Aop: 13


A descriptive title which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Binding of antagonist to NMDARs impairs cognition

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help


List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Magdalini Sachana, Sharon Munn, Anna Bal-Price

Joint Research Centre Institute for Health and Consumer Protection Systems Toxicology Unit Via E. Fermi 2749 - 21020 - Ispra (VA) -Italy

Corresponding author:

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Agnes Aggy   (email point of contact)


List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Anna Price
  • Agnes Aggy


The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment TFHA/WNT Endorsed 1.22 Included in OECD Work Plan
This AOP was last modified on April 05, 2021 18:16
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Binding of antagonist, NMDA receptors June 13, 2018 08:23
Decreased, Calcium influx June 13, 2018 08:26
Inhibition, NMDARs September 16, 2017 10:14
Impairment, Learning and memory March 16, 2020 09:20
Reduced levels of BDNF April 04, 2019 09:21
Aberrant, Dendritic morphology September 16, 2017 10:14
Decrease of synaptogenesis September 16, 2017 10:14
Decrease of neuronal network function May 28, 2018 11:36
Reduced, Presynaptic release of glutamate September 16, 2017 10:14
Cell injury/death September 11, 2020 08:27
Binding of antagonist, NMDA receptors leads to Inhibition, NMDARs June 13, 2018 08:48
Inhibition, NMDARs leads to Decreased, Calcium influx April 07, 2018 04:32
Synaptogenesis, Decreased leads to Neuronal network function, Decreased May 25, 2018 10:14
Decreased, Calcium influx leads to BDNF, Reduced June 13, 2018 09:00
BDNF, Reduced leads to Aberrant, Dendritic morphology November 29, 2016 20:07
BDNF, Reduced leads to Reduced, Presynaptic release of glutamate November 29, 2016 20:07
Cell injury/death leads to Synaptogenesis, Decreased November 29, 2016 20:07
Aberrant, Dendritic morphology leads to Synaptogenesis, Decreased November 29, 2016 20:07
Neuronal network function, Decreased leads to Impairment, Learning and memory December 03, 2019 04:44
BDNF, Reduced leads to Cell injury/death November 29, 2016 20:07
Reduced, Presynaptic release of glutamate leads to Synaptogenesis, Decreased November 29, 2016 20:07


In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

It is well documented and accepted that learning and memory processes rely on physiological functioning of the glutamate receptor N-methyl-D-aspartate (NMDAR). Both animal and human studies investigating NMDA itself, experiments with NMDAR antagonists and mutant mice lacking NMDAR subunits strongly support this statement (Rezvani, 2006). Activation of NMDARs results in long-term potentiation (LTP), which is related to increased synaptic strength, plasticity and memory formation in the hippocampus (Johnston et al., 2009). LTP induced by activation of NMDA receptors has been found to be elevated in the developing rodent brain compared to the mature brain, partially due to 'developmental switch' of the NMDAR 2A and 2B subunits (Johnston et al., 2009). Activation of the NMDAR also enhances brain derived neurotrophic factor (BDNF) release, which promotes neuronal survival, differentiation and synaptogenesis (Tyler et al., 2002; Johnston et al., 2009). Consequently, the blockage of NMDAR by chemical substances during synaptogenesis disrupts neuronal network formation resulting in the impairment of learning and memory processes (Toscano and Guilarte, 2005). This AOP is relevant to developmental neurotoxicity (DNT). The molecular initiating event (MIE) is described as the chronic binding of antagonist to NMDAR in neurons during synaptogenesis (development) in hippocampus (one of the critical brain structures for learning and memory formation). One of the chemicals that blocks NMDAR after chronic exposure is lead (Pb2+), a well-known developmental neurotoxicant.

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 201 Binding of antagonist, NMDA receptors Binding of antagonist, NMDA receptors
2 KE 52 Decreased, Calcium influx Decreased, Calcium influx
3 KE 195 Inhibition, NMDARs Inhibition, NMDARs
4 KE 381 Reduced levels of BDNF BDNF, Reduced
5 KE 382 Aberrant, Dendritic morphology Aberrant, Dendritic morphology
6 KE 385 Decrease of synaptogenesis Synaptogenesis, Decreased
7 KE 386 Decrease of neuronal network function Neuronal network function, Decreased
8 KE 383 Reduced, Presynaptic release of glutamate Reduced, Presynaptic release of glutamate
9 KE 55 Cell injury/death Cell injury/death
10 AO 341 Impairment, Learning and memory Impairment, Learning and memory

Relationships Between Two Key Events (Including MIEs and AOs)

TESTINGThis table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and WoE summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help


The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
During brain development High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. 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
Monkey sp. unidentified monkey High NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

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

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

The aim of the present AOP is to construct a linear pathway that captures the KEs and KERs that occur after binding of antagonist to NMDA receptor in neurons during development in hippocampus and cortex. All KEs of the AOP are characterised by STRONG essentiality for the AO (learning and memory impairment). Similarly, the biological plausibility in the majority of KERs is rated STRONG as there is extensive mechanistic understanding. However, the empirical support for the present KERs cannot be rated high as in most occasions the KEup and KEdowm of a KER have not been investigated simultaneously under the same experimental protocol.

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Life Stage Applicability: This AOP is applicable only for specific period of brain development that is the time of synaptogenesis. This vulnerable period of synaptogenesis appears to happen in different developmental stages across species. For example, in rodents primarily synaptogenesis occurs during the first two weeks after birth. For rhesus monkeys, this period ranges from approximately 115-day gestation up to PND 60. In humans, it starts from the third trimester of pregnancy and continues 2-3 years following birth (Bai et al., 2013). Furthermore, synaptogenesis does not happen in a uniform way in all brain regions and there are important differences between the times of appearance of the main two types of synapses (reviewed in Erecinska et al., 2004). For example, in rat hippocampus excitatory synapses are well established or fully mature within the two first postnatal weeks, whereas inhibitory synapses cannot be found prior to PND 18, after which it increases steadily to reach adult levels at PND 28. In addition, in rat neostriatal neurons the excitatory responses to both cortical and thalamic stimuli can be observed by PND 6, but the long-lasting hyperpolarization and late depolarization is never seen before PND 12.


Taxonomic Applicability: The data used to support the KERs in this AOP derives from experimental studies conducted in rats and mice or cell cultures of similar origin as well as from human epidemiological studies. The majority of the KEs in this AOP seem to be highly conserved across species. It remains to be proved if these KERs of the present AOP are also applicable for other species rather than human, primates, rats and mice.

Sex Applicability: The majority of the studies addressing the KEs and KERs of this AOP were carried out mainly in male laboratory animals. Few studies are available in females and some of them compare the effects between females and males. It appears that this AOP is applicable for both females and males.

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

1) Essentiality of the MIE: binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex



The MIE is defined and described above as the binding of antagonist to NMDA receptor in neurons during development in hippocampus and cortex (the critical brain structures for learning and memory formation). Activation of NMDA receptors results in long-term potentiation (LTP), which is related to increased synaptic strength and memory formation in the hippocampus (Johnston et al., 2009). LTP induced by activation of NMDA receptors has been found to be elevated in the developing rodent brain compared to the mature brain, partially due to "developmental switch" of the NMDAR 2A and 2B subunits (Johnston et al., 2009).

Essentiality of MIE (binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex) for AO (Impairment of learning and memory) is STRONG: It is well documented that learning and memory processes rely on physiological functioning of NMDA receptors. The essentiality of the MIE has been demonstrated in both animal and human studies investigating NMDA itself, NMDA receptors antagonists and mutant mice lacking NMDA receptor subunits (reviewed in Haberny et al., 2002; Rezvani, 2006 and Granger et al., 2011). NMDA systemically administered in rats, has been shown to potentiate cognitive functions (Rezvani, 2006). There are various studies dealing with specific NMDA receptor subunit gene knock-out leading to a variety of phenotypes. Depending on the endogenous levels of NMDAR subunits, the pattern of their expression and their importance in developmental processes, the loss of a subunit may lead from early embryonic lethality, to mild neurobehavioral impairment up to neuronal disorders that manifest learning and memory deficits (reviewed in Rezvani, 2006 and Granger et al., 2011). Mutant mice lacking NR1 gene have shown perinatal lethality, whereas transgenic mice lacking NR1 subunit in the CA1 region of the hippocampus show both defective LTP and severe deficits in both spatial and nonspatial learning (Shimizu et al., 2000; Tsien et al., 1996). A similar impairment of LTP, long-term depression (LTD), and spatial memory has been seen with CA1-specific NR2B deletion (Brigman et al. 2010). However, LTP has been normal in postnatal forebrain knock-out of NR2A in mice, even though spatial memory has been impaired, probably because of the severe reduction observed in overall excitatory transmission (Shimshek et al., 2006), while the inactivation of the same gene has led to reduced hippocampal LTP and spatial learning (Sakimura et al., 1995). Furthermore, a NR2B transgenic (Tg) line of mice has been developed, in which the NMDA-receptor function has been increased, showing both larger LTP in the hippocampus and superior learning and memory (Tang et al., 1999). Finally, depletion of both NR2A and NR2B in single neurons has shown alteration in synaptic development (Gray et al., 2011). Interestingly, during development, especially during postnatal days (PND) 7-14 in rodents, the central nervous system (CNS) exhibits increased susceptibility to toxic insults that affect NMDA receptors (Haberny et al., 2002). This increased susceptibility has been suggested to be related to the elevated expression of specific NMDA receptor subunits (Miyamoto et al., 2001). Because of the critical role of the NMDA receptor system in brain development, the exposure to antagonists of NMDA receptors can have long-lasting and severe effects (Behar et al., 1999). NMDA-receptor antagonists such as MK-801, ketamine, phencyclidine (PCP) and 2-amino-5-phosphonopentanoate (AP5 or APV) have been extensively used to study the role of NMDA in learning and memory in developing organisms. Both acute and subchronic administration of NMDA-receptor antagonists in several laboratory animals has been shown to impair performance on tasks that seem to depend upon hippocampal functions (reviewed in Rezvani, 2006; Haberny et al., 2002). The developmental neurotoxicity of several agents, including methylmercury, lead, and ethanol is also thought to result from interaction of these substances with the NMDA receptor system (Guilarte, 1997; Guilarte and McGlothan, 1998; Ikonomidou et al., 2000; Kumari and Ticku, 1998; Miyamoto et al., 2001).

Essentiality of MIE (binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex for KE (aberrant dendritic morphology) is MODERATE: The cortex-restricted knockout of NR1 causes refinement in dendritic arborisation in cortex and loss of patterning (Iwasato et al., 2000; Lee et al., 2005). Similar alteration in dendritic arbor has also been identified after depletion of both NR2A and NR2B subunits in isolated neurons (Espinosa et al., 2009). Blockade of NMDA receptor with APV has shown decrease of dendritic growth rate in some in vivo experimental approaches (Rajan et al., 1999; Rajan and Cline, 1998). However, other studies have reported increase in dendritic spine number and dendritic branching after chronic APV-treatment both in vivo and in vitro (Rocha and Sur, 1995; McAllister et al., 1996). This discrepancy is possibly attributed to the different developmental expression of NMDA receptor subunits that triggers distinct intracellular signaling pathways linking NMDAR function to different morphological findings.

Essentiality of MIE (binding of antagonist to NMDAR in neurons during synaptogenesis in hippocampus and cortex) for KE (cell death) is STRONG: The essential role of NMDA receptors in survival during early cortical development has been pointed out both in in vitro (Hwang et al., 1999; Yoon et al., 2003) and in vivo rodent studies (Ikonomidou et al., 1999). NMDA receptor deficient mice have revealed the importance of this receptor for neuronal survival during development as an approximately 2-fold increase in developmental cell death has been observed in these transgenic mice, which was caspase-3 and Bax dependent (Adams et al., 2004; Rivero Vaccari et al., 2006).

Essentiality of MIE for KE (decreased neuronal network function) is STRONG: The NMDA receptor is associated with circuit formation and function at the developmental stage of an organism as a number of antagonists of this receptor importantly disrupt the neuronal circuit (Simon et al., 1992). Hence, the nature of evidence for the essentiality of the MIE is High (Strong).

2) Essentiality of the KE (Inhibition of NMDA receptors)

Essentiality of KE (Inhibition of NMDA receptors) for AO (Impairment of learning and memory) is STRONG: The noncompetitive antagonist MK-801 has been shown to induce dose-dependent impairment of learning and memory (Wong et al., 1986) and data on rodent models has been recently reviewed in van der Staay et al. 2011. Learning impairments induced by NMDA receptor blockade using MK-801 have also been reported in nonhuman primates (Ogura and Aigner, 1993). Moreover there are human studies demonstrating that NMDA-receptor inhibition impairs learning and memory processes (reviewed in Rezvani, 2006).

3) Essentiality of the KE (Decreased Calcium influx)

Essentiality of KE (Decreased Calcium influx) for AO (Impairment of learning and memory) is STRONG: In the nervous system, many intracellular responses to modified Ca2+ levels are mediated by calcium/calmodulin-regulated protein kinases (CaMKs), a family of protein kinases that are initially modulated by binding of Ca2+ to CaM and subsequently by protein phosphorylation (Wayman et al., 2008). Multifunctional CaMKs, such as CaMKII and members of CaMK cascade (CaMKK, CaMKI and CaMKIV) are highly abundant in CNS and regulate different protein substrates (Soderling, 1999). Mice with a mutation in the alpha- CaMKII that is abundantly found in the hippocampus have shown spatial learning impairments, whereas some types of non-spatial learning peocesses have not been affected (Silva et al., 1992).

4) Essentiality of KE (Decreased levels of BDNF)

Essentiality of KE (Decreased levels of BDNF) for AO (Impairment of learning and memory) is STRONG: BDNF serves essential functions in the brain development and more specific in synaptic plasticity (Poo, 2001) and is crucial for learning and memory processes (Lu et al., 2008). The action of BDNF signaling on synapses happens within seconds of its release (Kovalchuk et al., 2004) and strengthens LTP processes, a cellular model for learning and memory, via sustained TrkB activation as a result of elevated transcription of BDNF (Kang and Schuman, 1996; Nagappan and Lu, 2005). This positive transcriptional feedback happens through TrkB-mediated CREB activation and increases gene transcription of BDNF (Lu et al., 2008). Furthermore, there are experimental evidence showing that loss of BDNF through transgenic models or pharmacological manipulation leads to impaired LTP (Patterson et al., 1996; Monteggia et al., 2004) and decreased learning and memory (Lu et al., 2008). The important role for BDNF in LTP and learning and memory is suggested from numerous studies in rodents. Hippocampal LTP is impaired in mice lacking BDNF in their neurons, and BDNF enhances LTP in the hippocampus and visual cortex (reviewed in Mattson, 2008). BDNF can also be released from neurons during LTP and possibly recycled and used for LTP maintenance. In learning and memory enhancement studies, it has been found that dietary energy restriction (which enhances synaptic plasticity) increases the production of BDNF and glial cells derived neurotrophic factor (reviewed in Mattson, 2008). In humans, a common single-nucleotide polymorphism in the Bdnf gene results in poor performance on memory tasks and may contribute to the pathogenesis of depression and anxiety disorders (reviewed in Cohen and Greenberg, 2008). Similarly, the transgenic mice with such mutation display defects in learning and memory tasks as well as anxiety-related behaviours (reviewed in Cohen and Greenberg, 2008). BDNF has also been shown to play pivotal role in a variety of learning paradigms in a variety of animal models such as mice, monkeys, zebra finches and chicks (reviewed in Tyler et al., 2002).

5) Essentiality of KE (Cell death)

Essentiality of KE (Cell death) for AO (Impairment of learning and memory) is STRONG: Several experimental studies dealing with postnatal administration of NMDA receptor antagonists such as MK-801, ketamine or ethanol have shown a devastating cell apoptotic degeneration in several brain regions of animals models, resulting in learning deficits (reviewed in Fredriksson and Archer, 2004; Creeley and Olney, 2013). The apoptosis induced in developing brain after exposure to NMDA receptor antagonists is not reversible although the developing brain has plasticity properties that may allow to a certain degree to compensate for neuronal losses. This severe bilaterally symmetrical neuronal losses in both hemispheres that occurs by treatment with NMDA receptor antagonists leads to neurobehavioral disorders including learning and memory deficits (Creeley and Olney, 2013).

6) Essentiality of the KE (Decreased presynaptic release of glutamate)

Essentiality of KE (Decreased presynaptic release of glutamate) for AO (Impairment of learning and memory) is STRONG: Riedel et al. 2003 have reviewed data available that is related to the understanding of the role of glutamate and its different receptor subtypes in learning and memory, focusing mainly in psychopharmacological in vivo studies conducted in rodents and primates. Furthermore, this review has included literature on long-term potentiation (LTP) and long-term depression (LTD), the most commonly used models for studying the cellular mechanisms underlying memory formation in relation to glutamate rather than exploring relevant mechanistic data. Classical conditioning of a tone-shock association (commonly used to study learning and memory) causes a lasting increase in glutamate release in dentate gyrus synaptosomes, whereas blockade of NMDA receptors during learning prevents conditioning and the change in glutamate release (Redini-Del Negro and Laroche, 1993). It is worth mentioning that there are two types of LTP, the NMDA receptor-dependent and the NMDA receptor-independent. The later type of LTP is induced presynaptically and strongly activates presynaptic Ca2+ channels, which results in an increase in cAMP and activation of protein kinase A that is believed to be involved in the long-lasting enhancement of glutamate release from the presynaptic terminal. This type of LTP has been observed at mossy fiber-CA3 synapses in the hippocampus or at parallel fiber-Purkinje cell synapses in the cerebellum (Manabe, 2009).

7) Essentiality of the KE (Aberrant dendritic morphology)

Essentiality of KE (Aberrant dendritic morphology) for AO (Impairment of learning and memory) is STRONG: Spine morphology is considered to be an important morphological unit for establishing learning and memory (Sekino et al., 2007). As dendrites are the postsynaptic site of most synaptic contacts, dendritic development determines the number and pattern of synapses received by each neuron (McAllistair, 2000). Defects induced in dendritic growth are often leading to severe neurodevelopmental disorders such as mental retardation (Purpura, 1975). Thus, the proper growth and arborization of dendrites are crucial for proper functioning of the nervous system. Changes in spine formation have been found to be involved in impairment of learning and memory in live animals (Yang et al. 2009; Roberts et al. 2010). Electrical activity-dependent changes in the number as well as in the size and shape of dendritic spines have been strongly related to some forms of learning (reviewed in Holtmaat and Svoboda, 2009). In mouse, motor cortex learning leads to dendritic spine remodeling associated with the degree of behavioral improvement suggesting a crucial role for structural plasticity during memory formation (Yang et al., 2009 and Fu et al., 2012). Furthermore, accumulating evidence indicates that experience-dependent plasticity of specific circuits in the somatosensory and visual cortex involves structural changes at dendritic spines (Holtmaat and Svoboda, 2009).

8) Essentiality of the KE (Decreased synaptogenesis)

Essentiality of KE (Decreased synaptogenesis) for AO (Impairment of learning and memory) is STRONG: Learning and memory result from plastic events that modify the way neurons communicate with each other (Bear, 1996). Plastic events are considered changes in the structure, distribution and number of synapses and it has been suggested that morphological events like these underlie memory formation (Rusakov et al., 1997; Woolf, 1998; Klintsova and Greenough, 1999). In mutant mice lacking PSD-95, it has been recorded increase of NMDA-dependent LTP, at different frequencies of synaptic stimulation that cause severe impaired spatial learning, without thought affecting the synaptic NMDA receptor currents, subunit expression, localization and synaptic morphology (Migaud et al., 1998). Furthermore, recent genetic screening in human subjects and neurobehavioural studies in transgenic mice have suggested that loss of synaptophysin plays important role in mental retardation and/or learning deficits (Schmitt et al., 2009; Tarpey et al., 2009).

9) Essentiality of the KE (Decreased neuronal network function)

Essentiality of KE (Decreased neuronal network function ) for AO (Impairment of learning and memory) is STRONG: It is well understood and documented that the ability of neurons to communicate with each other is based on neural network formation that relies on functional synapse establishment (Colón-Ramos, 2009). The connectivity and functionality of neural networks depends on where and when synapses are formed. Therefore, the decreased synapse formation during the process of synaptogenesis is detrimental and leads to decrease of neural network formation and function. The neuronal electrical activity dependence on synapse formation and is critical for proper neuronal communication. Alterations in synaptic connectivity lead to refinement of neuronal networks during development (Cline and Haas, 2008). Indeed, knockdown of PSD-95 (postsynaptic protein) blocks the functional and morphological development of glutamatergic synapses (Ehrlich et al., 2007).

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

The table provides a summary of the biological plausibility and empirical support for each KER described in this AOP based on "Annex 1: Guidance for assessing relative level of confidence in the overall AOP based on rank ordered elements" found in User's Handbook.

More information about the evidence that support these KERs and the relevant literature can be found in each KER description.

The main reason for the overall scoring is that for the majority of KERs, the KEup and KEdown have not been investigated simultaneously in the same study.

KERs WoE Biological plausibility Does KEup occurs at lower doses than KEdown? Does KEup occurs at earlier time points than KE down? Is there higher incidence of KEup than of KEdown? Inconsistencies/Uncertainties
NMDARs, Binding of antagonist Directly Leads to NMDARs, Inhibition Extensive understanding N/A Yes N/A Limited conflicting data
NMDARs, Inhibition Directly Leads to Calcium influx, Decreased Extensive understanding Same dose Yes Not investigated Limited conficting data
Calcium influx, Decreased Indirectly Leads to Release of BDNF, Reduced Extensive understanding Not investigated Not investigated Not investigated Limited conficting data
Release of BDNF, Reduced Indirectly Leads to Dendritic morphology, Aberrant Extensive understanding Not investigated Yes Not investigated No conflicting data
Release of BDNF, Reduced Indirectly Leads to Cell death, N/A Extensive understanding Not investigated Yes Not investigated Limited conficting data
Release of BDNF, Reduced Indirectly Leads to Presynaptic release of glutamate, Reduced Extensive understanding Not investigated Not investigated Not investigated Limited conficting data
Cell death, N/A Indirectly Leads to Synaptogenesis, Decreased Extensive understanding Not investigated Yes Not investigated Limited conficting data
Dendritic morphology, Aberrant Indirectly Leads to Synaptogenesis, Decreased Extensive understanding Not investigated Not always Not investigated No conflicting data
Presynaptic release of glutamate, Reduced Indirectly Leads to Synaptogenesis, Decreased Extensive understanding Not investigated Not investigated Not investigated No conflicting data
Synaptogenesis, Decreased Directly Leads to Neuronal network function, Decreased Extensive understanding Not investigated Not investigated Not investigated No conflicting data
Neuronal network function, Decreased Indirectly Leads to Learning and memory, Impairment Scientific understanding is not completely established Not investigated Yes Not investigated Limited conficting data

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

A quantitative structure activity relationship (QSAR) model has been developed based on various molecular parameters that have been calculated for a series of competitive NMDA antagonists with known activity values and these parameters have been applied to make a regression analysis which provides a model that relates the computationally calculated parameters to experimentally determined activity values (Korkut and Varnali, 2003).

Recently, a QSAR model for non- competitive antagonists of NMDA receptor based on a series of 48 substituted MK-801 derivatives has been established (Chtitaa et al., 2015). In this paper, a quantitative model has been proposed and there has also been an attempt to interpret the activity of the compounds relying on the multivariate statistical analyses. By this approach, they have been able to predict the inhibitory activity of a set of new designed compounds (Chtitaa et al., 2015).

2D- and 3D-QSAR models have also been developed to establish the structural requirements for pyrazine and related derivatives for being NR2B selective NMDA receptor antagonists (Zambre et al., 2015).

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

Exposure to xenobiotics can potentially affect the nervous system resulting in neurobehavioral alterations and/or neurological clinical symptoms. To assess the neurotoxic properties of compounds, current testing largely relies on neurobehavioural tests in laboratory animals, histopathological analysis, neurochemical and occasionally electrophysiological observations. Throughout the years, a significant number of methods have been developed to assess neurobehaviour in laboratory animals and a comprehensive summary of them can be found in OECD Series on testing and assessment, number 20, Guidance Document for Neurotoxicity Testing (2004). Learning and memory is an important endpoint and a wide variety of tests to assess chemical effects on cognitive functions is available and used for the study of neurotoxicity in adult and young laboratory animals. Some of these tests that allow the appreciation of cognitive function in laboratory animals are: habituation, ethologically based anxiety tests (elevated plus maze test, black and white box test, social interaction test), conditioned taste aversion (CTA), active avoidance, passive avoidance, spatial mazes (Morris water maze, Biel water maze, T-maze), conditional discrimination (simple discrimination, matching to sample), delayed discrimination (delayed matching-to-sample, delayed alternation) and eye-blink conditioning.

The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 and OECD 426, respectively) require testing of learning and memory. These DNT Guidelines have been used to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009). Also in the scope of the OECD Test Guideline for Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test (422) and OECD Test Guideline for Extended One-Generation Reproductive Toxicity Study (443), learning and memory testing may have potential to be applied in the context of DNT studies. These DNT guidelines are based entirely on in vivo experiments, which are costly, time consuming, and unsuitable for testing a larger number of chemicals. For these reasons, there is currently no regulatory request for DNT studies prior to registration of new chemicals and recommendations for DNT testing are only based on certain triggers such as structural similarity with known reproductive toxicants, concerns for endocrine disruption, results from other toxicity studies, and the anticipated use and human exposure patterns. At the same time the published data strongly suggest that environmental chemicals contribute to the observed increase in children neurodevelopmental disorders such as lowered IQ, learning disabilities, attention deficit hyperactivity disorder (ADHD) and, in particular, autism. This highlights the pressing need for standardised alternative methodologies that can more rapidly and cost-effectively screen large numbers of chemicals for their potential to cause cognitive deficit in children.

The present AOP can encourage the development of new in vitro assays test battery and the use of these alternatives to assess NMDAR inhibitors as chemicals with potential to induce impairment of children cognitive function and at the same time reduce the use of in vivo studies. Some of the KEs presented in this AOP have already been identified as endpoints to be measured during the mapping of available in vitro and alternative DNT testing methods by EFSA (Fritsche et al., 2015). In addition, the majority of KEs in this AOP has strong essentiality to induce the AO (impairment of learning and memory) and established indirect relationship with the AO that would allow not only the development of testing methods that address these specific KEs but also the understanding of the relationship between the measured KEs and the AO. The present AOP can potentially provide the basis for development of a mechanistically informed IATA for DNT. The construction of IATA for predicting DNT effects is expected to make use of more than one AOP within an interconnected network in order to take into consideration all possible biological processes that may contribute to impairment of learning and memory in developing organisms. Through this network, common KEs can emerge that should be considered during IATA construction and that may inform also assay development.

Results derived from assays based on the KEs of this AOP can serve to interpret and accept results that derive from non-standard test methods. Omics data from toxicogenomic, transcriptomic, proteomic, and metabolomic studies can be interpreted in a structured way following this AOP as a guide. Finally, this AOP could provide the opportunity to group chemicals using not only chemical properties but also mechanistic information that can later inform data gap filling by read-across.


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