AOP ID and Title:

Short Title: IGR binding leads to impairment of learning and memory (via loss of drebrin)

Authors

Yuko Sekino¹, Shihori Tanabe², Tomoaki Shirao³

1 the University of Tokyo, Japan

2 National Institute of Health Sciences, Japan

3 Gunma University, Japan

Status

Abstract

Summary of the AOP

Events

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

Key Event Relationships

Overall Assessment of the AOP

References

1. Adverse Outcome Pathway on binding of agonists to ionotropic glutamate receptors in adult brain leading to excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment, Sachana M, et al. OECD Series on Adverse Outcome Pathways (2016) No. 6, OECD Publishing, Paris, doi: 10.1787/5jlr8vqgm630-en.
2. Synapse pathology in Alzheimer’s disease, Griffiths J, Grant GN. Seminars in Cell and Developmental Biology (2022) doi: 10.1016/j.semcdb.2022.05.028. (review)
3. Dopamine Restores Limbic Memory Loss, Dendritic Spine Structure, and NMDAR-Dependent LTD in the Nucleus Accumbens of Alcohol-Withdrawn Rats Cannizzaro C, et al. J Neurosci. (2019) Jan 30;39(5):929-943. doi: 10.1523/JNEUROSCI.1377-18.2018.
4. Actin in dendritic spines: connecting dynamics to function, Hotulainen P, Hoogenraad C. J Cell Biol (2010) 17;189(4):619-29. doi: 10.1083/jcb. 201003008. (review)
5. Role of actin cytoskeleton in dendritic spine morphogenesis. Sekino Y, et al. Neurochem Int. (2007) 51(2-4):92-104. doi: 10.1016/j.neuint.2007.04.029. (review)
6. Drebrin, a dendritic spine protein, is manifold decreased in brains of patients with Alzheimer’s disease and Down syndrome Shim KS, Lubec G. Neurosci Lett. 2002 May 24;324(3):209-12. doi: 10.1016/s0304-3940(02)00210-0.
7. Differential expression of synaptic proteins in the frontal and temporal cortex of elderly subjects with mild cognitive impairment, Counts SE, et al.  J Neuropathol Exp Neurol. 2006 Jun;65(6):592-601. doi: 10.1097/00005072-200606000-00007.
8. High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons Hanamura K, et al. J Pharmacol Toxicol Methods. (2019) 99:106607. doi: 10.1016/j.vascn.2019.106607.
9. Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay Mitsuoka T, et al. J Pharmacol Toxicol Methods. (2019) 99:106583. doi: 10.1016/j.vascn.2019
10. Genetic disruption of the alternative splicing of drebrin gene impairs context-dependent fear learning in adulthood, Kojima N, et al. Neuroscience. (2010) 165(1):138-50. Doi: 10.1016/j.neuroscience.2009.10.016.
11. Drebrin A regulates hippocampal LTP and hippocampus-dependent fear learning in adult mice, Kojima N, et al. Neuroscience. (2016) Jun 2;324:218-26. doi: 10.1016/j.neuroscience.2016.03.015.
12. Effective expression of Drebrin in hippocampus improves cognitive function and alleviates lesions of Alzheimer’s disease in APP (swe)/PS1 (ΔE9) mice, Liu Y, et al. CNS Neurosci Ther. (2017) Jul;23(7):590-604. doi: 10.1111/cns.12706.

Appendix 1

List of MIEs in this AOP

Event: 875: Binding of agonist, Ionotropic glutamate receptors

Short Name: Binding of agonist, Ionotropic glutamate receptors

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

Evidence for Chemical Initiation of this Molecular Initiating Event

L-Glutamate and glycine (or D-serine) are endogenous agonists that bind to the LBD of specific NMDA receptor subunits. Here listed some known agonists for NMDA receptor, some of them are specific to the NR1 subunit and some others to the NR2 subunit (reviewed in Traynelis et al., 2010):

Specific to NR1

Glycine, l-Serine, d-Serine, l-Alanine, d-Alanine, d-Cycloserine, HA 966, (+)-(1-hydroxy-3-aminopyrrolidine-2-one,) β-Cl-d-Alanine, β-F-dl-Alanine, tri-F-dl-Alanine, ACPC, 1-aminocyclopropane-1-carboxylic acid, ACBC, 1-aminocyclobutane-1-carboxylic acid, GLYX-13.

Specific to NR2

l-Glutamate, d-Glutamate, l-Aspartate, d-Aspartate, N-Methyl-l-aspartate, N-Methyl-d-aspartate, SYM208,1 l-Homocysteinsulfinate, d-Homocysteinsulfinate, l-Homocysteate, d-Homocysteate, l-Cysteinesulfinate, l-Cysteate, d-Cysteate, Homoquinolinate, Ibotenate, (R,S)-(Tetrazol-5-yl)glycine, L-CCG-IV, (2S,3R,4S)-2-(carboxycyclopropyl)glycine, trans-ACBD, trans-1-aminocyclobutane-1,3-dicarboxylate, cis-ADA, cis-azetidine-2,4-dicarboxylic acid, trans-ADC, azetidine-2,4-dicarboxylic acid, cis-ACPD, (1R,3R)-aminocyclopentane-cis-dicarboxylate, cis-2,3-Piperidinedicarboxylic acid, (R)-NHP4G, 2-(N-hydroxylpyrazol-4-yl)glycine, (R,S)-Ethyl-NHP5G, 2-(N-hydroxypyrazol-5-yl)glycine, (R)-Propyl-NHP5G, 2-(N-hydroxypyrazol-5-yl)glycine.

Domoic acid (DomA) is structurally similar to kainic acid (KA) and both of them are analogues of the excitatory neurotransmitter L-glutamate. DomA induces excitotoxicity by an integrative action on ionotropic glutamate receptors at pre- and post-synaptic sides. DomA directly activates KA/AMPARs receptors followed by indirect activation of the NMDARs. Indeed, indirect activation of NMDARs by DomA is linked to the fact that KA and AMPA receptors activated by DomA induce increased levels of intracellular Ca2+ and Na+ which, in turn, causes endogenous glutamate release that subsequently potentiates activation of NMDARs (Berman and Murray, 1997; Berman et al., 2002; Watanabe et al., 2011). DomA has been demonstrated through both in vitro and in vivo approaches to indirectly activate the NMDARs (reviewed in Pulido et al., 2008).

Glufosinate (GLF)((RS)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid, phosphinothricin) is a phosphorus containing amino acid herbicide that is naturally occurring as a component of the bacteria-derived bactericidal and fungicidal tripeptides bialaphos and phosalacine (Lanz et al., 2014). There are studies suggesting that convulsive and amnesic effects of GLF are mediated through direct binding and activation of NMDAR (Lantz et al., 2014; Matsumura et al., 2001). GLF agonist action at the NMDAR is expected to occur through direct interaction with the glutamate binding site and requires binding of the glycine co-agonist as well as release of the magnesium block from the channel pore.

Domain of Applicability

Taxonomic Applicability

---

The major determinants for ligand e.g. for both co-agonist glycine binding and L-glutamate binding are well conserved between species from lower organism to mammals (reviewed in Xia and Chiang, 2009). PCR analysis, cloning and subsequent sequencing of the seal lion NMDA receptors showed 80% homology to those from rats, but more than 95% homologus to those from dogs (Gill et al., 2010).

Key Event Description

The MIE of this AOP can be triggered by direct binding of an agonist to NMDARs or indirectly through initial activation of KA/AMPARs. Indeed, binding of agonist to KA/AMPARs results in ion influx (Na+ and a small efflux of K+) and glutamate release from excitatory synaptic vesicles causing depolarization of the postsynaptic neuron (Dingledine et al. 1999). Upon this depolarization the Mg2+ block is removed from the pore of NMDARs, allowing sodium, potassium, and importantly, calcium ions to enter into a cell. At positive potentials NMDARs then show maximal permeability (i.e., large outward currents can be observed under these circumstances). Due to the time needed for the Mg2+ removal, NMDARs activate more slowly, having a peak conductance long after the KA/AMPAR peak conductance takes place. It is important to note that NMDARs conduct currents only when Mg2+ block is relieved, glutamate is bound, and the postsynaptic neuron is depolarized. For this reason the NMDA receptors act as “coincidence detectors” and play a fundamental role in the establishment of Hebbian synaptic plasticity which is considered the physiological correlate of associative learning (Daoudal and Debanne, 2003; Glanzman, 2005). Post-synaptic membrane depolarization happens almost always through activation of KA/AMPARs (Luscher and Malenka, 2012). Therefore, a MIE of this AOP is defined as binding of an agonist to these three types of ionotropic receptors (KA/AMPA and NMDA) that can result in a prolonged overactivation of NMDARs through (a) direct binding of an agonist or (b) indirect, mediated through initial KA/AMPARs activation. The excitotoxic neuronal cell death, triggered by sustained NMDARs overactivation in the hippocampus and/or cortex leads to the impaired learning and memory, defined as the adverse outcome (AO) of this AOP.

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?

1. Ex vivo: The most common assay used is the NMDA receptor (MK801 site) radioligand competition binding assay (Reynolds and Palmer, 1991; Subramaniam and McGonigle, 1991; 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). The saturation binding of radioligands are used to measure the affinity (Kd) and density (Bmax) of kainate and AMPA receptors in striatum, cortex and hippocampus (Kürschner et al., 1998).

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 (Neville and Lytton, 1999; Mazumder Borah, 2014)

References

(for Abstract and MIE)

Barenberg P, Strahlendorf H, Strahlendorf., Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons. J. Neurosci Res. 2001, 40(3): 245-54.

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

Berman F.W. and T. F. Murray, “Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of excitatory amino acid release,” Journal of Neurochemistry, 1997, 69: 693–703.

Berman W.F., K. T. LePage, and T. F. Murray, Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca2+ influx pathway,” Brain Research, 2002, 924: 20–29.

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.

Daoudal G, Debanne D, Long-term plasticity of intrinsic excitability: learning rules and mechanisms. Learn Mem., 2003, 10(6):456-65.

Dingledine R, Borges K, Bowie D, Traynelis SF., The glutamate receptor ion channels. Pharmacol Rev., 1999, 51: 7–61.

Evans, R.H., Francis, A.A., Jones, A.W., et al., 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., 1982, 75: 65-75.

Gill S, Goldstein T, Situ D, Zabka TS, Gulland FM, Mueller RW., Cloning and characterization of glutamate receptors in Californian sea lions (Zalophus californianus). Mar Drugs, 2010, 8: 1637-1649.

Glanzman DL., Associative learning: Hebbian flies. Curr Biol., 2005, 7: 15(11):R416-9.

Kürschner VC, Petruzzi RL, Golden GT, Berrettini WH, Ferraro TN., Kainate and AMPA receptor binding in seizure-prone and seizure-resistant inbred mouse strains. Brain Res. 1998, 5: 780-788.

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

Lefebvre KA, Robertson A., Domoic acid and human exposure risks: a review.Toxicon. 2010, 56: 218-30.

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

Matsumura N, Takeuchi C., Hishikawa K, Fujii T, Nakaki T., Glufosinate ammonium induces convulsion through N-methyl-D-aspartate receptors in mice. Neurosci Lett. 2001, 304: 123-5.

Mazumder MK, Borah A. 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. Med Hypotheses. 2014, 83(6): 740-6.

Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity: bridge to various triggers in neurodegenerative disorders. Eur J Pharmacol. 2013, 698: 6-18.

Morris, RJ. 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., 1989, 9: 3040-3057.

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

Neville KR, Lytton WW. Potentiation of Ca2+ influx through NMDA channels by action potentials: a computer model. Neuroreport., 1999, 10(17): 3711-6.

Pulido OM., Domoic acid toxicologic pathology: a review. Mar Drugs., 2008, 6: 180-219.

Reynolds IJ, Palmer AM. Regional variations in [3H]MK801 binding to rat brain N-methyl-D-aspartate receptors. J Neurochem. 1991, 56(5):1731-40.

Subramaniam S, McGonigle P. Quantitative autoradiographic characterization of the binding of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ([3H]MK-801) in rat brain: regional effects of polyamines. J Pharmacol Exp Ther. 1991, 256(2): 811-9.

Schrattenholz A, Soskic V., NMDA receptors are not alone: dynamic regulation of NMDA receptor structure and function by neuregulins and transient cholesterol-rich membrane domains leads to disease-specific nuances of glutamate-signalling.Curr Top Med Chem., 2006, 6(7):663-86.

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. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev., 2010, 62: 405-496.

Watanabe KH, Andersen ME, Basu N, Carvan MJ 3rd, Crofton KM, King KA, Suñol C, Tiffany-Castiglioni E, Schultz IR. Defining and modeling known adverse outcome pathways: Domoic acid and neuronal signaling as a case study. Environ Toxicol Chem., 2011, 30: 9-21.

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

Retrieved from https://aopkb.org/aopwiki/index.php/?oldid=27027

List of Key Events in the AOP

Event: 388: Overactivation, NMDARs

Short Name: Overactivation, NMDARs

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability

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

Key Event Description

Biological state: Please see MIE NMDARs, Binding of antagonist

Biological compartments: Please see MIE NMDARs, Binding of antagonist

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

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

Additional text, specific for this AOP:

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

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

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

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

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

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

How it is Measured or Detected

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

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

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

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

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

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

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

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

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

References

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Event: 2078: Loss of drebrin

Short Name: Loss of drebrin

AOPs Including This Key Event

Biological Context

Event: 1944: Synaptic dysfunction

Short Name: Dysfunctional synapses

AOPs Including This Key Event

Biological Context

List of Adverse Outcomes in this AOP

Event: 341: Impairment, Learning and memory

Short Name: Impairment, Learning and memory

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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).While the prefrontal cortex and frontostriatal neuronal circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014).

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., 2001).

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 Wechsler Adult Intelligence Scale (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 test (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).

In Honey Bees: For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012; LaLone et al., 2017). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012). After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca2+-calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa andSandoz, 2012)."

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 TG 426) as well as OECD TG 443 (OECD, 2018) 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.

Guirfa, M., Sandoz, J.C., 2012. Invertebrate learning and memory: fifty years of olfactory conditioning of the proboscis extension response in honeybees. Learn. Mem. 19 (2),
54–66.

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.

LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death. STOTEN. 584-585, 751-775.

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.(2009) A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect.  Jan;117(1):17-25.

Menzel, R., 2012. The honeybee as a model for understanding the basis of cognition. Nat. Rev. Neurosci. 13 (11), 758–768.

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.

Puig, M.V., Antzoulatos, E.G., Miller, E.K., 2014. Prefrontal dopamine in associative learning and memory. Neuroscience 282, 217–229.

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