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Event: 388

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Overactivation, NMDARs

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Overactivation, NMDARs

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
NMDA glutamate receptor activity NMDA selective glutamate receptor complex increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
ionotropic glutamatergic receptors and cognition KeyEvent Allie Always (send email) Open for citation & comment WPHA/WNT Endorsed
AChE Inhibition Leading to Neurodegeneration KeyEvent Allie Always (send email) Under development: Not open for comment. Do not cite


This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI
zebrafish Danio rerio High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Biological state: Please see MIE NMDARs, Binding of antagonist

Biological compartments: Please see MIE NMDARs, Binding of antagonist

General role in biology: Please see MIE NMDARs, Binding of antagonist

The above chapters belong to the AOP entitled: Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities since the general characteristic of the NMDA receptor biology is the same for both AOPs.

Additional text, specific for this AOP:

At resting membrane potentials, NMDA receptors are inactive. Depending on the specific impulse train received, the NMDA receptor activation triggers long term potentiation (LTP) or long-term depression (LTD) (Malenka and Bear, 2004; Luscher and Malenka, 2012). LTP (the opposing process to LTD) is the long-lasting increase of synaptic strength. For LTP induction both pre- and postsynaptic neurons need to be active at the same time because the postsynaptic neuron must be depolarized when glutamate is released from the presynaptic bouton to fully relieve the Mg2+ block of NMDARs that prevents ion flows through it. Sustained activation of AMPA or KA receptors by, for instance, a train of impulses arriving at a pre-synaptic terminal, depolarizes the post-synaptic cell, releasing Mg2+ inhibition and thus allowing NMDA receptor activation. Unlike GluA2-containing AMPA receptors, NMDA receptors are permeable to calcium ions as well as being permeable to other ions. Thus NMDA receptor activation leads to a calcium influx into the post-synaptic cells, a signal that is instrumental in the activation of a number of signalling cascades (Calcium-dependent processes are describe in Key Event Calcium influx, increased). Postsynaptic Ca2+ signals of different amplitudes and durations are able to induce either LPT or LTD.

Conversely to LTP, LTD is induced by repeated activation of the presynaptic neuron at low frequencies without postsynaptic activity (Luscher and Malenka, 2012). Therefore, under physiological conditions LTD is one of several processes that serves to selectively weaken specific synapses in order to make constructive use of synaptic strengthening caused by LTP. This is necessary because, if allowed to continue increasing in strength, synapses would ultimately reach a ceiling level of efficiency, which would inhibit the encoding of new information (Purves, 2008).

LTD is an activity-dependent reduction in the efficacy of neuronal synapses lasting hours or longer following a long patterned stimulus. It has also been found to occur in different types of neurons however, the most common neurotransmitter involved in LTD is L-glutamate that acts on the NMDARs, AMPAR, KARs and metabotropic glutamate receptors (mGluRs). It can result from strong synaptic stimulation (as occurs e.g. in the cerebellar Purkinje cells) or from persistent weak synaptic stimulation (as in the hippocampus) resulting mainly from a decrease in postsynaptic AMPA receptor density, although a decrease in presynaptic neurotransmitter release may also play a role. Moreover, cerebellar LTD has been hypothesized to be important for motor learning and hippocampal LTD may be important for the clearing of old memory traces (Nicholls et al., 2008; Mallere et al., 2010). The main molecular mechanism underlying-LTD is the phosphorylation of AMPA glutamate receptors and their synaptic elimination (Ogasawara et al., 2008).

It is now commonly understood in the field of spine morphology that long lasting NMDAR-dependent LTD causes dendritic spine shrinkage, reduces number of synaptic AMPA receptors (Calabrese et al., 2014), possibly leading to synaptic dysfunction, contributing to decreased neuronal network function and impairment of learning and memory processes.

Additional text, specific for the AOP “Acetylcholinesterase inhibition leading to neurodegeneration”:

              Seizures caused by cholinesterase dependent mechanisms result in an excess of glutamate release  that activates the NMDA receptors.  As a result, intracellular Ca2+ levels at the postsynaptic neuron can overload the calcium-control mechanisms, activating without control all the calcium-dependent enzymes (proteases, lipases…) (Deshpande et al., 2014; Garcia-Reyero et al., 2016). In cases of strong acetylcholinesterase inhibition of the CNS, the NMDAR overactivation initiated by cholinergic mechanisms can result, after the initial seizure activity (focal seizure), in the development of status epilepticus. This key event separates the initial toxicity, driven by cholinergic activity, from the secondary toxicity, which is cholinergic independent  (McDonough and Shih, 1997).

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

No OECD methods are available to measure the activation state of NMDA receptors.

The measurement of the activation or the inhibition of NMDA receptors is done indirectly by recording the individual ion channels that are selective to Na+, K+ and Ca2+ by the patch clamp technique. This method relies on lack of measurable ion flux when NMDA ion channel is closed, whereas constant channel specific conductance is recorded at the open state of the receptor (Blanke and VanDongen, 2009). Furthermore, this method is based on the prediction that activation or inhibition of an ion channel results from an increase in the probability of being in the open or closed state, respectively (Ogdon and Stanfield, 2009; Zhao et al., 2009).

The whole-cell patch clamp recording techniques have also been used to study synaptically-evoked NMDA receptor-mediated excitatory or inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) in brain slices and neuronal cells, allowing the evaluation of the activated or inhibited state of the receptor.

Microelectrode array (MEA) recordings are used to measure mainly spontaneous network activity of cultured neurons (Keefer et al., 2001, Gramowski et al., 2000 and Gopal, 2003; Johnstone et al., 2010). However, using specific agonists and antagonists of a receptor, including NMDAR, MEA technology can be used to measure evoked activity, including glutamatergic receptor function (Lantz et al., 2014). For example it has been shown that MEA-coupled neuronal cortical networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic transmission (Frega et al., 2012). MEAs can also be applied in higher throughput platforms to facilitate screening of numerous chemical compounds (McConnell et al., 2012).

Excessive excitability can be also measured directly by evaluating the level of the extracellular glutamate using the enzyme-based microelectrode arrays. This technology is capable of detecting glutamate in vivo, to assess the effectiveness of hyperexcitability modulators on glutamate release in brain slices. Using glutamate oxidase coated ceramic MEAs coupled with constant voltage amperometry, it is possible to measure resting glutamate levels and synaptic overflow of glutamate after K(+) stimulation in brain slices (Quintero et al., 2011).

Neuronal network function can be also measured using optical detection of neuronal spikes both in vivo and in vitro (Wilt et al., 2013).

Drebrin immunocytochemistry: drebrin, a major actin-filament-binding protein localized in mature dendritic spines is a target of calpain mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. The NMDA-induced degradation of drebrin in mature neurons occurres concomitantly with a loss of f-actin. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. These findings suggest that calpain-mediated degradation of drebrin is mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin (A and E) regulates the synaptic clustering of NMDARs. Therefore, degradation of drebrin can be used as a readout for excitotoxicity induced by NMDAR overactivation. Degradation of drebrin can be evaluated quantitatively by Western blot analysis (mRNA evel) or by immunocytochemistry (at protein level) (Chimura et al., 2015: Sekino et al., 20069.

NMDAR overactivation-induced long lasting LTD can be measured by the dendritic spine shrinkage by quantification of cofilin and phospho-cofilin in neurons expressing eGFP and combined with immunocytochemical techniques (Calabrese et al., 2014).

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

It is important to note that in invertebrates the glutamatergic synaptic transmission has an inhibitory and not an excitatory role like in vertebrates. This type of neurotransmission is mediated by glutamate-gated chloride channels that are members of the ‘cys-loop’ ligand-gated anion channel superfamily found only in invertebrates. The subunits of glutamate-activated chloride channel have been isolated from C. elegans and from Drosophila (Blanke and VanDongen, 2009).


List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide ( (OECD, 2015). More help

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. Available from:

Calabrese B, Saffin JM, Halpain S. Activity-dependent dendritic spine shrinkage and growth involve downregulation of cofilin via distinct mechanisms. PLoS One., 2014, 16;9(4):e94787.

Chimura T., Launey T., Yoshida N.,Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo PLOS ONE, 2015, |DOI:10.1371/journal.pone.0125119.

Deshpande, L. S., D. S. Carter, K. F. Phillips, R. E. Blair and R. J. DeLorenzo (2014), "Development of status epilepticus, sustained calcium elevations and neuronal injury in a rat survival model of lethal paraoxon intoxication”, NeuroToxicology 44: 17-26. DOI: 10.1016/j.neuro.2014.04.006.

Frega M, Pasquale V, Tedesco M, Marcoli M, Contestabile A, Nanni M, Bonzano L, Maura G, Chiappalone M., Cortical cultures coupled to micro-electrode arrays: a novel approach to perform in vitro excitotoxicity testing. Neurotoxicol Teratol. 2012: 34(1):116-27.

Garcia-Reyero, N., L. Escalon, E. Prats, M. Faria, A. M. V. M. Soares and D. Raldúa (2016), "Targeted Gene Expression in Zebrafish Exposed to Chlorpyrifos-Oxon Confirms Phenotype-Specific Mechanisms Leading to Adverse Outcomes”, Bulletin of Environmental Contamination and Toxicology 96(6): 707-713. DOI: 10.1007/s00128-016-1798-3.

Gopal K., Neurotoxic effects of mercury on auditory cortex networks growing on microelectrode arrays: a preliminary analysis. Neurotoxicol Teratol., 2003, 25: 69-76.

Gramowski A, Schiffmann D, Gross GW., Quantification of acute neurotoxic effects of trimethyltin using neuronal networks cultures on microelectrode arrays. Neurotoxicology, 2000, 21: 331-342.

Johnstone AFM, Gross GW, Weiss D, Schroeder O, Shafer TJ.,Use of microelectrode arrays for neurotoxicity testing in the 21st century Neurotoxicology, 2000, 31: 331-350.

Keefer E, Norton S, Boyle N, Talesa V, Gross G., Acute toxicity screening of novel AChE inhibitors using neuronal networks on microelectrode arrays. Neurotoxicology, 2001, 22: 3-12.

Lantz SR, Mack CM, Wallace K, Key EF, Shafer TJ, Casida JE. Glufosinate binds N-methyl-D-aspartate receptors and increases neuronal network activity in vitro. Neurotoxicology. 2014, 45:38-47.

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

Malenka RC, Bear MF., LTP and LTD: An embarrassment of riches. Neuron, 2004, 44: 5–21.

Malleret G, Alarcon JM, Martel G, Takizawa S, Vronskaya S, Yin D, Chen IZ, Kandel ER, Shumyatsky GP., Bidirectional regulation of hippocampal long-term synaptic plasticity and its influence on opposing forms of memory". J Neurosci., 2010, 30 (10): 3813–25.

McConnell ER, McClain MA, Ross J, LeFew WR, Shafer TJ., Evaluation of multi-well microelectrode arrays for neurotoxicity screening using a chemical training set Neurotoxicology, 2012, 33: 1048-1057.

McDonough, J. H., Jr. and T. M. Shih (1997), "Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology”, Neurosci Biobehav Rev 21(5): 559-579.

Nicholls RE, Alarcon JM, Malleret G, Carroll RC, Grody M, Vronskaya S, Kandel ER., Transgenic mice lacking NMDAR-dependent LTD exhibit deficits in behavioral flexibility". Neuron, 2008, 58 (1): 104–17.

Ogasawara H, Doi T, Kawato M. Systems biology perspectives on cerebellar long-term depression. Neurosignals, 2008, 16 (4): 300–17.

Ogdon D, Stanfield P., Patch clamp techniques for single channel and whole-cell recording. Chapter 4, pages 53-78, (

Paradiso MA, Bear MF, Connors BW., Neuroscience: exploring the brain. 2007, Hagerstwon, MD: Lippincott Williams & Wilkins. p. 718. ISBN 0-7817-6003-8.

Purves D., Neuroscience (4th ed.). Sunderland, Mass: Sinauer., 2008, pp. 197–200. ISBN 0-87893-697-1.

Sekino Y, Tanaka S, Hanamura K, Yamazaki H, Sasagawa Y, Xue Y, Hayashi K, Shirao T., Activation of N-methyl-D-aspartate receptor induces a shift of drebrin distribution: disappearance from dendritic spines and appearance in dendritic shafts. Mol Cell Neurosci. 2006, 31(3):493-504.

Quintero JE, Pomerleau F, Huettl P, Johnson KW, Offord J, Gerhardt GA. 2011. Methodology for rapid measures of glutamate release in rat brain slices using ceramic-based microelectrode arrays: basic characterization and drug pharmacology. Brain Res.2011, 1401:1-9.

Wilt BA, Fitzgerald JE, Schnitzer MJ., Photon shot noise limits on optical detection of neuronal spikes and estimation of spike timing. Biophys J. 2013, 8; 104(1):51-62.

Zhao Y, Inayat S, Dikin DA, Singer JH, Ruoff RS, Troy JB., Patch clamp techniques: review of the current state of art and potential contributions from nanoengineering. Proc. IMechE 222, Part N: J. Nanoengineering and Nanosystems, 2009, 149. DOI: 10.1243/17403499JNN149.