Upstream eventBinding of agonist, NMDARs
Key Event Relationship Overview
AOPs Referencing Relationship
Life Stage Applicability
Key Event Relationship Description
NMDARs can be activated indirectly through initial activation of KA/AMPARs as it happens in the case of DomA exposure. DomA is an agonist of presynaptic and postsynaptic KARs and sustained activation of these receptors by DomA results in massive ion flux and excessive release of glutamate from excitatory terminals causing depolarization of the postsynaptic neuron (as descibed in MIE). Upon this depolarization the Mg2+ block is removed from the pore of NMDARs, resulting in their activation allowing sodium, potassium, and, importantly, calcium ions to enter into a cell. The sustained exposure to DomA causes pathological overactivation of NMDARs.
In the case of exposure to glufosinate NMDARs activation is triggered by direct, sustained binding of glufosinate to the NMDARs.
Evidence Supporting this KER
NMDARs are unique among ligand-gated ion channels in that their activation requires binding of two co-agonists, glycine and endogenous neurotransmitter, L-glutamate. Physiologically, however, glycine and glutamate have distinct functions. While L-glutamate is released from specific presynaptic terminals, low concentrations of ambient glycine present at the synapse are thought to be sufficient to allow receptor activation.
There is a clear understanding that binding of glutamate or its analogue will activate NMDA receptor (accepted dogma). The prolonged activation of NMDARs will lead to a pathological over-activation of a receptor leading to excitotoxicity (minor role of KA/AMPARs), allowing high levels of calcium ions to enter the cell. However, KA/AMPARs play an important role for indirect NMDAR activation since (almost always) an initial activation of these receptors triggers depolarization of postsynaptic neurons that relieves the block of the channel pore by Mg2+, resulting in NMDAR activation.
NMDA receptors are formed by a ligand binding domain (LBD) and an ion channel that are considered the core structural and functional elements of the receptors. There is a clear understanding of how agonist binding leads to channel opening that relies on structural (e.g. crystallography or NMR) and functional (e.g. UV and IR spectrometric measurements) experimental studies of the water-soluble LBD combined with functional studies of the intact receptor. After the initial agonist binding, a conformational change—so-called clam shell closure—that prevents agonist dissociation occurs followed by a conformational change in the ion channel that is tightly coupled to that in the LBD (reviewed in Traynelis et al., 2010).
Consequently it can be stated that there is a clear structural and functional mechanistic understanding in this KER between MIE (Binding of agonist to glutamate ionotropic receptors) and KE1, NMDAR overactivation that, as explained above, can be triggered by direct binding to NMDAR or indirectly, through initial activation of KA/AMPARs as it happens in the case of exposure to glufosinate and DomA respectively, two stressors described in this AOP.
Indeed, domoic acid has a very strong affinity for the ionotropic glutamate receptors, the activation of which results in excitotoxicity, initiated by an integrative action of ionotropic receptors at both sides of the synapse blocking the channel from rapid desensitization. It has a synergistic effect with endogenous glutamate and it acts mainly as an agonist for presynaptic and postsynaptic kainate receptors. Activation of ionotropic receptors leads to the influx of Na+, K+ and Ca2+, particulary after activation of NMDARs. In combination with the inhibitory GABA neurotransmitter, glutamate contributes to the control of overall neuronal excitability.
Gufosinate (GLF) triggers alterations in glutamatergic signaling through direct binding and activation of NMDARs (Lantz et al., 2014: Matsumura et al., 2001). GLF agonist action at the NMDAR is expected to occur through interaction with the glutamate binding site and requires binding of the glycine co-agonist as well as release of the magnesium block from the channel pore. Additionally, the possible inhibition by GLF of the high affinity glutamate re-uptake transporter, especially GLT-I was studied to determine whether GLF could increase the levels of endogenous glutamate at the synaptic cleft, resulting in over activation of NMDARs. Such mechanism was excluded by Lantz (Lantz et al., 2014) but suggested by other studies (Watanabe and Sano, 1998).
Include consideration of temporal concordance here
There is well established understanding of NMDAR activation by endogenous agonist glutamate that happens in the absence of the Mg2+ block under conditions of depolarized post-synaptic membrane(accepted dogma) (Blanke et al., 2009; Enoki R, et al.,2004).
Single channel behavior of NMDARs from hippocampal CA1 neurons was studied using very low glutamate concentrations to improve temporal resolution of individual glutamate binding events. Openings resulting from individual receptor activations showed surprising complexity: they consist of a long cluster of bursts of openings. Furthermore, the NMDARs appeared to have different gating modes, occasionally entering periods of very high open probability (Gibb et al., 1991).
Single channel analysis also provided insight in how NMDARs function at the synapse. In response to a brief pulse of glutamate, mimicking synaptic release, NMDARs activate slowly over hundreds of milliseconds and continue activating long after all glutamate has been removed from the synaptic cleft, thereby briefly “memorizing” the occurrence of a synaptic input. Single channel analysis of NR1 and NR2A receptors indicates that after a brief pulse of glutamate, receptors enter a high affinity closed state from which either channel opening or agonist unbinding occurs with approximately equal probability (Popescu et al., 2004). A single synaptic event is therefore expected to only partially activate NMDARs. Consequently, a closely spaced second pulse of agonist is able to further increase the open probability, endowing the NMDAR with an ability to decode synaptic input frequency.
Domoic acid is an agonist for presynaptic and postsynaptic kainate receptors, however indirectly also activates NMDA receptors. Kainate receptors are localized both at presynaptic and postsynaptic sites. At presynaptic sites, they directly affect transmitter release from both excitatory and inhibitory neuron terminals. At postsynaptic sites, kainate receptors lead to cell depolarization, which would bring the neuron closer to its spike firing threshold. By having this dual localization, kainate receptors help in the control of neuronal excitability. However, sustained activation of postsynaptic kainate receptors by domoic acid results in massive ion flux and excessive release of glutamate from excitatory terminals. The released glutamate in turn activates NMDA receptors, which have lost their physiologic Mg2++ block because of domoic acid–induced depolarization. The final event is an increase of NMDA-mediated Ca2++ flux and subsequent activation of intracellular pro-oxidative cascades and ion imbalances, eventually leading to excitotoxicity-mediated neuronal death (Babot et al., 2005;Giordano et al., 2006).
Kainate receptors are widely expressed in the hippocampus. Glutamatergic granule cells in the hippocampus express these receptors, suggesting that cell death found after domoic acid intoxication may be produced by hyperstimulation of NMDA receptor after glutamate is released in excess. In agreement with this hypothesis, the seriously damaged CA3 area of the hippocampus receives projections from hippocampal granule cells. Qiu and Curras-Collazo (Qiu et al., 2006a) elegantly demonstrated that domoic acid first targets kainate receptors in the hippocampus by blocking its effects in vivo with a kainate receptor antagonist. The sequential involvement of distinct glutamate receptors was confirmed and further elucidated in rat mixed cortical cell and hippocampal slice cultures (Jakobsen et al., 2002; Qiu et al., 2006b).
Using primary cultures of rodent cerebellar granule cells, an in vitro model mainly constituted by glutamatergic neurons that express both NMDA and kainate receptors it was proved that domoic acid increased glutamate release, intracellular calcium, and cell death, which were prevented by kainate and NMDA receptor antagonists (Berman and Murray, 1997; Vale-Gonzalez et al., 2006) confirming that DomA toxicity is mediated by both KA and NMDARs.
Glufosinate (GLF) and its primary metabolite N-acetylglufosinate (NAcGLF) interaction with NMDARs was studied in the primary culture of rat cortical neurons by performing [3H]CGP 39653 binding experiments. The results showed that their binding affinity to NMDAR (IC50, GLF 668 uM and NAcGLF approximately 100 uM) corresponded to the concentration that produce the highest increase of mean firing rate. Furthermore, they produced biphasic MFR profile, specific to NMDAR agonists. The obtained results suggest that GLF and NAcGLF can produce both effects,excitatory and inhibitory on network activity through direct activation of NMDARs (Lantz et al., 2014).
Direct activation of NMDARs by GLF is also suggested by in vivo studies where three NMDA receptor antagonists, dizocilpine, LY235959, and Compound 40, and AMPA/KA antagonist, NBQX, were co-administrated with glufosinate ammonium (80 mg/kg, intraperitoneally) in mice. Statistical analyses showed that the NMDA receptor antagonists markedly inhibited the GLF-induced convulsions, while the AMPA/KA receptor antagonist had no effect. These results suggest that the convulsion caused by glufosinate ammonium were mediated through activation of NMDA receptors (Matsumura et al., 2001).
Uncertainties and Inconsistencies
The increase in MFR induced by GLF in neuronal networks was significantly blocked by MK-801 but not entirely suggesting that GLF can increase activity in the MEA system through non-synaptic NMDARs, since these are not blocked by MK-801.
It is not entirely clear whether GLF can work through an inhibition of the glutamate reuptake transporter, GLT-I, increasing the concentration of endogenous glutamate at the synaptic cleft and subsequently resulting in over activation of NMDARs (Lantz et al., 2014: Watanabe and Sano, 1998). Further studies are necessary to determine whether this alternative mechanism of GLF-induced NMDAR overactivation takes place.
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?
To predict how potent an agonist can be, it is usually based on the half maximal effective concentration (EC50) that induces the currents through NMDA receptors of brain slices and cells (or in recombinantly expressed proteins of these receptors). Traynelis et al. 2010 summarised the IC50 values for agonists of the different NMDA receptor subunits.
The activation effect (efficacy) of agonist on NMDA receptor have been found to be dependent on:
-the type of subunits that form the NMDA receptor
-the chemical structure of the agonist
-the binding site of a receptor that the agonist prefers
-how tightly an agonist binds to the receptor (affinity)
Glufosinate and its primary metabolite N-acetylglufosinate NAcGLF bind to the NMDAR with the following affinity: the IC50 value for GLF was 668 mM and for NAcGLF was about 100 mM.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Various studies suggest the existence of functional NMDA-like receptors in invertebrates (Xia et al., 2005). Fly and rodent NMDARs exhibit several important differences (Murphy and Glanzman, 1997). The expression and function of NMDA receptors in rodent and primates is well characterized in the existing literature.
Babot Z, Cristofol R, Sun˜ol C.,Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acidsensitive chloride channels. Eur J Neurosci., 2005, 21: 103–112.
Blanke ML, VanDongen AMJ., Activation Mechanisms of the NMDA Receptor. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press; 2009. Chapter 13. Frontiers in Neuroscience.
Berman FW, Murray TF., Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of excitatory amino acid release. J Neurochem., 1997, 69: 693–703.
Blanke ML., and Antonius M.J. VanDongen, Activation Mechanisms of the NMDA Receptor in Biology of the NMDA Receptor,2009, Chapter 13, Van Dongen AM, editor. Boca Raton (FL): CRC Press.
Enoki R, et al., NMDAR-mediated depolarizing after-potentials in the basal dendrites of CA1 pyramidal neurons. Neurosci Res., 2004, 48: 325-337.
Gibb AJ, Colquhoun D. Glutamate activation of a single NMDAR-channel produces a cluster of channel openings. Proc. R. Soc. Lond. (Biol.) 1991, 243: 39-47.
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.
Jakobsen B, Tasker A, Zimmer J., Domoic acid neurotoxicity in hippocampal slice cultures. Amino Acids, 2002, 23: 37–44.
Lantz Stephen R , Cina M. Mack , Kathleen Wallace, Ellen F. Key , Timothy J. Shafer , John E. Casida, Glufosinate binds to N-methyl-D-aspartate receptors and increases neuronal network activity in vitro. NeuroToxicology, 2014, 45: 38–47.
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.
Murphy GG, Glanzman DL., Mediation of classical conditioning in Aplysia californica by long-term potentiation of sensorimotor synapses. Science, 1997, 278: 467-78.
Popescu G, et al. Reaction mechanism determines NMDAR response to repetitive stimulation. Nature. 2004, 430: 790-799.
Qiu S, Curras-Collazo MC., Histopathological and molecular changes produced by hippocampal microinjection of domoic acid. Neurotoxicol Teratol., 2006a, 28: 354–362.
Qiu S, Pak CW, Curras-Collazo MC., Sequential involvement of distinct glutamate receptors in domoic acid-induced neurotoxicity in rat mixed cortical cultures: Effect of multiple dose/duration paradigms, chronological age, and repeated exposure. Toxicol Sci., 2006b, 89: 243–256.
Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R., Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev., 2010, 62(3):405-96.
Vale-Gonzalez C, Alfonso A, Sun˜ol C, Vieytes MR, Botana LM., Role of the plasma membrane calcium adenosine triphosphatase on domoate-induced intracellular acidification in primary cultures of cerebellar granule cells. J Neurosci Res., 2006, 84: 326–337.
Watanabe T1, Sano T., Neurological effects of glufosinate poisoning with a brief review. Hum Exp Toxicol. 1998, 17: 35-9.
Xia S, et al., NMDARs mediate olfactory learning and memory in Drosophila. Curr Biol., 2005, 15:603-618.