Graphical RepresentationClick to download graphical representation template
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: firstname.lastname@example.org
Point of Contact
Anna Price (email point of contact)
- Anna Price
|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 November 30, 2016 13:05
|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||June 13, 2018 08:45|
|Reduced levels of BDNF||June 13, 2018 08:31|
|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|
|N/A, Cell injury/death||September 16, 2017 10:14|
|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|
|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|
|N/A, Cell injury/death leads to Synaptogenesis, Decreased||November 29, 2016 20:07|
|Aberrant, Dendritic morphology leads to Synaptogenesis, Decreased||November 29, 2016 20:07|
|Synaptogenesis, Decreased leads to Neuronal network function, Decreased||May 25, 2018 10:14|
|Neuronal network function, Decreased leads to Impairment, Learning and memory||January 26, 2018 10:32|
|BDNF, Reduced leads to N/A, Cell injury/death||November 29, 2016 20:07|
|Reduced, Presynaptic release of glutamate leads to Synaptogenesis, Decreased||November 29, 2016 20:07|
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.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|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||N/A, Cell injury/death||N/A, Cell injury/death|
|10||AO||341||Impairment, Learning and memory||Impairment, Learning and memory|
Relationships Between Two Key Events
(Including MIEs and AOs)
|Binding of antagonist, NMDA receptors leads to Inhibition, NMDARs||adjacent||High|
|Inhibition, NMDARs leads to Decreased, Calcium influx||adjacent||Moderate|
|Synaptogenesis, Decreased leads to Neuronal network function, Decreased||adjacent||Low|
|Decreased, Calcium influx leads to BDNF, Reduced||non-adjacent||Low|
|BDNF, Reduced leads to Aberrant, Dendritic morphology||non-adjacent||Low|
|BDNF, Reduced leads to Reduced, Presynaptic release of glutamate||non-adjacent||Low|
|N/A, Cell injury/death leads to Synaptogenesis, Decreased||non-adjacent||Low|
|Aberrant, Dendritic morphology leads to Synaptogenesis, Decreased||non-adjacent||Low|
|Neuronal network function, Decreased leads to Impairment, Learning and memory||non-adjacent||Low|
|BDNF, Reduced leads to N/A, Cell injury/death||non-adjacent||Low|
|Reduced, Presynaptic release of glutamate leads to Synaptogenesis, Decreased||non-adjacent||Low|
Life Stage Applicability
|During brain development||High|
|Monkey sp.||unidentified monkey||High||NCBI|
Overall Assessment of the AOP
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
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
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).
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|
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)
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.
Adams SM, Rivero Vaccari JC, Corriveau RA. (2004) Pronounced cell death in the absence of NMDA receptors in the developing somatosensory thalamus. J Neurosci. 24: 9441-9450.
Bai X, Twaroski D, Bosnjak ZJ. (2013) Modeling anesthetic developmental neurotoxicity using human stem cells. Semin Cardiothorac Vasc Anesth. 17: 276-287.
Bear MF. (1996) A synaptic basis for memory storage in the cerebral cortex. Proc Natl Acad Sci USA 93: 13453-13459.
Behar TN, Scott CA, Greene CL, Wen X, Smith SV, Maric D, Liu Q-Y, Colton CA, Barker JL. (1999) Glutamate acting at NMDA receptors stimulates embryonic cortical neuronal migration. J Neurosci. 19, 4449–4461.
Brigman JL, Wright T, Talani G, Prasad-Mulcare S, Jinde S, Seabold GK, et al. (2010) Loss of GluN2B-containing NMDA receptors in CA1 hippocampus and cortex impairs long-term depression, reduces dendritic spine density, and disrupts learning. J Neurosci. 30:4590–4600.
Chtitaa S, Larifb M, Ghamalia M, Bouachrinec M, Lakhlifia T. (2015) DFT-based QSAR Studies of MK801 derivatives for non competitive antagonists of NMDA using electronic and topological descriptors. Journal of Taibah University for Science. 9: 143-154.
Cohen S, Greenberg ME. (2008) Communication between the synapse and the nucleus in neuronal development, plasticity and disease. Annu Rev Cell Dev Biol. 24: 183-209.
Colon-Ramos DA. (2009) Synapse formation in developing neural circuits. Current topics in developmental biology 87: 53-79.
Cline H, Haas K. (2008) The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis. J Physiol 586: 1509-1517.
Creeley CE, Olney JW. (2013) Drug-Induced Apoptosis: Mechanism by which Alcohol and Many Other Drugs can Disrupt Brain Development. Brain Sci. 3: 1153–1181.
Ehrlich I, Klein M, Rumpel S, Malinow R. (2007) PSD-95 is required for activity-driven synapse stabilization. Proceedings of the National Academy of Sciences of the United States of America 104: 4176-4181.
Erecinska M, Cherian S, Silver IA. (2004) Energy metabolism in mammalian brain during development. Prog Neurobiol. 73: 397-445.
Espinosa JS, Wheeler DG, Tsien RW, Luo L. (2009) Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron 62:205–217.
Fredriksson A, Archer T. (2004) Neurobehavioural deficits associated with apoptotic neurodegeneration and vulnerability for ADHD. Neurotox Res. 6: 435–456.
Fritsche E, Alm H, Baumann J, Geerts L, Håkansson H, Masjosthusmann S, Witters H. (2015) Literature review on in vitro and alternative Developmental Neurotoxicity (DNT) testing methods. EFSA supporting publication 2015:EN-778. http://www.efsa.europa.eu/en/supporting/pub/778e.
Fu M, Yu X, Lu J, Zuo Y. (2012) Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo. Nature 483: 92-95.
Granger AJ, Gray JA, Lu W, Nicol RA. (2011) Genetic analysis of neuronal ionotropic glutamate receptors subunits. J Physiol. 589: 4095–4101.
Gray JA, Shi Y, Usui H, During MJ, Sakimura K, Nicoll RA. (2011) Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo. Neuron. 71:1085-101.
Guilarte TR. (1997) Glutamatergic system and developmental lead neurotoxicity. Neurotoxicology 18, 665–672.
Guilarte TR, McGlothan, JL. (1998) Hippocampal NMDA receptor mRNA undergoes subunit-specific changes during developmental lead exposure. Brain Res. 790, 98–107.
Haberny KA, Paule MG, Scallet AC, Sistare FD, Lester DS, Hanig JP, Slikker W Jr. (2002) Ontogeny of the N-methyl-D-aspartate (NMDA) receptor system and susceptibility to neurotoxicity. Toxicol Sci. 68:9-17.
Holtmaat A, Svoboda K. (2009) Experience-dependent structural synaptic plasticity in the mammalian brain. Nat Rev Neurosci. 10: 647-658.
Hwang JY, Kim YH, Ahn YH, Wie MB, Koh JY. (1999) N-Methyl-D-aspartate receptor blockade induces neuronal apoptosis in cortical culture. Exp Neurol. 159: 124-130.
Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J., Dikranian, K., Tenkova, T. I., Stefovska, V., Turski, L., Olney, J. W. (1999). Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283, 70–74.
Ikonomidou, C., Bittigau, P., Ishimaru, M. J., Wozniak, D. F., Koch, C., Genz, K., Price, M. T., Stefovska, V., Horster, F., Tenkova, T., Dikranian, K., and Olney, J. W. (2000) Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 287: 1056–1060.
Iwasato T, Datwani A, Wolf AM, Nishiyama H, Taguchi Y, Tonegawa S., et al. (2000) Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex. Nature 406: 726–731.
Johnston MV, Ishida A, Ishida WN, Matsushita HB, Nishimura A, Tsuji M. (2009) Plasticity and injury in the developing brain. Brain Dev. 31:1-10.
Kang H, Schuman EM. (1996) A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 273:1402–1406.
Klintsova AY, Greenough WT. (1999) Synaptic plasticity in cortical systems. Curr Opin Neurobiol. 9: 203-208.
Korkut A, Varnali T. (2003) Quantitative structure activity relationship (QSAR) of competitive N-methyl-D-aspartate (NMDA) antagonists. Mol Phys 101: 3285-3291.
Kovalchuk Y, Holthoff K, and Konnerth A. (2004) Neurotrophin action on a rapid timescale. Curr Opin Neurobiol 14:558–563.
Kumari M, Ticku MK. (1998). Ethanol and regulation of the NMDA receptor subunits in fetal cortical neuron. J Neurochem: 70, 1467–1473.
Lee LJ, Iwasato T, Ithoara S, Erzurumlu RS. (2005). Exuberant thalamocortical axon arborization in cortex-specific NMDAR1 knockout mice. J Comp Neurol. 485: 280–292.
Lu Y, Christian K, Lu B. (2008) BDNF: a key regulator for protein synthesis dependent LTP and long-term memory? Neurobiol Learn Mem. 89: 312–323.
Makris SL, Raffaele K, Allen S, Bowers WJ, Hass U, Alleva E, Calamandrei G, Sheets L, Amcoff P, Delrue N, Crofton KM. (2009) A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect. 117:17-25.
Manabe T. (2009) LTP. Encyclopedia of neuroscience. M D. Binder, N. Hirokawa and U. Windhorst (Eds). Springer-Verlag GmbH Berlin Heidelberg. pp 2188-2190
Mattson MP. (2008) Glutamate and neurotrophic factors in neuronal plasticity and disease. Ann N Y Acad Sci. 1144: 97-112.
McAllister AK, Katz LC, Lo DC. (1996) Neurotrophin regulation of cortical dendritic growth requires activity. Neuron 17: 1057.
McAllistair AK. (2000) Cellular and molecular mechanisms of dendrite growth. Cereb Cortex 10: 963-973.
Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ, Grant SG. (1998) Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396: 433-439.
Miyamoto K, Nakanishi H, Moriguchi S, Fukuyama N, Eto K, Wakamiya J, Murao K, Arimura K, Osame M. (2001) Involvement of enhanced sensitivity of N-methyl-D-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity. Brain Res. 901: 252-258.
Monteggia LM, Barrot M, Powell CM, Berton O, Galanis V, Gemelli T, Meuth S, Nagy A, Greene RW, Nestler EJ. (2004) Essential role of brain-derived neurotrophic factor in adult hippocampal function. Proc Natl Acad Sci USA 101: 10827-10832.
Nagappan G, Lu B. (2005) Activity-dependent modulation of the BDNF receptor TrkB: mechanisms and implications. Trends Neurosci. 28: 464-471.
OECD (1996) Guideline 422 on Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
OECD (2007) Guideline 426 on Developmental Neurotoxicity.
OECD (2011) Guideline 443 on Extended One-Generation Reproductive Toxicity Study.
OECD (2004) Series on testing and assessment number 20, Guidance document for neurotoxicity testing.
Ogura H, Aigner TG. (1993) MK-801 impairs recognition memory in rhesus monkeys: comparison with cholinergic drugs. J Pharmacol Exp Ther. 266: 60-64.
Patterson SL, Abel T, Deuel TA, Martin KC, Rose JC, and Kandel ER. (1996) Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron 16:1137–1145.
Poo MM. (2001) Neurotrophins as synaptic modulators. Nat Rev Neurosci. 2: 24–32.
Purpura DP. (1975) Dendritic differentiation in human cerebral cortex: normal and aberrant developmental patterns. Adv Neurol. 12: 91–134.
Rajan I, Witte S, Cline HT. (1999) NMDAR activity stabilizes presynaptic retinotectal axons and postsynaptic optic tectal cell dendrites in vivo. J Neurobiol. 38:357.
Rajan I, Cline HT. (1998) Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. J Neurosci. 18: 7836.
Redini-Del Negro C, Laroche S. (1993) Learning-induced increase in glutamate release in the dentate gyrus is blocked by the NMDA receptor antagonist AP5. Neurosci Res Commun. 13:157-165.
Rezvani AH. (2006) Involvement of the NMDA System in Learning and Memory. In: Levin ED, Buccafusco JJ, editors. Animal Models of Cognitive Impairment. Boca Raton (FL): CRC Press; Chapter 4. Available from: http://www.ncbi.nlm.nih.gov/books/NBK2532/
Riedel G, Platt B, Micheau J. (2003) Glutamate receptor function in learning and memory. Behav Brain Res. 140: 1-47.
Rivero Vaccari JC, Casey GP, Aleem S, Park WM, Corriveau RA. (2006) NMDA receptors promote survival in somatosensory relay nuclei by inhibiting Bax-dependent developmental cell death. Proc Natl Acad Sci USA. 103: 16971-16976.
Roberts TF, Tschida KA, Klein ME, Mooney R. (2010) Rapid spine stabilization and synaptic enhancement at the onset of behavioural learning. Nature 463: 948-952.
Rocha M, Sur M. (1995) Rapid acquisition of dendritic spines by visual thalamic neurons after blockade of N-methyl-D-aspartate receptors. Proc Natl Acad Sc. USA. 92: 8026.
Rusakov DA, Davies HA, Harrison E, Diana G, Richter-Levin G, Bliss TVP, Stewart MG. (1997) Ultrastructural synaptic correlates of spatial learning in rat hippocampus. Neuroscience 80: 69-77.
Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, et al. (1995) Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature 373: 151–155.
Schmitt U, Tanimoto N, Seeliger M, Schaeffel F, Leube RE. (2009) Detection of behavioral alterations and learning deficits in mice lacking synaptophysin. Neuroscience 162: 234-243.
Sekino Y, Kojima N, Shirao T. (2007) Role of actin cytoskeleton in dendritic spine morphogenesis. Neurochem Int. 51: 92-104.
Shimizu E, Tang YP, Rampon C, Tsien JZ. (2000) NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation. Science 290: 1170–1174.
Shimshek DR, Jensen V, Celikel T, Geng Y, Schupp B, Bus T, et al. (2006) Forebrain-specific glutamate receptor B deletion impairs spatial memory but not hippocampal field long-term potentiation. J Neurosci. 26: 8428-8440.
Silva AJ, Paylor R, Wehner JM, Tonegawa S. (1992) Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice. Science 257: 206-211.
Simon DK, Prusky GT, O’Leary DD, Constantine-Paton M (1992) N-methyl-d-aspartate receptor antagonists disrupt the formation of a mammalian neural map. Proc Natl Acad Sci USA 89: 10593-10597.
Soderling TR. (1999) Ta Ca-calmodulin-dependent protein cascade. Trends Biochem Sci. 24: 232-236.
Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, Liu G, Tsien JZ. (1999) Genetic enhancement of learning and memory in mice. Nature 401: 63-69.
Tarpey PS, Smith R, Pleasance E, Whibley A, Edkins S, Hardy C, O’Meara S, Latimer C, Dicks E, Menzies A, et al. (2009) A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nat Genet. 41: 535-543.
Toscano CD, Guilarte TR. (2005) Lead neurotoxicity: From exposure to molecular effects. Brain Res Rev. 49: 529-554.
Tsien JZ, Huerta PT, Tonegawa S. (1996) The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 87: 1327–1338.
Tyler WJ, Alonso M, Bramham CR, Pozzo-Miller LD. (2002) From acquisition to consolidation: on the role of brain-derived neurotrophic factor signaling in hippocampal-dependent learning. Learn Mem. 9: 224–237.
van der Staay FJ, Rutten K, Erb C, Blokland A. (2011) Effects of the cognition impairer MK-801 on learning and memory in mice and rats. Behav Brain Res. 220: 215-229.
Wayman GA, Lee YS, Tokomitsu H, Silva A, Soderling TR. (2008) Calmodulin-kinases: modulators of neuronal development and plasticity. Neuron 59: 914-931.
Wong EH, Kemp JA, Priestley T, Knight AR, Woodruff GN, Iversen LL. (1986) The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci U S A. 83: 7104-7108.
Woolf NJ. (1998) A structural basis for memory storage in mammals. Prog Neurobiol. 55: 59-77.
Yang G, Pan F, Gan WB. (2009) Stably maintained dendritic spines are associated with lifelong memories. Nature 462: 920-924.
Yang J, Siao CJ, Nagappan G, Marinic T, Jing D, McGrath K. (2009) Neuronal release of proBDNF. Nat Neurosci. 12: 113-115.
Yoon WJ, Won SJ, Ryu BR, Gwag BJ. (2003) Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax- cytochrome C-caspase pathway: the causative role of Ca2+deficiency. J Neurochem. 85: 525-533.
Zambre VP, Hambarde VA, Petkar NN, Patela CN, Sawanta SD. (2015) Structural investigations by in silico modeling for designing NR2B subunit selective NMDA receptor antagonists. RSC Adv. 5: 23922-23940.