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AOP ID and Title:


AOP 12: Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development leads to neurodegeneration with impairment in learning and memory in aging
Short Title: Binding of antagonist to NMDARs can lead to neuroinflammation and neurodegeneration

Graphical Representation


Authors


Florianne Tschudi-Monnet, Department of Physiology, University of Lausanne, Switzerland, and Swiss Center for Applied Human Toxicology (SCAHT), Florianne.Tschudi-Monnet@unil.ch, corresponding author

Rex FitzGerald, SCAHT, Universität Basel, Missionsstrasse 64, CH-4055 Basel, Rex.FitzGerald@unibas.ch

Acknowledgments: The authors greatly acknowledged the contribution of Drs Anna Price and Magda Sachana who prepared the MIE and KE1-KE4 as well as the related KERs of this AOP.

Anna Price, Joint Research Centre Institute for Health and Consumer Protection Systems Toxicology Unit Via E. Fermi 2749 - 21020 - Ispra (VA) -Italy, e-mail address: PRICE Anna <Anna.PRICE@ec.europa.eu>

Magdalini Sachana, Joint Research Centre Institute for Health and Consumer Protection Systems Toxicology Unit Via E. Fermi 2749 - 21020 - Ispra (VA) -Italy, present e-mail address: "Magdalini.SACHANA@oecd.org" <Magdalini.SACHANA@oecd.org>


Status

Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Approved 1.13 Included in OECD Work Plan

Abstract


This AOP is an extension of AOP 13 linking NMDAR chronic inhibition during brain development to impairment of learning and memory. It links chronic NMDA receptors inhibition during brain development to Adverse Outcomes, i.e. neurodegeneration in hippocampus and cortex with amyloid plaque deposition and tau hyperphosphorylation and impairment of learning and memory, which are considered as hallmark of Alzheimer's disease. It introduces another KE, Neuroinflammation, which is involved in several neurodegenerative diseases. With Neuroinflammation and Neurodegeneration, this AOP connects to AOP 48, where in adult brain, « neuroinflammation » leads to « Neurodegeneration » ; « Neurodegeneration » leads to « Decreased neuronal network function », which finally leads to « Impairement of learning and memory ». Both neurodegeneration and cognitive deficits are observed in Alzheimer’s pathology. But as neurodegenerative diseases are complex and multifactorial, the authors proposed two Adverse outcomes: one at the organism level « Impairment of learning and memory», and one at the organ level, « neurodegeneration ». Both are regulatory endpoints. This AOP integrates in the network of AOPs relative to neurotoxicity testing.

This AOP is based on the hypothesis of Landrigan and coworkers (2005) proposing an early origin of neurodegenerative diseases in later life. The chemical initiator known to block NMDARs and used in this AOP for the empirical support is lead (Pb), which is a well-known developmental neurotoxicant. In epidemiological studies of adults, cumulative lifetime lead exposure has been associated with accelerated decline in cognition (Bakulski et al., 2012), suggesting that long term exposure to lead during brain development or occupational exposure in adulthood increases the risk to develop a neurodegenerative disease of Alzheimer's type. The long latency period between exposure and late-onset of neurodegeneration and cognitive deficits gives a very broad life-stage applicability, where developmental exposure has consequences in the aging brain. Such a long temporal delay between exposure and adverse outcome is a real difficulty and challenge for neurotoxicity testing. As the Key Event « Neuroinflammation » appears to play a crucial role in the neurodegenerative process, the authors propose to include the measurement of this apical KE in the battery of regulation-required neurotoxicity testing.



Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
1 MIE 201 Binding of antagonist, NMDA receptors Binding of antagonist, NMDA receptors
2 KE 195 Inhibition, NMDARs Inhibition, NMDARs
3 KE 52 Decreased, Calcium influx Decreased, Calcium influx
4 KE 381 Reduced levels of BDNF BDNF, Reduced
5 KE 55 N/A, Cell injury/death N/A, Cell injury/death
6 KE 188 N/A, Neuroinflammation N/A, Neuroinflammation
7 AO 352 N/A, Neurodegeneration N/A, Neurodegeneration
8 AO 341 Impairment, Learning and memory Impairment, Learning and memory

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Binding of antagonist, NMDA receptors adjacent Inhibition, NMDARs High
Inhibition, NMDARs adjacent Decreased, Calcium influx High
Decreased, Calcium influx adjacent Reduced levels of BDNF High
Reduced levels of BDNF adjacent N/A, Cell injury/death High
N/A, Cell injury/death adjacent N/A, Neuroinflammation Moderate
N/A, Neuroinflammation adjacent N/A, Neurodegeneration Moderate
N/A, Neurodegeneration adjacent Impairment, Learning and memory High
N/A, Neurodegeneration adjacent N/A, Neuroinflammation Moderate

Stressors


Name Evidence
Lead

Overall Assessment of the AOP

The aim of this AOP is to capture the KEs and KERs that occur after chronic binding of antagonist to NMDA receptors in neurons of hippocampus and cortex during brain development and that lead to neurodegeneration with impairment in learning and memory in later life.  Neurodegenreation with accumulation of amyloid plaques and hyperphosphorylated tau, as well as cognitive deficit are associated with Alzheimer-type neurodegeneration. Currently, the hypothesis of Landrigan et al., (2005) of developmental origins of neurodegenerative diseases has been demonstrated in monkeys, in rats, mice  and in zebrafish following Pb treatment (Zawia and Basha, 2005; Basha and Reddy, 2010; Bihaqui et al., 2013; Bihaqui et al., 2014 ; Lee and Freeman, 2014). There is strong agreement that Alzheimer's disease is progressive and that neurodegeneration is occuring mainly in hippocampus and cortex, associated with cognitive deficits (Schoemaker et al., 2014). This AOP uses the MIE and several KEs of the AOP 13 entitled "Binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities ", with an additional KE: neuroinflammation and two AOs: an AO at the organ level: Neurodegeneration in hippocampus and cortex and an AO at the organism level: Impairment of learning and memory.  Impairment of learning and memory is the same AO as in  AOP 13, but the point is that this AO is detected when the brain is aging, and it is due to neurodegeneration with accumulation of amyloid peptides and tau hyperphosphorylation. The recent review by Taratglione and coworkers (2016) is a very good summary of the challenges and experimental studies described in this AOP.

 

Developmental Pb exposure has adverse effects on cognitive functioning that can persist into adulthood and may be exacerbated with aging (Schneider et al., 2013). Such delayed effects may be due to epigenetic effects of developmental Pb exposure on DNA methylation mediated at least in part through dysregulation of methyltransferases observed often at the lowest level of exposure (Schneider et al., 2013). In addition, key neurodevelopmental events, such as neural differentiation, cell migration and network formation may be modulated by Pb exposure, predisposing the brain for alterations in higher brain functions, such as learning and memory, and this at different ages (for review, see Aschner et al., 2017). The fact that neuroinflammation trigggered during early brain development was shown to cause Alzheimer pathology when aging (Krstic et al., 2012), suggests that chronic neuroinflammation may play a causal role in cognitive decline in aging. A recent report described a mechanistic link between chronic inflammation and aging microglia; and a causal role of aging microglia in neurodegenerative cognitive deficits: A sirtuin 1 (SIRT1) deficiency was observed in aging microglia, leading to a selective activation of IL1-b transcription mediated through hypomethylation of IL-1b proximal promoter exacerbating aging or tau-associated cognitive deficits (Cho et al. 2015). Taken together, these data suggest that Pb-induced neuroinflammation during brain development may underlie the delayed effects on cognitive deficits in aging, as depicted in the proposed AOP


Domain of Applicability

Life Stage Applicability
Life Stage Evidence
During brain development
Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Low NCBI
Monkey Monkey High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus Moderate NCBI
zebrafish Danio rerio Moderate NCBI

This AOP is not sex dependent. Regarding the life stage applicability, MIE induced during brain development can have consequences when brain is aging, according to the hypothesis proposed by Landrigan and coworkers (2005). However, it is also possible that the AO does not depend exclusively on developmental exposure, since cumulative occupational exposure also decreased cognitive functions in aging (Stewart et al., 2006).

Essentiality of the Key Events

Table: Essentiality of KEs

2 Support for Essentiality of KEs

Defining Question

Are downstream KEs and/or the AO prevented if an upstream KE is blocked ?

High (Strong)

Moderate

Low (Weak)

Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, knock out models, etc.)

Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO

No or contradictory experimental evidence of the essentiality of any of the KEs

KE1

NMDARs inhibition

STRONG

Activation of NMDAR results in LTP, which is related to increase synaptic strength and memory formation in hippocampus (Johnston et al., 2009).

KE2

Calcium influx decreased

STRONG

In CNS, many intracellular responses to modified calcium level are mediated by calcium/calmoduline-regulated protein kinases (Wayman et al., 2008). Mice with a mutation of calmoduline kinase II, which is abundantly found in hippocampus, have shown spatial learning impairment (Silva et al., 1992)

KE3

Release of BDNF, reduced

STRONG

BDNF serves essential function in synaptic plasticity (Poo, 2001) and is crucial for learning and memory processes (Lu et al., 2008). Precursor form of BDNF and mature BDNF are decreased in the preclinical stages of Alzheimer's disease (Peng et al., 2005)

KE4

Cell Injury/death, increased

STRONG

Several studies dealing with postnatal administration of NMDAR antagonists such as MK 801, ketamine or ethanol have shown a devastating cell apoptotic degeneration in several brain areas of animal models resulting in learning deficits (Creeley and Olney, 2013)

KE5

Neuroinflammation

MODERATE

Rationale: Rats treated with Pb from PND 24 to 80 showed a neuroinflammatory response associated with neuronal death in hippocampus and LTP impairment. These effects were significantly reversed by administration of minocycline, an antibiotic known to block microglial reactivity (Liu et al., 2012), demonstrating the essentiality of neuroinflammation for neurodegeneration in hippocampus and impairment of memory processes. In addition, the fact that neuroinflammation triggered during brain development by a systemic immune challenge caused Alzheimer's like pathology (Krstic et al., 2012), showed the central role of neuroinflammation in this pathology. In addition, in a mouse model of Alzheimer's disease, the blockade of microglial cell proliferation and the shifting of the microglial inflammatory profile to an anti-inflammatory phenotype by inhibiting the colony-stimulating factor 1 receptor on microglial cells, prevented synaptic degeneration and improved cognitive functions (Olmos-Alonso et al., 2016). This latter experiment has not been done during brain development. But the hypothesis is that a chronic neuroinflammation during a prolonged period increased the risk to develop an Alzheimer's neurodegenerative disease in aging (Krstic and Knuesel, 2013).

However, as other mechanisms such epigenetic modifications can lead to accumulation of amyloid plaques- and tau hyperphosphorylation-related neurodegeneration, and due to some inconsistencies of anti-inflammatory treatments as protection against the neurodegenerative process, the essentiality of Neuroinflammation was considered as moderate.

AO (at organ level)

Neurodegeneration in

 hippocampus and cortex

STRONG

Several studies descibed Pb-induced accumulation of amyloid peptides and hyperphosphorylated and Pb-induced cell injury/deathin hippocampus or decrease in hippocampal volume, what are all well accepted landmarks of Alzheimer's pathology (Lloret et al., 2015). As described in AOP 48, neurodegeneration can lead to "Decreased neuronal network function" which in turn leads to "impairment of learning and memory", which is also considered as a hallmark of Alzheimer's pathology (Schoemaker et al., 2014).

However, there is some controversy about the relationship between increased accumulation of amyloid plaques and increased cognitive deficits:  Lichtenstein and coworkers (2010) described that accumulation of amyloid plaques reaches a plateau, whereas a temporal relationship is observed between increased microglial activation, widespread degeneration (decreased hippocampal volume) and increased cognitive deficits. Therefore the essentiality for accumulation of amyloid and tau to cognitive deficits should be considered as moderate. But, as cell injury/death in hippocampus and cortex or decrease in hippocampal volume due to widespread neurodegeneration is strongly associated to impairment in learning and memory, the essentiality of this KE has been rated as strong.

 AO (at organism level)

Impairment of learning and memory

STRONG

Neurodegenerative diseases are complex and multifactorial, depend on gene-environment interactions, and have a slow temporal evolution (Sherer et al., 2002; Steece-Collier et al., 2002; Tsang and Soong, 2003); Mutter et al., 2004). A direct association between Pb exposure during brain development and Alzheimer's pathology is not supported by epidemiological studies. However, two studies reported that past adult exposure is linked with neurodegeneration (Stewart et al., 2006) and decline in cognitive function (Schwartz et al., 2000), effects which were observed long after exposure ceases. Tibia lead levels were good predictors of these delayed effects. Another study showed an association between lead exposure early in life with cognitive and behavioral consequences in early adulthood (Agency for toxic substances, 1997). Despite the lack of specific epidemiological evidence, the principle of delayed effects occuring long after exposure, as well as strong evidence from experimental studies (for review, see Chin-Chan et al., 2015) suggest that long-term exposure to environmental toxicants such as Pb during brain development or exposure later in life can be considered as a risk factor for the development of neurodegenerative diseases in aging.

 

Weight of Evidence Summary

1. Concordance of dose-response and temporal concordance between KEs and the AO

It is difficult to analyze the dose-response relationships between the different KEs, (i) because of the long temporal delay between MIE and AOs ; (ii) because no study has analyzed them simultaneously, and (iii)  because of the difficulties in extrapolating in vitro to in vivo data. As the apical KEs and AO occur and can be measured years after exposure, even when Pb blood level has returned to normal, measurement of bone Pb content has been proposed as a measurement of historical Pb exposure in adults (Bakulski et al., 2012, 2014).  The following table gives an overview of the doses/concentrations and exposure duration at which the different KEs were measured.

 

KE1

KE2

KE3

KE4

K5

AO at organ level

AO at organism level

NMDAR inhibition

Calcium influx, decreased

BDNF release, decreased

Cell injury/death

Neuroinflammation

Neurodegeneration with amyloid plaques and tau hyperphosphorylation

Impairment of learning and memory

 

Pb 2.5-5 mM acute

inhibits

NMDAR whole cell and channel current in hippocampal neurons

 

(Alkondon et al., 1990)

Pb 100 nM 1h-24h

decrease Ca2+ in embryonic rat hippocampal neurons

 

(Ferguson et al., 2000)

No direct evidence

 

Pb 2mM in drinking water 3 weeks before mating till weaning (PND 21) resulting in

at PND 21

Pb blood 108.8 mg/L

Pb hippoc. 0.253 mg/g

at PND 91

Pb blood 39.27 mg/L

Pb hippoc. 0.196 mg/g

 

about 35% decrease in synapses in hippocampus

 

about 30% decrease of hippocampal neurons

 

(Xiao et al., 2014)

 

In vivo

0.22 ppm (together with As and Cd) from gestational day 5 till day 180

 

in adulthood: IL-1b, TNF-a, IL-6 increased 2x

 

Ahsok et al., 2015

 

Rats exposed to Pb 100 ppm for 8 weeks (from PND 24 to 80) caused at the end of treatment microglial activation in hippocampus.

(Liu et al., 2012

 

 

In vitro 

10-6-10-4 M for 10 days

in 3D cultures of fetal rat brain cells

 

microglial and astrocyte reactivities

 

(Zurich et al., 2002)

 

co-cultures of hippocampal neurons with microglial cells treated with Pb (50 micomol/L for 48h) caused microglial activation and upregulation of IL-1beta, TNF-alpha and i_NOS

(Liu et al., 2012)

Monkeys exposed to

Pb 1.5 mg/kg/day

from birth to 400 days

 

at 23 years of age

 

Tau accumulation

Overexpression of amyloid-beta protein precursor and of amyloid-beta

enhanced pathologic neurodegeneration

 

(Bihaqi et al., 2011; Bihaqi and Zawia, 2013)

 

Mice exposed to

Pb 0.2% in drinking water from PND 1-20 or from PND 1-20 + From 3-7 months

 

at 700 days of age

 

elevated protein and mRNA for tau

and

aberrant site-specific tau hyperphosphorylation

 

(Bihaqi et al., 2014)

 

Human Tg-SWDI APP transgenic mice , PB 50 mg/kg by gavage for 6 weeks exhibit increase AB in CSF, cortex and hippocampus and increased amyloid plaque load (Gu et al., 2012)

 

Rats exposed to Pb 100 ppm for 8 weeks (from PND 24 to 80) caused at the end of treatment neuronal death in hippocampus.

(Liu et al., 2012)

 

Mice exposed to

Pb 0.2% in drinking water from PND 1-20 or from PND 1-20 and from 3-7 months

 

Tested at 700 days of age

 

Decrease in cognitive functions (Morris water maze, Y maze testing for spatial memory and memory, a hippocampal formation-dependent task)

 

(Bihaqi et al., 2014)

 

Rats exposed to Pb 100 ppm for 8 weeks (from PND 24 to 80) reduced hippocampal LTP level at the end of the treatment

(Liu et al., 2012)

 

 

Human Tg-SWDI APP transgenic mice , PB 50 mg/kg by gavage for 6 weeks showed an impaired spatial learning (Gu et al., 2012)

 

 


2. Strength, consistency and association of AO and MIE

The accepted molecular mechanism of action of the chemical initiator Pb is inhibition of NMDARs (Alkondon et al., 1990; Gavazzo et al., 2001, 2008; Guilarte et al., 1992; Omelchenko et al., 1997) and several experimental studies in rat, monkey and zebrafish linked chronic exposure to Pb during brain development to Alzheimer's-like neurodegeneration with cognitive deficits (Zawia and Basha, 2005; Basha and Reddy, 2010; Bihaqui et al., 2013; Bihaqui et al., 2014 ; Lee and Freeman, 2014). This AOP is defined by a single environmental chemical, Pb. However, other NMDAR antagonists used as general anesthetics (MK 801, phenylcyclidine, ketamine) applied during brain development may also lead to functional impairments in cognitive domains relevant to memory. The effects of these anesthetics on brain function appear to have a delayed onset, and can be very long-lasting if not permanent. In general, longer durations, higher concentrations and longer or repeated exposures tend to exacerbate impairments (for review, see Walters and Paule, 2017). The mechanisms underlying anesthetic-induced neurotoxicity are unclear, but several hypotheses have been proposed: impairment of mitochondrial integrity and function, dysregulation of intracellular calcium and neuroinflammation have all been implicated (Lei et al., 2012). Some of these mechanisms are common to the KEs described in this AOP, suggesting that such delayed effects on memory processes can be a general consequence of developmental brain exposure to NMDAR inhibitors. However, no studies have yet reported that these other NMDAR inhibitors cause amyloid plaque deposition or tau hyperphosphorylation associated with Alzheimer-like neurodegeneration when aging.

 

Interestingly, memantine, a NMDAR antagonist used in the treatment of Alzheimer's disease, was shown to improve cognitive functions (for review, see Dekundy, 2006). This might be considered as a discrepancy with the described AOP considering Pb as an antagonist of NMDAR and its potential risk to cause cognitive deficits and amyloid plaque accumulation, which are hallmarks of Alzheimer's disease. However, memantine antagonism of NMDAR is quite different (low affinity and voltage-dependent) and the window of exposure differs completely, since memantine is applied in aged patients when the disease has broken out; whereas the risk of delayed neurodegeneration described in this AOP is due to NMDAR inhibition during brain development.


3. Biological Plausibility, and empirical support

 

Defining Question

High /Strong

Moderate

Low/weak

Support for Biological Plausibility of KERs

Is there a mechanistic (i.e. structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?

Extensive understanding of the KER based on extensive previous documentation and broad acceptance

The KER is plausible based on analogy to accept biological relationship but scientific understanding is not completely established

There is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood

MIE to KE inhibition of NMDARs

 

Extensive understanding

Limited conflicting data

 

 

KE NMDAR inhibition to KE calcium influx, decreased

 

Extensive understanding

Limitied conflicting data

 

 

KE calcium influx, decreased to KE release of BDNF, decreased

 

Extensive understanding

Limited conflicting data

 

 

KE release of BDNF, decreased to KE Cell  Injury/death

 

Extensive understanding

Limited conflicting data

 

 

KE Cell injury/death to KE Neuroinflammation

 

 

The general mechanisms linking cell injury/death to neuroinflammation is well accepted. However, it is mainly descibed in adult brain. However, a neuroinflammatory response was found following Pb exposure of 3D cultures during synaptogenesis and myelination (Zurich et al., 2002). A controversy exists about apoptosis and neuroinflammation, but some empirical evidences has been provided.

 

The fact that cell injury/deat leads to neuroinflammation and that neuroinflammation leads to neurodegeneration is known as avicious circle and is involved in neurodegenerative diseases, suggesting that neuroinflammation exacerbates the neurodegenerative process (Griffin et al., 1998; 2006)

 

 

 

KE Neuroinflammation to AO Neurodegeneration in Hippocampus and cortex

 

 

In adult, the early involvement of neuroinflammation in the neurodegenerative process is widely accepted.

 

In immature brain, one study in mice link gestational induction of neuroinflammation to late neurodegeneration with accumulation of aberrant amyloid and tau (Kristic et al., 2012).

 

 

 

There is in vitro experimental data following Pb exposure linking neuroinflammation to extensive neuronal death in immature cells.

In vivo, There are several studies linking early Pb exposure to late neurodegeneration in several species. However, the mechanisms involved is epignenetic modifications of genes involved in the amyloid cascade. Such epigenetic modifications may be due to ROS released by the neuroinflammatory process (Bolin et al., 2006).

Therefore the link may be indirect and needs further analyses.

 

AO Neurodegeneration in hippocampus and cortex to KE Neuroinflammation 

Concept of vicious circle where neuroinflammation lead to neurodegeneration and vice versa (Griffin et al., 1998, 2006) 

 

 

 There are no specific empirical data for the chemical initiator Pb.

 AO Neurodegeneration in hippocampus and cortex  to AO Impairment of learning and memory

 

 The role of hippocampus in memory processes is well accepted. Alterations of LTP in hippocampus of rats exposed to Pb has been described (Liu et al., 2012), as well as preferential accumulation of hyperphosphorylated tau in frontal cortex of mice exposed during development to Pb. These mice exhibited cognitive deficit when aging (Bihaqi et al., 2014).

 

 

Quantitative Consideration

With an Adverse Outcome occurring after such a long delay after the MIE, it is extremely difficult to make a quantitative link, since the AO can occur when serum Pb levels have returned to normal. Bakulski and coworkers (2012) therefore proposed measuring Pb bone content as an index of historical Pb exposure. Similarly, Schwartz and coworkers (2000) showed that tibia Pb levels were good predictors of delayed cognitive decline of former organolead workers. Thus, Pb blood level is rather representative of acute exposure, whereas Pb bone level represent long-term accumulation.

Regarding the KER "cell injury/death to neuroinflammation", it is accepted that neuronal injury may be sufficient to trigger a neuroinflammatory response. But, because of the neuroprotective or neuroreparative potential of neuroinflammation, it is possible that the consequences of neuroinflammation will be in a first step positive, with microglia expressing the M2 phenotype. After an exposure arrest and a temporal delay (Sandström et al., 2014), or in the presence of cell death (Nakajima and Kohsaka, 2004; Hanish and Kettenmann, 2007), microglia can acquire the M1 neurodegenerative phenotype. Therefore, it is rather the qualitative phenotype of neuroinflammation that will induce neurodegeneration. However, a possible correlation of increased microglial reactivity, measured by PET, and a decrease in hippocampal volume, measured by MRI, suggests, in advanced Alzheimer's disease, a possible link between the intensity of neuroinflammation and the neurodegenerative consequences (Lichtenstein et al., 2010).

Considerations for Potential Applications of the AOP (optional)


This AOP aims at giving a conceptual framework to mechanistically understand an apical hazard, which can occur long after initial exposure; this hazard is not captured in standard regulatory neurotoxicity testing.

The KE "neuroinflammation", which is shared with other AOPs, appears to play an early and central role in the neurodegenerative process (Eikelenboom et al., 2000; Whitton, 2007; Krstic et al., 2012). Neuroinflammation is observed in most neurodegenerative diseases including Alzheimer's disease  (Whitton, 2007 ; Tansey and Goldberg, 2009 ; Niranjan, 2014 ; Verthratiky et al., 2014). Neuroinflammation can also be triggered by several classes of toxicants (Monnet-Tschudi et al., 2007). Any toxicant able to trigger a neuroinflammatory response expressing the neurodegenerative phenotype should be considered as a risk factor for neurodegenerative diseases. Therefore, testing for toxicant-induced neuroinflammation should be used as an endpoint in regulatory toxicology. The standard neurotoxicity testing does not require measurement of any marker of neuroinflammation, except for fuel additives, where testing for a potential increase in glial fibrillary acidic protein (GFAP), as marker of astrocyte reactivity, is mandatory according to US EPA (40 CFR 79 67).

The evolution of regulation towards mechanistically-driven approaches for supporting hazard identification implies also the development of in vitro testing. Three-dimensional cultures, prepared from fetal rat brain cells, exhibiting an histotypic organization comprising all types of brain cells (specifically microglial cells and astrocytes, as effector cells of neuroinflammation) and allowing long-term maintenance for repeated exposure and for studying the evolution of neuroinflammatory phenotypes, are already available (Alépée et al., 2014; Monnet-Tschudi et al., 2007 ; Sandström et al., 2014). Similar 3D cultures prepared from human pluripotent stem cells are in development (Schwartz et al., 2016; Stoppini et al., 2017).

References


Alepee N, Bahinski A, Daneshian M, De Wever B, Fritsche E, Goldberg A, et al. 2014. State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. Altex 31(4): 441-477.

Alkondon M, Costa AC, Radhakrishnan V, Aronstam RS, Albuquerque EX. 1990. Selective blockade of NMDA-activated channel currents may be implicated in learning deficits caused by lead. FEBS Lett 261(1): 124-130.

Aschner M, Ceccatelli S, Daneshian M, Fritsche E, Hasiwa N, Hartung T, et al. 2017. Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use. Altex 34(1): 49-74.

Ashok A, Rai NK, Tripathi S, Bandyopadhyay S. 2015. Exposure to As-, Cd-, and Pb-mixture induces Abeta, amyloidogenic APP processing and cognitive impairments via oxidative stress-dependent neuroinflammation in young rats. Toxicol Sci 143(1): 64-80.

Bakulski KM, Rozek LS, Dolinoy DC, Paulson HL, Hu H. 2012. Alzheimer's disease and environmental exposure to lead: the epidemiologic evidence and potential role of epigenetics. Curr Alzheimer Res 9(5): 563-573.

Basha R, Reddy GR. 2010. Developmental exposure to lead and late life abnormalities of nervous system. Indian journal of experimental biology 48(7): 636-641.

Bihaqi SW, Huang H, Wu J, Zawia NH. 2011. Infant exposure to lead (Pb) and epigenetic modifications in the aging primate brain: implications for Alzheimer's disease. J Alzheimers Dis 27(4): 819-833.

Bihaqi SW, Zawia NH. 2013. Enhanced taupathy and AD-like pathology in aged primate brains decades after infantile exposure to lead (Pb). Neurotoxicology 39: 95-101.

Bihaqi SW, Bahmani A, Adem A, Zawia NH. 2014a. Infantile postnatal exposure to lead (Pb) enhances tau expression in the cerebral cortex of aged mice: relevance to AD. Neurotoxicology 44: 114-120.

Bihaqi SW, Bahmani A, Subaiea GM, Zawia NH. 2014b. Infantile exposure to lead and late-age cognitive decline: relevance to AD. Alzheimer's & dementia : the journal of the Alzheimer's Association 10(2): 187-195.

Bolin CM, Basha R, Cox D, Zawia NH, Maloney B, Lahiri DK, et al. 2006. Exposure to lead and the developmental origin of oxidative DNA damage in the aging brain. Faseb J 20(6): 788-790.

Cho SH, Chen JA, Sayed F, Ward ME, Gao F, Nguyen TA, et al. 2015. SIRT1 deficiency in microglia contributes to cognitive decline in aging and neurodegeneration via epigenetic regulation of IL-1beta. J Neurosci 35(2): 807-818.

Dekundy A.2006. Coadministration of memantine with Acetylcholinesterase Inhibitors: Preclinical and Clinical Evidence. In: Toxicology of Organophosphate and Carbamate Compounds. Gupta RC, editor. Elsevier. Amsterdam. Chap. 4. pp 35-46.

Eikelenboom P, Rozemuller AJ, Hoozemans JJ, Veerhuis R, van Gool WA. 2000. Neuroinflammation and Alzheimer disease: clinical and therapeutic implications. Alzheimer Dis Assoc Disord 14 Suppl 1: S54-61.

Ferguson C, Kern M, Audesirk G. 2000. Nanomolar concentrations of inorganic lead increase Ca2+ efflux and decrease intracellular free Ca2+ ion concentrations in cultured rat hippocampal neurons by a calmodulin-dependent mechanism. Neurotoxicology 21(3): 365-378.

Hanisch UK, Kettenmann H. 2007. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10(11): 1387-1394.

Gavazzo P, Gazzoli A, Mazzolini M, Marchetti C. 2001. Lead inhibition of NMDA channels in native and recombinant receptors. Neuroreport 12(14): 3121-3125.

Gavazzo P, Zanardi I, Baranowska-Bosiacka I, Marchetti C. 2008. Molecular determinants of Pb2+ interaction with NMDA receptor channels. Neurochem Int 52(1-2): 329-337.

Griffin WST, Sheng JG, Royston MC, Gentleman SM, McKenzie JE, Graham DI, et al. 1998. Glial-neuronal interactions in Alzheimer's disease:  The potential role of a 'cytokine cycle' in disease progression. Brain Pathol 8: 65-72.

Griffin WS. 2006. Inflammation and neurodegenerative diseases. Am J Clin Nutr 83(2): 470S-474S.

Gu H, Robison G, Hong L, Barrea R, Wei X, Farlow MR, et al. 2012. Increased beta-amyloid deposition in Tg-SWDI transgenic mouse brain following in vivo lead exposure. Toxicol Lett 213(2): 211-219.

Guilarte TR, Miceli RC. 1992. Age-dependent effects of lead on [3H]MK-801 binding to the NMDA receptor-gated ionophore: in vitro and in vivo studies. Neurosci Lett 148(1-2): 27-30.

Krstic D, Madhusudan A, Doehner J, Vogel P, Notter T, Imhof C, Manalastas A, Hilfiker M, Pfister S, Schwerdel C, et al: Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice. J Neuroinflammation 2012, 9:151.

Krstic D, Knuesel I: Deciphering the mechanism underlying late-onset Alzheimer disease. Nat Rev Neurol 2013, 9:25-34.

Landrigan PJ, Sonawane B, Butler RN, Trasande L, Callan R, Droller D. 2005. Early environmental origins of neurodegenerative disease in later life. Environ Health Perspect 113(9): 1230-1233.

Lee J, Freeman JL. 2014. Zebrafish as a model for investigating developmental lead (Pb) neurotoxicity as a risk factor in adult neurodegenerative disease: a mini-review. Neurotoxicology 43: 57-64.

Lei X, Guo Q, Zhang J. 2012. Mechanistic insights into neurotoxicity induced by anesthetics in the developing brain. International journal of molecular sciences 13(6): 6772-6799.

Lichtenstein MP, Carriba P, Masgrau R, Pujol A, Galea E. 2010. Staging anti-inflammatory therapy in Alzheimer's disease. Frontiers in aging neuroscience 2: 142.

Monnet-Tschudi F, Zurich MG, Honegger P. 2007. Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26(4): 339-346.

Nakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc & Haematol Disorders 4: 65-84.

Niranjan R. 2014. The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson's disease: focus on astrocytes. Mol Neurobiol 49(1): 28-38.

Omelchenko IA, Nelson CS, Allen CN. 1997. Lead inhibition of N-methyl-D-aspartate receptors containing NR2A, NR2C and NR2D subunits. J Pharmacol Exp Ther 282(3): 1458-1464.

Sandström von Tobel, J., D. Zoia, et al. (2014a). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001

Schoemaker D, Gauthier S, Pruessner JC. 2014. Recollection and familiarity in aging individuals with mild cognitive impairment and Alzheimer's disease: a literature review. Neuropsychology review 24(3): 313-331.

Schneider JS, Kidd SK, Anderson DW. 2013. Influence of developmental lead exposure on expression of DNA methyltransferases and methyl cytosine-binding proteins in hippocampus. Toxicol Lett 217(1): 75-81.

Schwartz BS, Stewart WF, Bolla KI, Simon PD, Bandeen-Roche K, Gordon PB, et al. 2000. Past adult lead exposure is associated with longitudinal decline in cognitive function. Neurology 55(8): 1144-1150.

Schwartz MP, Hou Z, Propson NE, Zhang J, Engstrom CJ, Santos Costa V, et al. 2015. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity. Proc Natl Acad Sci U S A 112(40): 12516-12521.

Stewart WF, Schwartz BS, Davatzikos C, Shen D, Liu D, Wu X, et al. 2006. Past adult lead exposure is linked to neurodegeneration measured by brain MRI. Neurology 66(10): 1476-1484.

Sandstrom J, Eggermann E, Charvet I, Roux A, Toni N, Greggio C, et al. 2017. Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing. Toxicol In Vitro 38: 124-135.

Tansey MG, Goldberg MS. 2009. Neuroinflammation in Parkinson's disease: Its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis.

Verkhratsky A, Parpura V, Pekna M, Pekny M, Sofroniew M. 2014. Glia in the pathogenesis of neurodegenerative diseases. Biochemical Society transactions 42(5): 1291-1301.

Walters JL, Paule MG. 2017. Review of preclinical studies on pediatric general anesthesia-induced developmental neurotoxicity. Neurotoxicol Teratol 60: 2-23.

Whitton PS. 2007. Inflammation as a causative factor in the aetiology of Parkinson's disease. Br J Pharmacol 150(8): 963-976.

Xiao Y, Fu H, Han X, Hu X, Gu H, Chen Y, et al. 2014. Role of synaptic structural plasticity in impairments of spatial learning and memory induced by developmental lead exposure in Wistar rats. PLoS One 9(12): e115556.

Zawia NH, Basha MR. 2005. Environmental risk factors and the developmental basis for Alzheimer's disease. Rev Neurosci 16(4): 325

Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.

 


Appendix 1

List of MIEs in this AOP

Event: 201: Binding of antagonist, NMDA receptors

Short Name: Binding of antagonist, NMDA receptors

Stressors

Name
Lead

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
neuron

Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

Glu and glycine are endogenous agonists that bind to LBD of specific NMDA receptor subunits. In this binding site numerous competitive exogenous antagonists have been identified to cause closure of binding site and inhibition of NMDA receptor (reviewed in Traynelis et al., 2010). Here, are listed some known competitive antagonists for NMDA receptor, some of them are specific to NR1 subunit and some to NR2 subunit:

α-AA, α-aminoadipate;

5,7-DCKA, 5,7-dichlorokynurenic acid;

7-CKA, 7-chlorokynurenic acid;

ACEA-1011, 5-chloro-7-trifluoromethyl-1,4-dihydro-2,3-quinoxalinedione;

ACEA-1021, licostinel;

AP5, 2-amino-5-phosphonopentanoate;

AP7, 2-amino-7-phosphonopentanoate;

CGP-61594, (±)-trans-4-[2-(4-azidophyenyl)acetylamino]-5,7-dichloro-1,2,3,4-tetrahydroquinoline-2-carboxylic acid;

CGP-40116, d-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid;

CGP-43487, d-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid methyl ester;

CGP-58411, 7-chloro-4-hydroxy-3-phenyl-1H-quinolin-2-one;

CGS-19755, (2R,4S)-4-(phosphonomethyl)piperidine-2-carboxylic acid;

CPP, 4-(3-phosphonopropyl) pizerazine-2-carboxylic acid;

GV150,526A, gavestinel;

GV196,771A, (E)-4,6-dichloro-3-[(2-oxo-1-phenyl-3-pyrrolidinylidene)methyl]-1H-indole-2-carboxylic acid;

L-689,560, 4-trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline;

L-701,324, 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolone;

MDL105,519, (E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid;

PBPD, (2S,3R)-1-(biphenyl-4-carbonyl)piperazine-2,3-dicarboxylic acid;

PMPA, (R,S)-4-(phosphonomethyl)-piperazine-2-carboxylic acid;

PPDA, (2S,3R)-1-(phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid

Besides competitive antagonists, noncompetitive antagonists have also been designed like phenylethanolamine ifenprodil that interacts with the NR2B extracellular amino-terminal domain. It has been suggested that they act by stabilizing an agonist-bound state in which the receptor has a low open probability. Other more potent derivatives of ifenprodil are: α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propanol (Ro 25-6981), 1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol (Ro 63-1908), besonprodil (CI-1041), and traxoprodil mesylate (CP-101,606). Ethanol has been proposed to be a noncompetitive antagonist of NMDA receptors, binding to NR2 subunit (Nagy, 2008). Inhibition of NMDA receptor function by ethanol and interactions between ethanol and the noncompetitive NMDA receptor antagonist ifenprodil have been examined in neocortical neurons from rat and human embryonic kidney (HEK) 293 cells expressing recombinant NMDA receptors (Lovinger, 1995). Recently, a structural model has been suggested that predicts the presence of four sites of ethanol action on the NMDA receptor, each containing four pairs of positions in the NR1/NR2 subunits (reviewed in Chandrasekar, 2013). Some other antagonists can become trapped in the pore of the NMDA receptor after channel closure and these antagonists are called uncompetitive or trapping blockers. The most well studied NMDA receptor uncompetitive antagonists are Mg2+, polyamines, phencyclidine, ketamine, MK-801, memantine, amantadine, pentamidine, 9-tetrahydroaminoacridine, dextromethorphan, and its metabolite dextrorphan. MK-801 has been shown to prevent toluene-induced alterations in pattern-elicited visual-evoked potentials in vivo, suggesting the possibility that the binding site of toluene might be common with the one of MK-801 (Bale et al., 2007). However, another study suggests that toluene interference with the NMDA receptor might not be exclusively because of the binding to the channel pore (Smothers and Woodward, 2007) but it may involve some other binding sites. Lead (Pb2+) is considered a voltage independent antagonist of NMDA receptors and it is believed that possibly shares the same binding site with Zn2+ (reviewed in Neal and Guilarte, 2010; Traynelis et al., 2010). However, studies done in recombinant NR2A- and NR2B- containing NMDA receptors with mutated Zn2+ binding sites exhibit that additional structural elements, different from those important for Zn2+ binding are involved in Pb2+ binding site (reviewed in Neal and Guilarte, 2010). Similarly, there are contradicting experimental evidence and disagreement about Pb2+'s role as competitive or non-competitive antagonist (Neal and Guilarte, 2010).



Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI

The evolution of NMDAR subunits (NR1, NR2, NR3) is well-conserved throughout different species from lower organism to mammals, including humans (Ewald and Cline, 2009; Tikhonov and Magazanik, 2009; Koo and Hampson, 2010; Teng et al., 2010; Flores-soto et al., 2012).

Many of the binding sites for the noncompetitive or competitive antagonists e.g. for binding of dizocilpine (MK-801), phencyclidine, D-2-amino-5-phosphonopentanoate (AP5) and 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP) are also conserved in Drosophila (reviewed in Xia and Chiang, 2009).

Cellular membranes can be prepared from different brain areas of distinct species. Using [3H]MK-801, high affinity binding sites for MK-801 were detected in membranes of the rat brain (Woodruff et al., 1987). The same binding assay has been used in preparations from human brains mostly by patients with neurodegenerative disorders (Slater et al., 1993) as well as from different marine, avian species (Scheuhammer et al., 2008) and insects (Eldefrawi et al., 1993).


Key Event Description

Biological state: L-glutamate (Glu) is a neurotransmitter with important role in the regulation of brain development and maturation processes. Two major classes of Glu receptors, ionotropic and metabotropic, have been identified. Due to its physiological and pharmacological properties, Glu activates three classes of ionotropic receptors named: α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA receptors), 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (kainate receptors) and N-methyl-D-aspartate (NMDA receptors, NMDARs), which transduce the postsynaptic signal. Ionotropic glutamate receptors are integral membrane proteins formed by four large subunits that compose a central ion channel pore. In case of NMDA receptors, two NR1 subunits are combined with either two NR2 (NR2A, NR2B, NR2C, NR2D) subunits and less commonly are assembled together with a combination of NR2 and NR3 (A, B) subunits (reviewed in Traynelis et al., 2010). To be activated NMDA receptors require simultaneous binding of both glutamate to NR2 subunits and of glycine to either NR1 or NR3 subunits that provide the specific binding sites named extracellular ligand-binding domains (LBDs). Apart from LBDs, NMDA receptor subunits contain three more domains that are considered semiautonomous: 1) the extracellular amino-terminal domain that plays important role in assembly and trafficking of these receptors; 2) the transmembrane domain that is linked with LBD and contributes to the formation of the core of the ion channel and 3) the intracellular carboxyl-terminal domain that influences membrane targeting, stabilization, degradation and post-translation modifications.

Biological compartments: The genes of the NMDAR subunits are expressed in various tissues and are not only restricted to the nervous system. The level of expression of these receptors in neuronal and non-neuronal cells depends on: transcription, chromatin remodelling, mRNA levels, translation, stabilization of the protein, receptor assembly and trafficking, energy metabolism and numerous environmental stimuli (reviewed in Traynelis et al., 2010).

In hippocampus region of the brain, NR2A and NR2B are the most abundant NR2 family subunits. NR2A-containing NMDARs are mostly expressed synaptically, while NR2B-containing NMDARs are found both synaptically and extrasynaptically (Tovar and Westbrook, 1999).

General role in biology: NMDA receptors, when compared to the other Glu receptors, are characterized by higher affinity for Glu, slower activation and desensitisation kinetics, higher permeability for calcium (Ca2+) and susceptibility to potential-dependent blockage by magnesium ions (Mg2+). NMDA receptors are involved in fast excitatory synaptic transmission and neuronal plasticity in the central nervous system (CNS). Functions of NMDA receptors:

1. They are involved in cell signalling events converting environmental stimuli to genetic changes by regulating gene transcription and epigenetic modifications in neuronal cells (Cohen and Greenberg, 2008).

2. In NMDA receptors, the ion channel is blocked by extracellular Mg2+ and Zn2+ ions, allowing the flow of Na+ and Ca2+ ions into the cell and K+ out of the cell which is voltage-dependent. Ca2+ flux through the NMDA receptor is considered to play a critical role in pre- and post-synaptic plasticity, a cellular mechanism important for learning and memory (Barria and Malinow, 2002).

3. The NMDA receptors have been shown to play an essential role in the strengthening of synapses and neuronal differentiation, through long-term potentiation (LTP), and the weakening of synapses, through long-term depression (LTD). All these processes are implicated in the memory and learning function (Barria and Malinow, 2002).


How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? There is no OECD advised method for measuring NMDA receptor binding of antagonists. However, there are methods described in the scientific literature that allow measuring:

1. Ex vivo: The most common assay used is the NMDA receptor (MK801 site) radioligand competition binding assays (Reynolds, 2001; Gao et al., 2013; http://pdsp.med.unc.edu/UNC-CH%20Protocol%20Book.pdf; http://www.currentprotocols.com/WileyCDA/CPUnit/refId-ph0120.html). This assay is based on the use of the most potent and specific antagonist of this receptor, MK801 that is used to detect and differentiate agonists and antagonists (competitive and non-competitive) that bind to this specific site of the receptor. Also radioligand competition binding assay can be performed using D, L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity selective antagonist at the glutamate site of NMDA receptor, which is a quantitative autoradiography technique (Mugnaini et al., 1996). D-AP5, a selective N-methyl-D-aspartate (NMDA) receptor antagonist that competitively inhibits the glutamate binding site of NMDA receptors, can be studied by evoked electrical activity measurements. AP5 has been widely used to study the activity of NMDA receptors particularly with regard to researching synaptic plasticity, learning, and memory (Evans et al.,1982; Morris, 1989).

2. In silico: The prediction of NMDA receptor targeting is achievable by combining database mining, molecular docking, structure-based pharmacophore searching, and chemical similarity searching methods together (Korkut and Varnali, 2003; Koutsoukos et al., 2011; Gao et al., 2013; Mazumber and Borah, 2014; Chtitaa et al., 2015).


References

Bale AS, Jackson MD, Krantz QT, Benignus VA, Bushnell PJ, Shafer TJ, Boyes WK. (2007) Evaluating the NMDA-glutamate receptor as a site of action for toluene, in vivo. Toxicol Sci. 98: 159-66.

Barria A, Malinow R. (2002) Subunit-specific NMDA receptor trafficking to synapses. Neuron 35: 345-353.

Chandrasekar R. (2013) Alcohol and NMDA receptor: current research and future direction. Front Mol Neurosci. 6: 14.

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. Ann Rev Cell Dev Biol 24: 183-209.

Eldefrawi ME, Anis NA, Eldefrawi AT. (1993) Glutamate receptor inhibitors as potential insecticides. Arch Insect Biochem Physiol. 22: 25-39.

Evans RH, Francis AA, Jones AW, Smith DA, Watkins JC. (1982) The Effects of a Series of ω-Phosphonic α-Carboxylic Amino Acids on Electrically Evoked and Excitant Amino Acid-Induced Responses in Isolated Spinal Cord Preparations. Br J Pharmac. 75: 65-75.

Ewald RC, Cline HT. (2009) Cloning and phylogentic analysis of NMDA receptor subunits NR1, NR2A and NR2B in Xenopus laevis tadpoles. Front Mol Neurosci. 2: 4.

Flores-soto ME, Chaparro-Huerta V, Escoto-Delgadillo M, Vazuez-Valls E, Gonzalez-Castaneda RE, Beas-Zarate C. (2012) Structure and function of NMDA-type glutamate receptor subunits. Neurologia 27: 301-310.

Gao L, Fang JS, Bai XY, Zhou D, Wang YT, Liu AL, Du GH. (2013) In silico Target Fishing for the Potential Targets and Molecular Mechanisms of Baicalein as an Antiparkinsonian Agent: Discovery of the Protective Effects on NMDA Receptor-Mediated Neurotoxicity. Chem Biol Drug Des. 81: 675-87.

Koo JCP, Hampson DR. (2010) Phylogenic and evolutionary analysis of glutamate receptor based on extant invertebrate genes. JULS 1: 42-48.

Korkut A, Varnali T. (2003) Quantitative structure activity relationship (QSAR) of competitive N-methyl-D-aspartate (NMDA) antagonists. Mol Phys 101: 3285-3291.

Koutsoukas A, Simms B, Kirchmair J, Bond PJ, Whitmore AV, Zimmer S, Young MP, Jenkins JL, Glick M, Glen RC, Bender A. (2011) From in silico target prediction to multi-target drug design: current databases, methods and applications. J Proteomics 74: 2554-2574.

Lovinger DM. (1995) Developmental decrease in ethanol inhibition of N-methyl-D-aspartate receptors in rat neocortical neurons: relation to the actions of ifenprodil. J Pharmacol Exp Ther. 274: 164-172.

Mazumder MK, Borah A. (2014) Piroxicam inhibits NMDA receptor-mediated excitotoxicity through allosteric inhibition of the GluN2B subunit: An in silico study elucidating a novel mechanism of action of the drug. Medical Hypotheses 83: 740–746.

Morris RJ. (1989) Synaptic Plasticity and Learning: Selective Impairment of Learning in Rats and Blockade of Long-Term Potentiation in vivo by the N-Methyl-D-Aspartate Receptor Antagonist AP5. J Neurosci 9: 3040-3057.

Mugnaini M, van Amsterdam FT, Ratti E, Trist DG, Bowery NG. (1996) Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain. Br J Pharmacol 119: 819–828.

Nagy J. (2008) Alcohol related changes in regulation of NMDA receptor functions. Curr Neuropharmacol 6: 39-54.

Neal AP, Guilarte TR. (2010) Molecular Neurobiology of Lead (Pb2+): Effects on Synaptic Function. Mol Neurobiol. 42: 151-160.

Nihei MK, Guilarte TR. (1999) NMDAR-2A subunit protein expression is reduced in the hippocampus of rats exposed to Pb2+ during development. Brain Res Mol Brain Res. 66: 42-49.

Reynolds IJ. (2001) [3H](+)MK801 radioligand binding assay at the N-methyl-D-aspartate receptor. Curr Protoc Pharmacol. Chapter 1:Unit 1.20. doi: 10.1002/0471141755.ph0120s11.

Scheuhammer AM, Basu N, Burgess NM, Elliott JE, Campbell GD, Wayland M, Champoux L, Rodrigue J. (2008) Relationships among mercury, selenium, and neurochemical parameters in common loons (Gavia immer) and bald eagles (Haliaeetus leucocephalus). Ecotoxicology 17: 93-101.

Slater P, McConnell SE, D'Souza SW, Barson AJ. (1993) Postnatal changes in N-methyl-D-aspartate receptor binding and stimulation by glutamate and glycine of [3H]-MK-801 binding in human temporal cortex. Br J Pharmacol. 108: 1143-1149.

Smothers CT, Woodward JJ. (2007) Pharmacological characterization of glycine-activated currents in HEK 293 cells expressing N-methyl-d-aspartate NR1 and NR3 subunits. J Pharmacol Exp Ther. 322: 739-748.

Teng H, Cai W, Zhou L, Zhang J, Liu Q, Wang Y, et al. (2010) Evolutionary mode of functional divergence of vertebrate NMDA receptor subunit 2 Genes. PLoS ONE. 5(10): e13342.

Tikhonov DB, Magazanik LG. (2009) Origin and molecular evolution of ionotropic glutamate receptors. Neurosci Behav Physiol. 39: 763-772.

Tovar KR, Westbrook GL. (1999) The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci. 19: 4180–4188.

Traynelis S, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R. (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 62: 405-496.

Woodruff GN, Foster AC, Gill R, Kemp JA, Wong EH, Iversen LL. (1987) The interaction between MK-801 and receptors for N-methyl-D-aspartate: functional consequences. Neuropharmacology 26(7B): 903-909.

Xia S, Chiang AS. (2009) NMDA Receptors in Drosophila. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press; Chapter 10. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5286/


List of Key Events in the AOP

Event: 195: Inhibition, NMDARs

Short Name: Inhibition, NMDARs

Key Event Component

Process Object Action
NMDA glutamate receptor activity NMDA selective glutamate receptor complex decreased

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
neuron

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI

The cellular expression of the NMDAR subunits has been studied in both adult human cortex and hippocampus (Scherzer et al., 1998) as well as during the development of the human hippocampal formation (Law et al., 2003). The whole-cell patch clamp recording techniques have been used in NMDA receptors expressed in human TsA cells (derivative of the human embryonic kidney cell line HEK-293) (Ludolph et al., 2010). Cell-attached single-channel recordings of NMDA channels has been carried out in human dentate gyrus granule cells acutely dissociated from slices prepared from hippocampi surgically removed from human patients (Lieberman and Mody, 1999).

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


Key Event Description

Biological state: L-glutamate (Glu) is a neurotransmitter with important role in the regulation of brain development and maturation processes. Two major classes of Glu receptors, ionotropic and metabotropic, have been identified. Due to its physiological and pharmacological properties, Glu activates three classes of ionotropic receptors named: α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA receptors), 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (kainate receptors) and N-methyl-D-aspartate (NMDA receptors, NMDARs), which transduce the postsynaptic signal. Ionotropic glutamate receptors are integral membrane proteins formed by four large subunits that compose a central ion channel pore. In case of NMDA receptors, two NR1 subunits are combined with either two NR2 (NR2A, NR2B, NR2C, NR2D) subunits and less commonly are assembled together with a combination of NR2 and NR3 (A, B) subunits (reviewed in Traynelis et al., 2010). To be activated NMDA receptors require simultaneous binding of both glutamate to NR2 subunits and of glycine to either NR1 or NR3 subunits that provide the specific binding sites named extracellular ligand-binding domains (LBDs). Apart from LBDs, NMDA receptor subunits contain three more domains that are considered semiautonomous: 1) the extracellular amino-terminal domain that plays important role in assembly and trafficking of these receptors; 2) the transmembrane domain that is linked with LBD and contributes to the formation of the core of the ion channel and 3) the intracellular carboxyl-terminal domain that influences membrane targeting, stabilization, degradation and post-translation modifications.

Biological compartments: The genes of the NMDAR subunits are expressed in various tissues and are not only restricted to the nervous system. The level of expression of these receptors in neuronal and non-neuronal cells depends on: transcription, chromatin remodelling, mRNA levels, translation, stabilization of the protein, receptor assembly and trafficking, energy metabolism and numerous environmental stimuli (reviewed in Traynelis et al., 2010).

In hippocampus region of the brain, NR2A and NR2B are the most abundant NR2 family subunits. NR2A-containing NMDARs are mostly expressed synaptically, while NR2B-containing NMDARs are found both synaptically and extrasynaptically (Tovar and Westbrook, 1999).

General role in biology: NMDA receptors, when compared to the other Glu receptors, are characterized by higher affinity for Glu, slower activation and desensitisation kinetics, higher permeability for calcium (Ca2+) and susceptibility to potential-dependent blockage by magnesium ions (Mg2+). NMDA receptors are involved in fast excitatory synaptic transmission and neuronal plasticity in the central nervous system (CNS). Functions of NMDA receptors:

1. They are involved in cell signalling events converting environmental stimuli to genetic changes by regulating gene transcription and epigenetic modifications in neuronal cells (Cohen and Greenberg, 2008).

2. In NMDA receptors, the ion channel is blocked by extracellular Mg2+ and Zn2+ ions, allowing the flow of Na+ and Ca2+ ions into the cell and K+ out of the cell which is voltage-dependent. Ca2+ flux through the NMDA receptor is considered to play a critical role in pre- and post-synaptic plasticity, a cellular mechanism important for learning and memory (Barria and Malinow, 2002).

3. The NMDA receptors have been shown to play an essential role in the strengthening of synapses and neuronal differentiation, through long-term potentiation (LTP), and the weakening of synapses, through long-term depression (LTD). All these processes are implicated in the memory and learning function (Barria and Malinow, 2002).


How it is Measured or Detected

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

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

The measurement of the activation or the inhibition of NMDA receptors is done indirectly by recording the individual ion channels that are selective to Na+, K+ and Ca+2 by the patch clamp technique. This method relies on lack of measurable ion flux when NMDA ion channel is closed, whereas constant channel specific conductance is recorded at the open state of the receptor (Blanke and VanDongen, 2009). Furthermore, this method is based on the prediction that activation or inhibition of an ion channel results from an increase in the probability of being in the open or close state, respectively.

The whole-cell patch clamp recording techniques have also been used to study synaptically-evoked NMDA receptor-mediated excitatory or inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) in brain slices and neuronal cells, allowing the evaluation of the activated or inhibited state of the receptor (Ogdon and Stanfield, 2009; Zhao et al., 2009).

Microelectrode array (MEA) recordings are used to measure electrical activity in cultured neurons in response to NMDA receptor activation or inactivation (Keefer et al., 2001, Gramowski et al., 2000 and Gopal, 2003; Johnstone et al., 2010). MEAs can also be applied in higher throughput platforms to facilitate screening of numerous chemical compounds based on electrical activity measurements (McConnell et al., 2012).


References


Barria A, Malinow R. (2002) Subunit-specific NMDA receptor trafficking to synapses. Neuron 35: 345-353.

Blanke ML, VanDongen AMJ. (2009) Activation Mechanisms of the NMDA Receptor. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press; Chapter 13. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5274/

Cohen S, Greenberg ME. (2008) Communication between the synapse and the nucleus in neuronal development, plasticity, and disease. Ann Rev Cell Dev Biol 24: 183-209.

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

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

Johnstone AFM, Gross GW, Weiss D, Schroeder O, Shafer TJ. (2010). Use of microelectrode arrays for neurotoxicity testing in the 21st century Neurotoxicology 31: 331-350.

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

Law AJ, Weickert CS, Webster MJ, Herman MM, Kleinman JE, Harrison PJ. (2003) Expression of NMDA receptor NR1, NR2A and NR2B subunit mRNAs during development of the human hippocampal formation. Eur J Neurosci. 18: 1197-1205.

Lieberman DN, Mody I. (1999) Properties of single NMDA receptor channels in human dentate gyrus granule cells. J Physiol. 518: 55-70.

Ludolph AG, Udvardi PT, Schaz U, Henes C, Adolph O, Weigt HU, Fegert JM, Boeckers TM, Föhr KJ. (2010) Atomoxetine acts as an NMDA receptor blocker in clinically relevant concentrations. Br J Pharmacol. 160: 283-291.

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

Ogdon D, Stanfield P. (2009) Patch clamp techniques for single channel and whole-cell recording. Chapter 4, pages 53-78. http://www.utdallas.edu/~tres/microelectrode/microelectrodes_ch04.pdf

Scherzer CR, Landwehrmeyer GB, Kerner JA, Counihan TJ, Kosinski CM, Standaert DG, Daggett LP, Veliçelebi G, Penney JB, Young AB. (1998) Expression of N-methyl-D-aspartate receptor subunit mRNAs in the human brain: hippocampus and cortex. J Comp Neurol. 390: 75-90.

Tovar KR, Westbrook GL. (1999) The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci. 19: 4180–4188.

Traynelis S, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R. (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 62: 405-496.

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


Event: 52: Decreased, Calcium influx

Short Name: Decreased, Calcium influx

Key Event Component

Process Object Action
calcium ion transport calcium ion decreased

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
neuron

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mice Mus sp. High NCBI
zebrafish Danio rerio High NCBI

Ca2+ homeostatic system is known to be highly conserved throughout evolution and is present from humans to invertebrates (Case et al., 2007).


Key Event Description

Biological state: Under physiological resting conditions of the cell, the free intracellular Ca2+ reaches around 100 nM, whereas the extracellular Ca2+ can be found at higher concentrations of 1.2 mM that under certain stimulus may invade the cell (Berridge et al, 2000). Six to seven oxygen atoms surround Ca2+, whereas the protein chelator of Ca2+ is the EF motif that is present in many proteins such as calmodulin (Clapham, 2007). The EF-hand is a helix-loop-helix calcium-binding motif in which two helices pack together at an angle of approximately 90 degrees (Lewit-Bentley and Réty, 2000). The two helices are separated by a loop region where calcium actually binds. The EF notation for the motif is derived from the notation applied to the structure of parvalbumin, in which the E and F helices were originally identified as forming this calcium-binding motif.

Biological compartments: Ca2+ ions accumulate in the cytoplasm, cellular organelles (e.g. mitochondria and endoplasmic reticulum) and nucleus in response to diverse classes of stimuli.

General role in biology: In order to adapt to altered stimulus from exposure to different environmental factors, cells require signal transmission. However, signalling needs messengers whose concentration is modified upon stimulus (Clapham, 2007). Ca2+ ions act as an important intracellular messenger playing the role of ubiquitous signalling molecules and consequently regulate many different cellular functions (Berridge, 2012; Hagenston and Bading, 2011). Given its important role in processes that are fundamental to all cell types, Ca2+ homeostasis is tightly regulated by intracellular and extracellular mechanisms (Barhoumi et al., 2010). Intracellular Ca2+ concentration is regulated by opening or closing channels in the plasma membrane. Additionally, the Ca2+ ions can be released from intracellular stores of the endoplasmic reticulum (ER) through ryanodine receptors (RYRs) or inositol 1,4,5-trisphosphate receptors (InsP3Rs). Ca2+ homeostasis is also regulated by the mechanisms that remove Ca2+ from the cytosol, for example pumps in both cell membrane and ER membrane. In addition, cytosolic Ca2+ regulation involves accumulation of Ca2+ in mitochondria that have the capacity to buffer the excess of cytoplasmic Ca2+ ions. In neurons, Ca2+ ions regulate many critical functions. Firstly, they contribute to dendritic electrical signalling, producing postsynaptic depolarization by the current carried by Ca2+ ions. Secondly, Ca2+ activates Ca2+-sensitive proteins such as different kinases, calcineurin and calpain, triggering signalling pathways critical for cell physiology. Modification of the gene transcription is the final outcome of the Ca2+ ions impact on long-term modifications affecting neurotransmitters release (reviewed in Neher and Sakaba, 2008), neuronal differentiation, synapse function and cell viability (Clapham, 2007; Higley and Sabatini, 2012). Thus, the Ca2+ that enters and accumulates in cytoplasm and nucleus is a central signalling molecule that regulates synapse and neuronal cell function, including learning and memory processes (Berridge, 2012; Hagenston and Bading, 2011).


How it is Measured or Detected

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

No OECD method is available to measure intracellular Ca2+.

The gold standard method for measuring Ca2+ current through NMDA receptor is patch clamp electrophysiology (Blanke and VanDongen, 2009).

In vitro, well-established flow cytometric or high content imaging analysis with specific fluorescent dyes (Ca2+-sensitive fluorophores) such as Fura-2, Oregon Green-BAPTA, Fluo-4 and X-Rhod exist for determination of intracellular Ca2+ concentration. The use of different fluorometric calcium indicators in neuroscience and neurotoxicology have been recently reviewed by Grienberger and Konnerth (2012) and Calvo et al (2015).

Barhoumi et al. 2010 summarised all the methods to measure cytosolic Ca2+ alterations due to exposure to neurotoxic compounds, including steady state, short-term kinetic measurements of stimulated Ca2+ transients and dynamic measurements. This paper further discusses the strengths and weaknesses of each approach in intracellular Ca2+ measurements and its applicability in high throughput screening.

For quantitative estimation of Ca2+ in dendritic spines, besides of Ca2+-sensitive fluorophores the use of two-photon released caged neurotransmitters has been suggested as it allows direct stimulation of visualized spines (Higley and Sabatini, 2012). In Higley and Sabatini 2012 further technical information can be found in relation to study Ca2+ in dendritic spines.

Furthermore, there are three methods for measuring Ca2+ influx in NMDA receptors that involve the measurement of 1) relative Ca2+ permeability, 2) channel blockage by Ca2+, and 3) fractional Ca2+ currents from whole-cell currents determined in the presence of high concentrations of intracellular Fura-2 (Traynelis et al., 2010).

In vivo, two-photon Ca2+ imaging using Ca2+-sensitive fluorescent indicators that measure changes in intracellular Ca2+ concentration as a readout for suprathreshold and subthreshold neuronal activity has also been used to study learning and memory in live rodents (Chen et al., 2013) The last two decades the neuronal function of the larval and adult zebrafish has been extensively studied using Ca2+ imaging methods. By applying simple Ca2+ indicators such as dextran or acetoxymethyl esters to more powerful genetically encoded Ca2+ indicators, zebrafish provides a transparent model where live Ca2+ imaging can be successfully achieved (Kettunen, 2012).

Fluorescent Ca2+ indicators have been also used as Pb2+ sensors in order to resolve spatiotemporal changes in intracellular Pb2+ in relation to cellular signaling and intracellular divalent metal homeostasis (Vijveberg and Westerink, 2012).

Intra-cellular calcium concentration can be measured in cell cultures with the calcium sensitive fluorescent dye Fura-2 AM and fluorescence microscopy. This technique appeared to be more sensitive than the plate-reader based assay Meijer et al., 2014).


References

Barhoumi R, Qian Y, Burghardt RC, Tiffany-Castiglioni E. (2010) Image analysis of Ca2+ signals as a basis for neurotoxicity assays: promises and challenges. Neurotoxicol Teratol. 32: 16-24.

Berridge MJ, Lipp P, Bootman MD. (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol. 1:11-21.

Berridge MJ. (2012) Calcium signalling remodelling and disease. Biochem Soc Trans. 40: 297-309.

Blanke ML, VanDongen AMJ. (2009) Activation Mechanisms of the NMDA Receptor. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press; Chapter 13. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5274/

Calvo M, Villalobos C, Núñez L. (2015) Calcium imaging in neuron cell death. Methods Mol Biol. 1254: 73-85.

Case RM, Eisner D, Gurney A, Jones O, Muallem S, Verkhratsky A. (2007) Evolution of calcium homeostasis: from birth of the first cell to an omnipresent signalling system. Cell Calcium 42: 345-350.

Chen JL, Andermann ML, Keck T, Xu NL, Ziv Y. (2013) Imaging neuronal populations in behaving rodents: paradigms for studying neural circuits underlying behavior in the mammalian cortex. J Neurosci. 33: 17631-17640.

Clapham DE. (2007) Calcium signaling. Cell 131: 1047-1058.

Grienberger C, Konnerth A. (2012) Imaging calcium in neurons. Neuron 73: 862-885.

Hagenston AM, Bading H. (2011) Calcium Signaling in Synapse-to-Nucleus Communication. Cold Spring Harb Perspect Biol. 3: a004564.

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

Kettunen P. (2012) Calcium imaging in the zebrafish. Adv Exp Med Biol. 740: 1039-1071.

Meijer M, Hendriks HS, Heusinkveld HJ, Langeveld WT, Westerink RH. 2014. Comparison of plate reader-based methods with fluorescence microscopy for measurements of intracellular calcium levels for the assessment of in vitro neurotoxicity. Neurotoxicology 45: 31-37.

Neher E., Sakaba T. (2008). Multiple roles of calcium ions in the regulation of neurotransmitter release. Neuron 59: 861-872.

Traynelis S, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R. (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 62: 405-496.

Vijverberg HP, Westerink RH. 2012. Sense in Pb2+ sensing. Toxicol Sci 130(1): 1-3.


Event: 381: Reduced levels of BDNF

Short Name: BDNF, Reduced

Key Event Component

Process Object Action
gene expression brain-derived neurotrophic factor decreased
secretion brain-derived neurotrophic factor decreased

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
neural cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
Life Stage Applicability
Life Stage Evidence
During brain development High
Sex Applicability
Sex Evidence
Mixed High

BDNF plays a critical role in normal brain development in most vertebrates, primarily documented empirically in mammalian species. Klein et al. (2011) examined blood, serum, plasma and brain-tissue and measured BDNF levels in three different mammalian species: rat, pig, and mouse, using an ELISA method (Aid et al., 2007), whereas Trajkovska et al. 2007 determined BDNF levels in human blood.

There is compelling data that demonstrates the role  of  BDNF  in brain development for many other taxa, including fish where it acts as neurotrophic factor in controlling cell proliferation (D'Angelo L et al., 2014; Heinrich and Pagtakhan, 2004) and  birds where BDNF influences development of the brain area that involved in the song control (Brenowitz 2013) and  the addition of new neurons to a cortical nucleus in adults . In the Xenopus visual system, BDNF acts as neurotrophic factor that mediates synaptic differentiation and maturation of the retinotectal circuit through cell autonomous TrkB signaling on retinal ganglion cells (Sanchez et al., 2006; Marshak et al., 2007).


Key Event Description

Biological state: BDNF belongs to a family of closely related neurotrophic factors named neurotrophins and is widely expressed in the developing and mature CNS. In the rodent cortex, postnatal BDNF expression is initially low but slowly increases to reach high levels around weaning. Therefore, BDNF expression peaks at a time when both structural and functional maturation of cortical circuitry occurs. During postnatal development, BDNF levels are dynamically regulated, in part by neuronal activity dependent mechanisms (Waterhouse and Xu, 2009). Glutamate has been shown to increase the transcription and release of BDNF. Indeed, BDNF is synthesized, stored and released from glutamatergic neurons (Lessmann et al., 2003).

Biological compartments: BDNF initially is synthesized as precursor proteins (proBDNF), which is processed intracellularly to be transformed in its mature form (mBDNF) after proteolytically cleaved in the synaptic cleft by plasmin which is a protease activated by tissue plasminogen activator (tPA) (Cohen-Cory et al., 2010). proBDNF is constantly secreted while tPA release and mBDNF production depends on neuronal excitation (Head et al., 2009). Storage and activity-dependent release of BDNF has been demonstrated in both dendrites and axon terminals (Waterhouse and Xu, 2009). More specifically, in hippocampus, BDNF appears to be stored in dendritic processes of neurons (Balkowiec and Katz, 2002). BDNF is abundant in cerebellum and cortex and has also been measured in cerebrospinal fluid (CSF) (Zhang et al., 2008), whole blood, plasma, serum (plasma without clotting factors) and platelets (Trajkovska et al., 2007). BDNF has been found to be produced by astrocytes under both physiological and pathological conditions (Endo, 2005; Coco et al., 2013; Nelson and Alkon, 2014).

In humans, mBDNF is sequestered in platelets, consequently BDNF can reach all tissues and organs. Lymphocytic cells have been shown to express BDNF in vitro similarly to eosinophils, dendritic cells, and endothelial cells. The visceral and airway epithelium are also significant sources of BDNF. Female reproductive system including ovaries, placenta and uterus also express BDNF (Wessels et al., 2014).

General role in biology: The biological functions of mBDNF are mediated by binding to tyrosine kinase B (TrkB) receptor that leads to the activation of three major intracellular signalling pathways, including MAPK, PI3K and PLCγ1 (Soulé et al., 2006). TrkB-mediated signaling regulates gene transcription in the nucleus through the activation of several transcription factors. These genes are involved in neurite outgrowth, synaptogenesis, synapse maturation and stabilization (Pang et al., 2004; Lu et al., 2005; Nelson and Alkon, 2014).

On the other hand, proBDNF binds to the p75 neurotrophin receptor (p75NTR) and activates RhoA, a small GTPase that regulates actin cytoskeleton polymerization leading to inhibition of axonal elongation, growth cone collapse, and apoptosis (Dubreuil et al., 2003; Yamauchi et al., 2004; Head et al., 2009).


How it is Measured or Detected

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


No OECD methods are available to measure BDNF protein and mRNA levels. Depending on the tissue or fluid measurements distinct methods are used.

Brain tissue: BDNF protein levels can be measured by commercial available antibody sandwich ELISA kits, Western blotting, immunohistochemistry and immunofluorescence. BDNF primers for different exons are available to determine mRNA levels by RT-PCR. The Bdnf gene consists of multiple alternative exons (ten in human, eight in rodents and six in lower vertebrates), and a single exon coding for the entire pro-BDNF protein (Cohen-Cory et al., 2010).

Cerebro-spinal fluid (CSF): There are available commercial antibody sandwich ELISA kits (Trajkovska et al., 2007) and immunobead-based multiplex assays for high throughput screening (Zhang et al., 2008).

Whole blood, serum, plasma and platelets: There are several commercial double antibody sandwich ELISA kits that can be used for identification of BDNF levels in biological fluids (Trajkovska et al., 2007).

Methodological considerations that have to be taken into account during sample preparation and measurement of BDNF by ELISA have been recently reviewed in Elfving et al. 2010. A study measuring BDNF by a commercially available ELISA kit in various tissues and biological liquids derived from distinct species revealed that BDNF is undetectable in mouse blood and pig plasma (Klein et al., 2011). This study also showed that in most cases BDNF levels are comparable to levels reported in humans and that there is positive correlation between blood BDNF levels and hippocampal BDNF levels in rats and pigs (Klein et al., 2011).


References


Aid T, Kazantseva A, Piirsoo M, Palm K, Timmusk T. (2007) Mouse and rat BDNF gene structure and expression revisited. J Neurosci Res. 85: 525-535.

Balkowiec A, Katz DM. (2002) Cellular mechanisms regulating activity-dependent release of native brain-derived neurotrophic factor from hippocampal neurons. J Neurosci. 22: 10399-10407.

Brenowitz EA. (2013) Testosterone and brain-derived neurotrophic factor interactions in the avian song control system. Neuroscience 239: 115-123.

Coco M, Caggia S, Musumeci G, Perciavalle V, Graziano AC, Pannuzzo G, Cardile V. (2013) Sodium L-lactate differently affects brain-derived neurothrophic factor, inducible nitric oxide synthase, and heat shock protein 70 kDa production in human astrocytes and SH-SY5Y cultures.J Neurosci Res. 91: 313-320.

Cohen-Cory S, Kidane AH, Shirkey NJ, Marshak S. (2010) Brain-derived neurotrophic factor and the development of structural neuronal connectivity. Dev Neurobiol. 70: 271-288.

D'Angelo L, De Girolamo P, Lucini C, Terzibasi ET, Baumgart M, Castaldo L, Cellerino A (2014). Brain-derived neurotrophic factor: mRNA expression and protein distribution in the brain of the teleost Nothobranchius furzeri. J Comp Neurol. 1;522(5):1004-30.

Dubreuil CI, Winton MJ, McKerracher L. (2003) Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system. J Cell Biol. 162: 233-243.

Elfving B, Plougmann PH, Wegener G. (2010) Detection of brain-derived neurotrophic factor (BDNF) in rat blood and brain preparations using ELISA: pitfalls and solutions. J Neurosci Methods 187: 73-77.

Endo T. (2005) Glycans and glycan-binding proteins in brain: galectin-1-induced expression of neurotrophic factors in astrocytes. Curr Drug Targets. 6:427-436.

Head BP, Patel HH, Niesman IR, Drummond JC, Roth DM, Patel PM. (2009) Inhibition of p75 neurotrophin receptor attenuates isoflurane-mediated neuronal apoptosis in the neonatal central nervous system. Anesthesiology 110: 813-825.

Heinrich G, Pagtakhan CJ. (2004) Both 5' and 3' flanks regulate Zebrafish brain-derived neurotrophic factor gene expression. BMC Neurosci. 5: 19.

Klein AB, Williamson R, Santini MA, Clemmensen C, Ettrup A, Rios M, Knudsen GM, Aznar S. (2011) Blood BDNF concentrations reflect brain-tissue BDNF levels across species. Int J Neuropsychopharmacol. 14: 347-353.

Lessmann V, Gottmann K, Malcangio M. (2003) Neurotrophin secretion: current facts and future prospects. Prog Neurobiol. 69: 341-374.

Lu B, Pang PT, Woo NH. (2005) The yin and yang of neurotrophin action. Nat Rev Neurosci. 6: 603-614.

Marshak S, Nikolakopoulou AM, Dirks R, Martens GJ, Cohen-Cory S (2007)Cell-autonomous TrkB signaling in presynaptic retinal ganglion cells mediates axon arbor growth and synapse maturation during the establishment of retinotectal synaptic connectivity. J Neurosci 27:2444 –2456.

Nelson TJ, Alkon DL. (2014) Molecular regulation of synaptogenesis during associative learning and memory. Brain Res. pii: S0006-8993(14)01660-6. doi: 10.1016/j.brainres.2014.11.054.

Pang PT, Teng HK, Zaitsev E, Woo NT, Sakata K, Zhen S, Teng KK, Yung WH, Hempstead BL, Lu B. (2004) Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science. 306: 487-491.

Pruunsild P, Kazantseva A, Aid T, Palm K, Timmusk T. (2007) Dissecting the human BDNF locus: bidirectional transcription, complex splicing, and multiple promoters. Genomics. 90: 397-406.

Sanchez AL, Matthews BJ, Meynard MM, Hu B, Javed S, Cohen Cory S (2006) BDNF increases synapse density in dendrites of developing tectal neurons in vivo. Development 133:2477–2486.

Soule´ J, Messaoudi E, Bramham CR. (2006) Brain-derived neurotrophic factor and control of synaptic consolidation in the adult brain. Biochem Soc Trans. 34 :600-604.

Trajkovska V, Marcussen AB, Vinberg M, Hartvig P, Aznar S, Knudsen GM. (2007) Measurements of brain-derived neurotrophic factor: methodological aspects and demographical data. Brain Res Bull. 73: 143-149.

Waterhouse EG, Xu B. (2009) New insights into the role of brain-derived neurotrophic factor in synaptic plasticity. Mol Cell Neurosci. 42: 81-89.

Wessels JM, Wu L, Leyland NA, Wang H, Foster WG. (2014) The Brain-Uterus Connection: Brain Derived Neurotrophic Factor (BDNF) and Its Receptor (Ntrk2) Are Conserved in the Mammalian Uterus. PLoS ONE 9: e94036.

Yamauchi J, Chan JR, Shooter EM. (2004) Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases. Proc Natl Acad Sci U S A. 101: 8774-8779.

Zhang J, Sokal I, Peskind ER, Quinn JF, Jankovic J, Kenney C, Chung KA, Millard SP, Nutt JG, Montine TJ. (2008) CSF multianalyte profile distinguishes Alzheimer and Parkinson diseases. Am J Clin Pathol. 129: 526-529.


Event: 55: N/A, Cell injury/death

Short Name: N/A, Cell injury/death

Key Event Component

Process Object Action
cell death increased

AOPs Including This Key Event


Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
eukaryotic cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
human and other cells in culture human and other cells in culture High NCBI
Rattus norvegicus Rattus norvegicus High NCBI
mouse Mus musculus High NCBI

Cell death is an universal event occurring in cells of any species. [11]


Key Event Description

Two types of cell death can be distinguished by morphological features, although it is likely that these are two ends of a spectrum with possible intermediate forms. Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of the cell into discrete bodies with intact plasma membranes. These are rapidly phagocytosed by neighbouring cells. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. In contrast, necrotic cell death is characterized by cell swelling and lysis. This is usually a consequence of profound loss of mitochondrial function and resultant ATP depletion, leading to loss of ion homeostasis, including volume regulation, and increased Ca2+. The latter activates a number of nonspecific hydrolases (i.e., proteases, nucleases, and phospholipases) as well as calcium dependent kinases. Activation of calpain I, the Ca2+-dependent cysteine protease cleaves the death-promoting Bcl-2 family members Bid and Bax which translocate to mitochondrial membranes, resulting in release of truncated apoptosis-inducing factor (tAIF), cytochrome c and endonuclease in the case of Bid and cytocrome c in the case of Bax. tAIF translocates to cell nuclei, and together with cyclophilin A and phosphorylated histone H2AX (γH2AX) is responsible for DNA cleavage, a feature of programmed necrosis. Activated calpain I has also been shown to cleave the plasma membrane Na+–Ca2+ exchanger, which leads to build-up of intracellular Ca2+, which is the source of additional increased intracellular Ca2+. Cytochrome c in cellular apoptosis is a component of the apoptosome.

DNA damage activates nuclear poly(ADP-ribose) polymerase-1(PARP-1), a DNA repair enzyme. PARP-1 forms poly(ADP-ribose) polymers, to repair DNA, but when DNA damage is extensive, PAR accumulates, exits cell nuclei and travels to mitochondrial membranes, where it, like calpain I, is involved in AIF release from mitochondria. A fundamental distinction between necrosis and apoptosis is the loss of plasma membrane integrity; this is integral to the former but not the latter. As a consequence, lytic release of cellular constituents promotes a local inflammatory reaction, whereas the rapid removal of apoptotic bodies minimizes such a reaction. The distinction between the two modes of death is easily accomplished in vitro but not in vivo. Thus, although claims that certain drugs induce apoptosis have been made, these are relatively unconvincing. DNA fragmentation can occur in necrosis, leading to positive TUNEL staining. Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis.

Two alternative pathways - either extrinsic (receptor-mediated) or intrinsic (mitochondria-mediated) - lead to apoptotic cell death. The initiation of cell death begins either at the plasma membrane with the binding of TNF or FasL to their cognate receptors or within the cell. The latter is due to the occurrence of intracellular stress in the form of biochemical events such as oxidative stress, redox changes, covalent binding, lipid peroxidation, and consequent functional effects on mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, or DNA. The intrinsic mitochondrial pathway involves the initiator, caspase-9, which, when activated, forms an “apoptosome” in the cytosol, together with cytochrome c, which translocates from mitochondria, Apaf-1 and dATP. The apoptosome activates caspase-3, the central effector caspase, which in turn activates downstream factors that are responsible for the apoptotic death of a cell [1]. Intracellular stress either directly affects mitochondria or can lead to effects on other organelles, which then send signals to the mitochondria to recruit participation in the death process [1][2]Constitutively expressed nitric oxide synthase (nNOS) is a Ca2+-dependent cytosolic enzyme that forms nitric oxide (NO) from L-arginine, and NO reacts with the free radical such as superoxide (O2−) to form the very toxic free radical peroxynitrite (ONOO−). Free radicals such as ONOO−, O2 − and hydroxyl radical (OH−) damage cellular membranes and intracellular proteins, enzymes and DNA [1], [2] [3][4]


How it is Measured or Detected

Necrosis:

LDH is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor. The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample. [5]

The MTT assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light [6].

Propidium iodide (PI) is an intercalating agent and a fluorescent molecule used to stain necrotic cells. It is cell membrane impermeant so it stains only those cells where the cell membrane is destroyed. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm [7] .

Alamar Blue (resazurin) fluorescent dye. The oxidized blue non fluorescent Alamar blue is reduced to a pink fluorescent dye in the medium by cell activity (O'Brien et al., 2000) (12).

Neutral red uptake, which is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in lysosomes (Repetto et al., 2008)(13).

ATP assay: Quantification of ATP, signaling the presence of metabolically active cells (CellTiter-Glo; Promega).


Apoptosis:

TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage.

Caspase activity assays measured by fluorescence. During apoptosis, mainly caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. Antibodies to the 85 kDa fragment of cleaved PARP or to caspase-3 both serve as markers for apoptotic cells that can be monitored using immunofluorescence [8].

Hoechst 33342 staining: Hoechst dyes are cell-permeable and bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. The stained, condensed or fragmented DNA is a marker of apoptosis. [9] [10]

Acridine Orange/Ethidium Bromide staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis.


References

  1. 1.0 1.1 1.2 Fujikawa, D.G. (2015), The role of excitotoxic programmed necrosis in acute brain injury, Comput Struct Biotechnol J, vol. 13, pp. 212-221.
  2. 2.0 2.1 Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.
  3. Kaplowitz, N. (2002), Biochemical and Cellular Mechanisms of Toxic Liver Injury, Semin Liver Dis, vol. 22, no. 2, http://www.medscape.com/viewarticle/433631 (accessed on 20 January 2016).
  4. Kroemer, G. et al., (2009), Classification of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death Differ, vol. 16, no. 1, pp. 3-11.
  5. Chan, F.K., K. Moriwaki and M.J. De Rosa (2013), Detection of necrosis by release of lactate dehydrogenase (LDH) activity, Methods Mol Biol, vol. 979, pp. 65–70.
  6. Berridge, M.V., P.M. Herst and A.S. Tan (2005), Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, vol. 11, pp 127-152.
  7. Moore, A, et al.(1998), Simultaneous measurement of cell cycle and apoptotic cell death,Methods Cell Biol, vol. 57, pp. 265–278.
  8. Li, Peng et al. (2004), Mitochondrial activation of apoptosis, Cell, vol. 116, no. 2 Suppl,pp. S57-59, 2 p following S59.
  9. Loo, D.T. (2002), TUNEL Assay an overview of techniques, Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage, chapter 2, Didenko VV (ed.), Humana Press Inc.
  10. Kubbies, M. and P.S. Rabinovitch (1983), Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes, Cytometry, vol. 3, no. 4, pp. 276–281.
  11. Fink, S.L. and B.T. Cookson (2005), Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells, Infect Immun, vol. 73, no. 4, pp.1907-1916.
  12. O'Brien J, Wilson I, Orton T, Pognan F. 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European journal of biochemistry / FEBS 267(17): 5421-5426.
  13. Repetto G, del Peso A, Zurita JL. 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols 3(7): 1125-1131.

Event: 188: N/A, Neuroinflammation

Short Name: N/A, Neuroinflammation

Key Event Component

Process Object Action
brain inflammation microglial cell pathological
brain inflammation astrocyte pathological

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
brain

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
human Homo sapiens Moderate NCBI

Neuroinflammation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure. Some references (non-exhaustive list) are given below for illustration:

In human: Vennetti et al., 2006

In monkey (Macaca fascicularis): Charleston et al., 1994, 1996

Iin rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002

In mouse: Liu et al., 2012

In zebrafish: Xu et al., 2014.


Key Event Description

Neuroinflammation or brain inflammation differs from peripheral inflammation in that the vascular response and the role of peripheral bone marrow-derived cells are less conspicuous. The most easily detectable feature of neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signalling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001), as well as in the production of reactive oxygen (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signalling and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004).

Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006 ; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells scan the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defence), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been descibed recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1Þ), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.


How it is Measured or Detected

Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:

  1. Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.
  2. The most frequently used astrocyte marker is GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflamatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for staining of astrocytes (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.
  3. All immunocytochemical methods can also be applied to cell culture models.
  4. In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).
  5. Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of activation markers. This can for instance be done by PCR quantification of inflammatory factors, by measurement of the respective mediators, e.g. by ELISA-related immuno-quantification. Such markers include:
  • Pro- and anti-inflammatory cytokine expression (IL-1β; TNF-α, Il-6, IL-4); or expression of immunostimmulatory proteins (e.g. MHC-II)
  • Itgam, CD86 expression as markers of M1 microglial phenotype
  • Arg1, MRC1, as markers of M2 microglial phenotype

(for descriptions of techniques, see also  Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al.,  2014)

Regulatory example using the KE:Measurement of glial fibrillary acidic protein (GFAP) in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67), but is optional for other toxicant evaluations..


References

Aschner M (1998) Immune and inflammatory responses in the CNS: modulation by astrocytes. ToxicolLett 103: 283-287

Banati, R. B. (2002). "Visualising microglial activation in vivo." Glia 40: 206-217.     

Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355

Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.

Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.

Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190

Eng LF, Ghirnikar RS, Lee YL (2000) Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000). NeurochemRes 25: 1439-1451

Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.

Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004  Jan;88(1):181-93.

Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907.

Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.

Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34.

Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35

Graeber MB, Streit WJ (1990) Microglia: immune network in the CNS. Brain Pathol 1: 2-5

Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444

Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42

Kreutzberg GW (1995) Microglia, the first line of defence in brain pathologies. Arzneimttelforsch 45: 357-360

Kreutzberg GW (1996) Microglia : a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318

Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638): 481-487.

Little AR, Miller DB, Li S, Kashon ML, O'Callaghan JP. 2012. Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis. Neurotoxicol Teratol 34(1): 72-82.

Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.

Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87.

Maresz K, Ponomarev ED, Barteneva N, Tan Y, Mann MK, Dittel BN (2008) IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages. J Reprod Immunol 78: 140-148

Monnet-Tschudi F, Zurich MG, Honegger P (2007) Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26: 339-346

Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19.         

Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958-969

Nakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc & Haematol Disorders 4: 65-84.

Perego C, Fumagalli S, De Simoni MG (2011) Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation 8: 174

Ponomarev ED, Maresz K, Tan Y, Dittel BN (2007) CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci 27: 10714-10721

Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81: 374-389

Ransohoff RM. 2016. A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19(8): 987-991.

Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001

Struzynska L, Dabrowska-Bouta B, Koza K, Sulkowski G (2007) Inflammation-Like Glial Response in Lead-Exposed Immature Rat Brain. Toxicol Sc 95:156-162

von Tobel, J. S., P. Antinori, et al. (2014). "Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype." Neurotoxicology 44C: 61-70.

Venneti S, Lopresti BJ, Wiley CA. 2006. The peripheral benzodiazepine receptor (Translocator protein 18kDa) in microglia: from pathology to imaging. Prog Neurobiol 80(6): 308-322.

Xu DP, Zhang K, Zhang ZJ, Sun YW, Guo BJ, Wang YQ, et al. 2014. A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-kappaB and the PKCalpha/PI3-K/Akt pathways. Neurochem Int 78: 76-85.

Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.

 


List of Adverse Outcomes in this AOP

Event: 352: N/A, Neurodegeneration

Short Name: N/A, Neurodegeneration

Key Event Component

Process Object Action
neurodegeneration increased

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
brain

Domain of Applicability


The necrotic and apoptotic cell death pathways are quite well conserved throughout taxa (Blackstone and Green, 1999, Aravind et al., 2001). It has been widely suggested that apoptosis is also conserved in metazoans, although despite conservation of Bcl-2 proteins, APAF-1, and caspases there is no biochemical evidence of the existence of the mitochondrial pathway in either C. elegans or Drosophila apoptosis (Baum et al., 2007; Blackstone and Green, 1999).


Key Event Description

The term neurodegeneration is a combination of two words - "neuro," referring to nerve cells and "degeneration," referring to progressive damage. The term "neurodegeneration" can be applied to several conditions that result in the loss of nerve structure and function, and neuronal loss by necrosis and/or apoptosis

Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of “neurodegenerative diseases" including Huntington's, Alzheimer’s and Parkinson’s disease. All of these conditions lead to progressive brain damage and neurodegeneration.

Alzheimer's disease is characterised by loss of neurons and synapses in the cerebral cortex and certain subcortical regions, with gross atrophy of the affected regions; symptoms include memory loss.

Parkinson's disease (PD) results from the death of dopaminergic neurons in the midbrain substantia nigra pars compacta; symptoms include bradykinesia, rigidity, and resting tremor.

Several observations suggest correlative links between environmental exposure and neurodegenerative diseases, but only few suggest causative links:

Only an extremely small proportion (less than 5%) of neurodegenerative diseases are caused by genetic mutations. The remainders are thought to be caused by the following:

•A build up of toxic proteins in the brain

•A loss of mitochondrial function that leads to the oxidative stress and creation of neurotoxic molecules that trigger cell death (apoptotic, necrotic or autophagy)

•Changes in the levels and activities of neurotrophic factors

•Variations in the activity of neural networks

Protein aggregation: the correlation between neurodegenerative disease and protein aggregation in the brain has long been recognized, but a causal relationship has not been unequivocally established (Lansbury et al., 2006). The dynamic nature of protein aggregation mean that, despite progress in understanding its mechanisms, its relationship to disease is difficult to determine in the laboratory.

Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered pre-symptomatically, drastically reduce the incidence of these diseases.

Loss of mitochondrial function: many lines of evidence suggest that mitochondria have a central role in neurodegenerative diseases (Lin and Beal, 2006). Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Dysfunction of mitochondria induces oxidative stress, production of free radicals, calcium overload, and mutations in mitochondrial DNA that contribute to neurodegenerative diseases. In all major examples of these diseases there is strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis. Moreover, an impressive number of disease- specific proteins interact with mitochondria. Thus, therapies targeting basic mitochondrial processes, such as energy metabolism or free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise.

Decreased level of neurotrophic factors: decreased levels and activities of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), have been described in a number of neurodegenerative disorders, including Huntington's disease, Alzheimer disease and Parkinson disease (Zuccato and Cattaneo, 2009). These studies have led to the development of experimental strategies aimed at increasing BDNF levels in the brains of animals that have been genetically altered to mimic the aforementioned human diseases, with a view to ultimately influencing the clinical treatment of these conditions. Therefore BDNF treatment is being considered as a beneficial and feasible therapeutic approach in the clinic.

Variations in the activity of neural networks: Patients with various neurodegenerative disorders show remarkable fluctuations in neurological functions, even during the same day (Palop et al., 2006). These fluctuations cannot be caused by sudden loss or gain of nerve cells. Instead, it is likely that they reflect variations in the activity of neural networks and, perhaps, chronic intoxication by abnormal proteins that the brain is only temporarily able to overcome.


How it is Measured or Detected

The assays for measurements of necrotic or apoptotic cell death are described in the Key Event: Cell injury/Cell death

Recent neuropathological studies have shown that Fluoro-Jade, an anionic fluorescent dye, is a good marker of degenerating neurons. Fluoro-Jade and Fluoro-Jade B were found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death (Schmued et al., 2005). More recently, Fluoro-Jade C was shown to be highly resistant to fading and compatible with virtually all histological processing and staining protocols (Schmued et al., 2005). In addition, Fluoro-Jade C is a good tool for detecting acutely and chronically degenerating neurons (Ehara and Ueda, 2009).


Regulatory Significance of the AO

Currently the four available OECD Test Guidelines (TGs) for neurotoxicity testing are entirely based on in vivo neurotoxicity studies: (1)Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure (TG 418); (2) Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study (TG 419); (3) Neurotoxicity Study in Rodents (TG 424) involves daily oral dosing of rats for acute, subchronic, or chronic assessments (28 days, 90 days, or one year or longer); (4) Developmental Neurotoxicity (DNT) Study (TG 426) evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. One of the endpoints required by all four of these OECD TGs is evaluation of neurodegeneration that, so far, is performed through in vivo neuropathological and histological studies. Therefore, neurodegeneration described in this AOP as a key event, has a regulatory relevance and could be performed using in vitro assays that allow a reliable evaluation of neurodegeneration using a large range of existing assays, specific for apoptosis, necrosis and autophagy ( see also KE Cell injury/Cell death).


References

Aravind, L., Dixit, V. M., and Koonin, E. V. (2001). Apoptotic Molecular Machinery: Vastly Increased Complexity in Vertebrates Revealed by Genome Comparisons. Science 291, 1279-1284.

Baum, J. S., Arama, E., Steller, H., and McCall, K. (2007). The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis. Cell Death Differ 14, 1508-1517.

Blackstone, N. W., and Green, D. R. (1999). The evolution of a mechanism of cell suicide. Bioessays 21, 84-88.

Corvino V, Marchese E, Michetti F, Geloso MC (2013) Neuroprotective strategies in hippocampal neurodegeneration induced by the neurotoxicant trimethyltin. Neurochem Res 38: 240-253

Ehara A, Ueda S. 2009. Application of Fluoro-Jade C in acute and chronic neurodegeneration models: utilities and staining differences. Acta histochemica et cytochemica 42(6): 171-179.

Falluel-Morel A, Lin L, Sokolowski K, McCandlish E, Buckley B, DiCicco-Bloom E (2012) N-acetyl cysteine treatment reduces mercury-induced neurotoxicity in the developing rat hippocampus. J Neurosci Res 90: 743-750

Lansbury1 PT & Lashuel HA, Review Article A century-old debate on protein aggregation and neurodegeneration enters the clinic. 2006, Nature 443, 774-779.

Lin1 MT & Beal MF, Review Article Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, 2006, Nature 443, 787-795

Palop JJ, Chin1 J & Mucke L, Review Article A network dysfunction perspective on neurodegenerative diseases. 2006, Nature 443, 768-773

Schmued LC, Stowers CC, Scallet AC, Xu L. 2005. Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res 1035(1): 24-31.

Zuccato C & Cattaneo E, Brain-derived neurotrophic factor in neurodegenerative diseases.2009, Nature Reviews Neurology 5, 311-3

 


Event: 341: Impairment, Learning and memory

Short Name: Impairment, Learning and memory

Key Event Component

Process Object Action
learning decreased
memory decreased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:13 - Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities AdverseOutcome
Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment. AdverseOutcome
Aop:54 - Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment AdverseOutcome
Aop:77 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony death/failure 1 KeyEvent
Aop:78 - Nicotinic acetylcholine receptor activation contributes to abnormal role change within the worker bee caste leading to colony death failure 1 KeyEvent
Aop:87 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure KeyEvent
Aop:88 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure via abnormal role change within caste KeyEvent
Aop:89 - Nicotinic acetylcholine receptor activation followed by desensitization contributes to abnormal foraging and directly leads to colony loss/failure KeyEvent
Aop:90 - Nicotinic acetylcholine receptor activation contributes to abnormal roll change within the worker bee caste leading to colony loss/failure 2 KeyEvent
Aop:12 - Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development leads to neurodegeneration with impairment in learning and memory in aging AdverseOutcome
Aop:99 - Histamine (H2) receptor antagonism leading to reduced survival KeyEvent
Aop:17 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins during brain development leads to impairment of learning and memory AdverseOutcome

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
fruit fly Drosophila melanogaster High NCBI
zebrafish Danio rerio High NCBI
gastropods Physa heterostropha High NCBI
Life Stage Applicability
Life Stage Evidence
During brain development High
Sex Applicability
Sex Evidence
Mixed High

Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013).


Key Event Description

Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non-associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behaviour. On the other hand, non-associative learning can be defined as an alteration in the behavioural response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning.

The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterised by the non-conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer).

Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. For example, the hippocampus has been shown to be critical for spatial-temporal memory, visio-spatial memory, verbal and narrative memory, and episodic and autobiographical memory (Burgess et al., 2000; Vorhees and Williams, 2014). However, there is substantial evidence that fundamental learning and memory functions are not mediated by the hippocampus alone but require a network that includes, in addition to the hippocampus, anterior thalamic nuclei, mammillary bodies cortex, cerebellum and basal ganglia (Aggleton and Brown, 1999; Doya, 2000; Mitchell et al., 2002, Toscano and Guilarte, 2005; Gilbert et al., 2006, 2016). Thus, damage to variety of brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).

For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioural tests described below.


How it is Measured or Detected

In laboratory animals: in rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, passive avoidance and Spontaneous alternation and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows.

1) RAM, Barnes, MWM are examples of spatial tasks, animals are required to learn the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze), or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014).

2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention – I have seen one of these objects before, but not this one (Cohen and Stackman, 2015).

3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009).

4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2004).

In humans:  A variety of standardized learning and memory tests have been developed for human neuropsychological testing, including children (Rohlman et al., 2008). These include episodic autobiographical memory, perceptual motor tests, short and  long term memory tests, working memory tasks, word pair recognition memory; object location recognition memory. Some have been incorporated in general tests of intelligence (IQ) such as the WAIS and the Wechsler. Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include:

1) Rey Osterieth Complex Figure (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006).

2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986).

3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994).

4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986).

5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011).

6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).


Regulatory Significance of the AO

A prime example of impairments in learning and memory as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD 426) both require testing of learning and memory (USEPA, 1998; OECD, 2007) advising to use the following tests passive avoidance, delayed-matching-to-position for the adult rat and for the infant rat, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and acquisition and retention of schedule-controlled behaviour.  These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009).

Also in the frame of the OECD GD 43 (2008) on reproductive toxicity, learning and memory testing may have potential to be applied in the context of developmental neurotoxicity studies. However, many of the learning and memory tasks used in guideline studies may not readily detect subtle impairments in cognitive function associated with modest degrees of developmental thyroid disruption (Gilbert et al., 2012).


References

Aggleton JP, Brown MW. (1999) Episodic memory, amnesia, and the hippocampal-anterior thalamic axis. Behav Brain Sci. 22: 425-489.

Alexander RD (1990) Epigenetic rules and Darwinian algorithms: The adaptive study of learning and development. Ethology and Sociobiology 11:241-303.

Bellinger DC (2012) A strategy for comparing the contributions of environmental chemicals and other risk factors to neurodevelopment of children. Environ Health Perspect 120:501-507.

Burgess N (2002) The hippocampus, space, and viewpoints in episodic memory. Q J Exp Psychol A 55:1057-1080. Cohen, SJ and Stackman, RW. (2015). Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav. Brain Res. 285: 105-1176.

Cohen, SJ and Stackman, RW. (2015). Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav. Brain Res. 285: 105-1176.

Curzon P, Rustay NR, Browman KE. Cued and Contextual Fear Conditioning for Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009.

D'Hooge R, De Deyn PP (2001) Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 36:60-90.

Doya K. (2000) Complementary roles of basal ganglia and cerebellum in learning and motor control. Curr Opin Neurobiol. 10: 732-739.

Eichenbaum H (2000) A cortical-hippocampal system for declarative memory. Nat Rev Neurosci 1:41-50.

Fivush R. The development of autobiographical memory. Annu Rev Psychol. 2011;62:559-82.

Gilbert ME, Sanchez-Huerta K, Wood C (2016) Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.

Gilbert ME, Rovet J, Chen Z, Koibuchi N. (2012) Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology 33: 842-52.

Gilbert ME, Sui L (2006) Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.

Herold, C, Lässer, MM, Schmid, LA, Seidl, U, Kong, L, Fellhauer, I, Thomann,PA, Essig, M and Schröder, J. (2015). Neuropsychology, Autobiographical Memory, and Hippocampal Volume in “Younger” and “Older” Patients with Chronic Schizophrenia. Front. Psychiatry, 6: 53.

Lezak MD (1984) Neuropsychological assessment in behavioral toxicology--developing techniques and interpretative issues. Scand J Work Environ Health 10 Suppl 1:25-29.

Lezak MD (1994) Domains of behavior from a neuropsychological perspective: the whole story. Nebr Symp Motiv 41:23-55.

Makris SL, Raffaele K, Allen S, Bowers WJ, Hass U, Alleva E, Calamandrei G, Sheets L, Amcoff P, Delrue N, Crofton KM. A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect. 2009 Jan;117(1):17-25.

Mitchell AS, Dalrymple-Alford JC, Christie MA. (2002) Spatial working memory and the brainstem cholinergic innervation to the anterior thalamus. J Neurosci. 22: 1922-1928.

OECD. 2007. OECD guidelines for the testing of chemicals/ section 4: Health effects. Test no. 426: Developmental neurotoxicity study. www.Oecd.Org/dataoecd/20/52/37622194.Pdf [accessed may 21, 2012].

OECD (2008) Nr 43 GUIDANCE DOCUMENT ON MAMMALIAN REPRODUCTIVE TOXICITY TESTING AND ASSESSMENT. ENV/JM/MONO(2008)16

Ono T. (2009) Learning and Memory. Encyclopedia of neuroscience. M D. Binder, N. Hirokawa and U. Windhorst (Eds). Springer-Verlag GmbH Berlin Heidelberg. pp 2129-2137.

Roberts AC, Bill BR, Glanzman DL. (2013) Learning and memory in zebrafish larvae. Front Neural Circuits 7: 126.

Rohlman DS, Lucchini R, Anger WK, Bellinger DC, van Thriel C. (2008) Neurobehavioral testing in human risk assessment. Neurotoxicology. 29: 556-567.

Shin, MS, Park, SY, Park, SR, Oeol, SH and Kwon, JS. (2006). Clinical and empirical applications of the Rey-Osterieth complex figure test. Nature Protocols, 1: 892-899.

Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372-376.

Stanton ME, Spear LP (1990) Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol Teratol 12:261-267.

Talley, JL. (1986). Memory in learning disabled children: Digit span and eh Rey Auditory verbal learning test. Archives of Clinical Neuropsychology, Elseiver.

Toscano CD, Guilarte TR. (2005) Lead neurotoxicity: From exposure to molecular effects. Brain Res Rev. 49: 529-554.

U.S.EPA. 1998. Health effects guidelines OPPTS 870.6300 developmental neurotoxicity study. EPA Document 712-C-98-239.Office of Prevention Pesticides and Toxic Substances.

Vorhees CV, Williams MT (2014) Assessing spatial learning and memory in rodents. ILAR J 55:310-332.

Willoughby KA, McAndrews MP, Rovet JF. Accuracy of episodic autobiographical memory in children with early thyroid hormone deficiency using a staged event. Dev Cogn Neurosci. 2014 Jul;9:1-11.

 


Appendix 2

List of Key Event Relationships in the AOP