatini, 2012). Calcium flux through NMDA receptors is also thought to be critical in synaptic plasticity, a cellular mechanism for learning and memory. Indeed, NMDA receptor–dependent synaptic potentiation (LTP) and depression (LTD) are two forms of activity-dependent long-term changes in synaptic efficacy that are believed to represent cellular correlates of learning and memory processes. The best characterized form of NMDA receptor-dependent LTP and LTD occurs between CA3 and CA1 pyramidal neurons of the hippocampus (Luscher and Malenka, 2012). It is now well established that modest activation of NMDARs leads to modest increases in postsynaptic calcium, triggering LTD, whereas much stronger activation of NMDARs leading to much larger increases in postsynaptic calcium are required to trigger LTP (Luscher and Malenka, 2012). The high-frequency stimulation causes a strong temporal summation of the excitatory postsynaptic potentials, and depolarization of the postsynaptic cell is sufficient to relieve the Mg2+ block of the NMDAR and allow a large amount of calcium to enter into the post-synaptic cells.

Weight of Evidence

Biological Plausibility

There is structural and functional mechanistic understanding supporting this relationship between KE1 and KE2.

The relationship between KE1 and KE2 is plausible as the expression of the functional NMDA receptors is commonly carried out or assessed by Ca2+ imaging method. Calcium imaging techniques have been extensively utilized in the literature to investigate the potential interactions between NMDA-evoked Ca2+ influx and NMDA receptor activation. Approximately 15% of the current through NMDA receptors is mediated by Ca2+ under physiological conditions (Higley and Sabatini, 2012).

It has been shown that less than five and, occasionally, only a single NMDA receptor opens under physiological conditions, causing a total Ca2+ influx of about 6000 ions into a spine head reaching a concentration of ∼10 µm (Higley and Sabatini, 2012). However, the majority of the ions are rapidly eliminated by binding to Ca2+ proteins, reaching ∼1 µM of free Ca2+ concentration (Higley and Sabatini, 2012).

It has been shown that in rat primary forebrain cultures the intracellular Ca2+ increases after activation of the NMDA receptor through administration of NMDA but this increase in Ca2+ is blocked when the cells are cultured under Ca2+ free conditions, demonstrating that the NMDA-evoked increase in intracellular Ca2+ derives from extracellular and not intracellular sources (Liu et al., 2013).

Indirect mechanism of domoic acid (DA) induced overactivation of NMDARs that result in Ca2+ overload: depolarization of the pre-synaptic cell activates the release of endogenous Ca2+ which mobilizes vesicles containing GLU to the membrane surface. Glutamate (GLU) is then released into the synaptic cleft by exocytosis where it is able to interact with cell surface receptors. Exogenous DA can interact within the synaptic cleft with each of the three ionotropic receptor subtypes including the kainate, AMPA, and NMDA receptors on cell membranes. Activation of the kainate and AMPA receptors results in release of Ca2+ via coupled ion channels, into the post-synaptic cell. DA is also able to bind to NMDA receptors that are linked to both Ca2+ and NA/K+ ion channels and results in a cellular influx of both Na+ and Ca2+. Unlike GLU, DA induces prolonged receptor activation causing a constant influx of cations into the cell and the appropriate chemical cues for desensitization are blocked. The excess intracellular Ca2+ causes disruption of cellular function, cell swelling and ultimately cell death (Lefebvre and Robertson,2010).

Glufosinate (GLF) is the methylphosphinate analog of glutamate that directly can activate NMDARs (Lantz et al., 2014, Matsumura et al., 2001, Faro et al., 2013) (as described in KE: NMDARs, Binding of agonist). It is well established in the existing literature that activation of NMDARs leads to the intra-cellular Ca2+ overload and based on this assumption it can be suggested that an exposure to GLF leads to increased intra-cellular calcium levels.

Empirical Support for Linkage

Include consideration of temporal concordance here

Domoic acid (DomA)

  • Treatment of mouse cerebellar granule neurons (CGNs) with 1 or 10 µM DomA causes increase of intracellular Ca2+ by approximately 5 or 8 fold compared to controls, respectively (Giordano et al., 2006). Interestingly, when the cells are exposed simultaneously to DomA and the NMDA receptor antagonist MK-801, the Ca2+ levels measured are close to control levels, indicating that the Ca2+ elevation evoked by DomA involves activation of NMDA receptors (Giordano et al., 2006).
  • The same research group has performed a time course study by applying a high and a low DomA concentration and using CGNs from Gclm (+/+) and Gclm (−/−) mice lacking glutathione (Giordano et al., 2007). The low DomA dose (0.1μM) causes a small and delayed increase in intracellular Ca2+ concentration with a full recovery by 20 min. When the experiment is performed in the absence of extracellular calcium, this increase of intracellular Ca2+ levels in the presence of DomA is abolished, indicating that this change in homeostasis of Ca2+ is due to ion entry from outside the cell. However, this recording of intracellular Ca2+ is antagonised only by NBQX (AMPA receptor antagonist), but not by MK-801 (NMDA receptor antagonist). On the other hand, the higher DomA concentration (10μM) causes a rapid and robust increase in intracellular Ca2+, which lasts even after 25 min. This effect is antagonized by both NBQX and MK-801, suggesting that not only AMPA but also NMDA receptors are involved in Ca2+ elevation evoked by DomA at high doses (Giordano et al., 2007).
  • In an earlier study, the time course and concentration dependence of the increase in intracellular Ca2+ stimulated by DomA has been examined in 10-13 day-in-culture CGNs (Berman et al., 2002). DomA produces a rapid and concentration-dependent increase in intracellular Ca2+, showing the maximal increase at 10 μM DomA (Berman et al., 2002). At this concentration, fluo-3 fluorescence that is used to measure Ca2+ elevates rapidly during the first 40 s of exposure, increases more slowly before peaking at 3.5 min, after which the signal diminishes steadily over the 30 min course of the experiment to 55% of peak values. The EC50 for DomA-induced increase in intracellular Ca2+ is 0.61 μM. In the same study, the NMDA receptor antagonist MK-801 significantly reduced both peak and final plateau of intracellular Ca2+ by 30 and 70%, respectively (Berman et al., 2002).
  • These three studies (Giordano et al., 2006; 2007; Berman et al., 2002) do not provide a simultaneous measurement of NMDA receptor activation by DomA and intracellular Ca2+ levels. However, they do provide indirect evidence of NMDA receptor activation involvement in increased intracellular Ca2+ concentrations induced by DomA as they have used known antagonists of the NMDA receptors that reverses the situation in both KEs (blocking upstream KE will block downstream KE).
  • In an in vivo study it was indirectly shown that the microinjection to adult male Sprague Dawley rats of 10 μM DomA increased intracellular Ca2+ levels . A significant upregulation of phosphorylated CaMKII and phosphorylated CREB levels was recorded, possibly due to increased intracellular Ca2+ levels induced by DomA (Qiu and Currás-Collazo, 2006).

In CGNs, the co-treatment with 10 µM DomA and the kainate/AMPA receptor antagonist NBQX maintains Ca2+ levels near to control levels, suggesting that the Ca2+ elevation evoked by DomA is mediated by the activation of both AMPA/kainate and of NMDA receptors (Giordano et al., 2006).

The voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine (5 μM) and NBQX (10 μM), a competitive AMPA/kainate receptor antagonist reduces the peak and final intracellular Ca2+ concentration in CGNs (Berman et al., 2002), strengthening the view that the increase of Ca2+ influx is not only mediated by NMDA receptors but also by AMPA/kainate receptors and VSCCs.


Experimental Model

Tested concentrations

Exposure route

Exposure duration

Overactivation of NMDAR (KE up) (measurements, quantitative if available)

Increased intracelllular Ca 2+ levels (KE down) (measurements, quantitative if available)


Temporal Relationship

Dose-response relationship




Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice

0.01 to 10 µM

Time course (15 to 120 min)

5 and 8 fold increase of [Ca2+]i compared to controls.

Giordano et al., 2006

The cells were exposed simultaneously to DA and the NMDA receptor antagonist MK-801 and the Ca2+ levels were found to be close to control levels, indicating that the Ca2+ elevation evoked by DA involves activation of NMDA receptors.


CGNs from Gclm (+/+) and Gclm (−/−) mice

0.01 to 10 µM

Time course (0 to 25 min)

0.1μM domoic acid caused a small and delayed increase (4 fold) in [Ca2+]i, with a full recovery by 20 min.In contrast, the higher concentration of domoic acid (10μM) caused a rapid and robust increase (8 fold) in [Ca2+]i, which was still elevated after 25 min.

0.1μM DA increases [Ca2+]M by about 3 fold, with a delay of about 15 min. In contrast, no changes in [Ca2+]M were observed following 10μM of DA.

Giordano et al., 2007

At the low concentration (0.1μM), the recording of intracellular Ca2+ was antagonized only by NBQX (AMPA receptor antagonist), but not by MK-801 (NMDA receptor antagonist). On the other hand, the higher DA concentration (10μM) caused a rapid and robust increase in intracellular Ca2+ . This effect was antagonized by both NBQX and MK-801, suggesting the importance of NMDA receptors in Ca2+ elevation evoked by DA but only at high doses


10-13 DIV CGNs obtained from 8-day-old Sprague–Dawley rats

0.1 to 30 µM

Time course (0 to 45 min)

EC50 for DA-induced increase in intracellular Ca2+ was 0.61 μM

Berman et al., 2002

The NMDA receptor antagonist MK-801 significantly reduced both peak and final plateau of intracellular Ca2+ by 30 and 70%, respectively


Adult male Sprague Dawley rats

10 µM 

Brain microinjection

Increased phosphorylated CaMKII and phosphorylated CREB levels

Qiu and Currás-Collazo, 2006

Glufosinate (GLF)

There are no data showing that an exposure to GLF causes an increase in intra-cellular calcium. Such assumption can be proposed based on a fact that GLF directly activates NMDR as described in the MIE and other relevant KEs of this AOP.

Uncertainties or Inconsistencies

A case of a 59-yr-old woman who ingested a herbicide containing glufosinate was suffering from severe intoxication of this herbicide, however, she did not develop convulsions, which experimentally occurs in rats treated with glufosinate (Koyama et al., 1994) and is described in other human cases (Watanabe and Sano 1998).

Quantitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

The experiments describing semi-quantitative effects for these KERs are described in the table above.

Evidence Supporting Taxonomic Applicability

Data not available


Berman FW, LePage KT, Murray TF., Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca(2+) influx pathway. Brain Res., 2002, 924: 20-29.

Faro LR, Ferreira Nunes BV, Alfonso M, Ferreira VM, Durán R., Role of glutamate receptors and nitric oxide on the effects of glufosinate ammonium, an organophosphate pesticide, on in vivo dopamine release in rat striatum. Toxicology., 2013, Sep 15, 311: 154-61.

Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG., Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol. 2006., 70: 2116-2126.

Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG., Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.

Higley MJ, Sabatini BL., Calcium signalling in dendritic spines. Cold Spring Harb Perspect Biol., 2012, 4: a005686.

Koyama K, Andou Y, Saruki K, Matsuo H., Delayed and severe toxicities of a herbicide containing glufosinate and a surfactant. Vet Hum Toxicol., 1994, 36: 17-8.

Lantz Stephen R , Cina M. Mack , Kathleen Wallace, Ellen F. Key , Timothy J. Shafer , John E. Casida., Glufosinate binds N-methyl-D aspartate receptors and increases neuronal network activity in vitro. NeuroToxicology, 2014, 45: 38–47.

Lefebvre KA, Robertson A. Domoic acid and human exposure risks: a review. Toxicon. 2010 Aug 15;56(2):218-30.

Liu F, Patterson TA, Sadovova N, Zhang X, Liu S, Zou X, Hanig JP, Paule MG, Slikker W Jr, Wang C., Ketamine-induced neuronal damage and altered N-methyl-D-aspartate receptor function in rat primary forebrain culture. Toxicol Sci., 2013, 131: 548-557.

Luscher C. and Robert C. Malenka. NMDA Receptor-Dependent Long-Term Potentiation and Long-Term Depression (LTP/LTD). Cold Spring Harb Perspect Biol., 2012;4:a005710.

Matsumura N1, Takeuchi C, Hishikawa K, Fujii T, Nakaki T., Glufosinate ammonium induces convulsion through N-methyl-D-aspartate receptors in mice. Neurosci Lett., 2001, 304(1-2): 123-5.

Qiu S, Currás-Collazo MC., Histopathological and molecular changes produced by hippocampal microinjection of domoic acid. Neurotoxicol Teratol., 2006, 28: 354-362.

Watanabe T1, Sano T., Neurological effects of glufosinate poisoning with a brief review. Hum Exp Toxicol. 1998, 17: 35-9.