AOP ID and Title:

Authors

Yuko Sekino¹, Shihori Tanabe², Noriko Koganezawa³, Tomoaki Shirao³

1 the University of Tokyo, Japan

2 National Institute of Health Sciences, Japan

3 AlzMed, Inc,, Japan

Status

Abstract

Neurotoxicity risk assessment is crucial for regulatory agencies, as current methods rely on time-consuming and costly animal testing. With thousands of chemicals lacking neurotoxicity data, there is an urgent need for in vitro methods to rapidly evaluate potential risks. Chemicals that impair learning and memory are linked to neurodegenerative diseases such as Parkinson's and Alzheimer's, underscoring the importance of effective risk assessment. The proposed AOP highlights a cascade where the loss of drebrin from dendritic spines induces spine morphological abnormalities, leading to synaptic dysfunction. Notably, synaptic dysfunction alone, even in the absence of neuronal death, can result in learning and memory impairments. This provides a novel framework for evaluating neurotoxicity and developmental neurotoxicity.

Dendritic spines are specialized structures that serve as the primary sites of excitatory synaptic transmission, primarily mediated by glutamate receptors. Spine formation and functional maturation are governed by drebrin expression. Drebrin, an actin-binding protein discovered and named by Shirao's team, has two isoforms: drebrin E (DE) and drebrin A (DA). DE, a non-neuronal protein involved in cell motility and protrusion formation, is predominantly expressed during early brain development. It is gradually replaced by DA, a neuron-specific protein that stabilizes actin filaments during synaptogenesis and synaptic maturation. The protein levels and subcellular localization of drebrin reflect its distinct roles in neuronal development and synaptic function. Loss of drebrin triggers spine abnormalities and synaptic dysfunction, ultimately impairing learning and memory as the adverse outcome (AO). This AOP builds on the molecular initiating event (MIE) of AOP 48—“Binding of agonists to ionotropic glutamate receptors”—but uniquely highlights drebrin loss as a critical KE.

Empirical evidence supports this AOP. Studies show that glutamate induces drebrin loss and dendritic spine morphological changes. Compounds that directly bind to NMDA receptors, such as glutamate and NMDA, and compounds that indirectly enhance NMDA receptor activity, have been shown to induce drebrin loss, linking such exposure to synaptic dysfunction and learning impairments. The detection of drebrin gene expression levels, subcellular localization, and protein levels provides valuable insights into brain development and higher-order functions across species. To develop effective in vitro testing methods, it is essential to have biomarkers that are simple, reproducible, and allow for quantitative data analysis. We determined that drebrin is highly suitable as a biomarker for these purposes.

The proposed AOP promotes alternative testing methods aligned with the 3Rs (Replacement, Reduction, Refinement), using cryopreserved hippocampal neurons to reduce animal use. Quantification of drebrin via immunocytochemistry and ELISA enables reproducible and scalable chemical screening. This AOP provides a foundation for in vitro prediction models, advancing chemical safety evaluation for humans and the environment. By integrating dynamic drebrin expression patterns and functional properties, this AOP framework offers a robust tool for regulatory decision-making and assessing neurotoxicity risks.

Background

Drebrin, identified by our group in 1985, has been a central focus of our research for many years. This actin-binding protein is known to exist in two isoforms: drebrin E and drebrin A. Drebrin E is expressed in both neuronal and certain non-neuronal cells, and it plays a crucial role during fetal and early postnatal stages of brain development. In neurons, drebrin E is involved in processes essential for cell motility, neurite outgrowth, and the extension of axons and dendrites, which together contribute to the establishment of neural networks. In contrast, drebrin A is a neuron-specific isoform whose expression is initiated during the synaptic formation stage. Drebrin A is indispensable for the formation and maintenance of dendritic spines, which serve as the primary sites for excitatory synaptic transmission, largely mediated by glutamate receptors. During brain development, drebrin E, which predominates in the early stages, is gradually replaced by drebrin A during synaptogenesis, reflecting their distinct roles in neuronal development, excitatory synapse formation and synaptic plasticity.

Based on the dynamic expression patterns and functional properties of drebrin, we have proposed an Adverse Outcome Pathway (AOP) framework for assessing developmental neurotoxicity and neurotoxicity. Monitoring drebrin gene expression levels, subcellular localization in neurons, and protein levels provides valuable insights into brain development and higher-order brain functions across various species.

In 2017, we summarized our research findings in a monograph titled Drebrin: From Structure and Function to Physiological and Pathological Roles, published as part of Springer’s Advances in Experimental Medicine and Biology series.

Between 2020 and 2023, we initiated the development of an AOP for neurotoxicity and developmental neurotoxicity caused by glutamate receptor-binding agonists, supported by a three-year research grant from the Japan Chemical Industry Association (JCIA) Long-range Research Initiative (LRI). This project, entitled "Proposal of a new AOP for the neurotoxicity and developmental neurotoxicity assessment of glutamate receptor binding agonists that cause learning and memory impairment," identified a novel adverse outcome (AO): learning and memory impairment. This AO is characterized by key events, including the loss of drebrin from dendritic spines, leading to thin and elongated spine morphology and subsequent synaptic dysfunction. Drebrin, as a key regulator of dendritic spine morphology, plays an essential role in the structural plasticity associated with learning and memory. Furthermore, the subcellular localization of drebrin is dynamically influenced by glutamate receptor activity.

To advance these studies, we optimized Banker’s method for low-density neuronal cultures and developed an immunocytochemical evaluation system for drebrin clusters in hippocampal neurons using a 96-well plate format and frozen embryonic hippocampal neurons from rats. In addition, we designed a high-content imaging algorithm for the quantitative analysis of neuronal parameters, including neuron count, dendrite length, and drebrin clustering, using confocal image cytometry. Notably, our brightness distribution analysis of drebrin clusters demonstrated exceptional sensitivity, allowing for precise quantification of structural changes in neurons. These methodological advancements have enabled us to establish standardized protocols (SOPs) for both neuronal culture techniques and analytical procedures.

We plan to extend this research by developing an experimental system utilizing neurons derived from human-induced pluripotent stem cells (iPSCs), thereby enhancing the relevance and applicability of our findings to human brain development and function.

Summary of the AOP

Events

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

Key Event Relationships

Stressors

Overall Assessment of the AOP

The overall assessment of AOP475 emphasizes its relevance in evaluating neurodevelopmental toxicity caused by chemical exposures. The pathway links disruption of dendritic spine morphology to impaired synaptic plasticity and cognitive functions, such as learning and memory. Its robust mechanistic framework integrates molecular, cellular, and behavioral endpoints. AOP475 serves as a critical tool for regulatory risk assessment, particularly in identifying early indicators of synaptic dysfunction.

Domain of Applicability

Taxa:
Drebrin has been primarily studied in mammals, with its expression confirmed in vertebrates such as humans, mice, rats and C-elegans.

Sex:
No sex differences in the expression or function of drebrin have been reported. Therefore, its applicability is not influenced by sex.

Life Stage:
Drebrin has two main isoforms: drebrin E, which is predominantly expressed during the fetal and juvenile stages, and drebrin A, which is specific to the mature stage. This suggests that drebrin’s role varies depending on the life stage.

Essentiality of the Key Events

AOP475 describes a series of biological changes leading from the molecular initiating event (MIE), where glutamate receptor agonists bind to their receptors, to the adverse outcome (AO) of learning and memory impairment. Each Key Event (KE) plays an essential role in the progression of this pathway, and the causal relationships between these events are supported by experimental evidence and published literature. The following summarizes the Essentiality of Key Events in AOP475:

Molecular Initiating Event (MIE): Binding of agonist, Ionotropic glutamate receptors : Event ID MIE 875 

This is the primary trigger for all subsequent Key Events, initiating changes in calcium homeostasis and cellular signaling. The involvement of NMDAR in neurotoxicity has been extensively documented in the literature.

KE1: Overactivation, NMDAR  (KE 388)

NMDAR overactivation is a well-characterized phenomenon in excitotoxicity and has been supported by both experimental studies and reviews discussing its role in calcium dysregulation and neurodegeneration.

KE2: Increase, Intracellular Calcium overload   (KE389)

Overactivation of NMDAR causes a persistent increase in intracellular calcium levels, disrupting calcium homeostasis. This dysregulation impacts cytoskeletal dynamics, including actin remodeling, and is critical for the subsequent loss of Drebrin from dendritic spines. Evidence for calcium's role in synaptic and structural stability has been widely reported in the literature.

KE3: Loss of Drebrin from Dendritic Spines ( KE 2078 : a new key event )

Drebrin is essential for maintaining spine morphology and synaptic plasticity. Its loss from dendritic spines has been consistently observed in experimental models, and its significance is well-supported by publications emphasizing its role in spine stability and cognitive function.

KE4:Abnormalities Dendritic Spine Morphological (KE2242: a new key event)　

Dendritic spines become thin and elongated, losing their structural stability. Morphological changes in spines impair excitatory synaptic transmission and plasticity. These abnormalities are well-documented in both experimental findings and studies on neurodegenerative diseases.

KE5: Synaptic Dysfunction (KE1944: a new key event ) 

Becasue denddritc spine is a structure that receive glutamate signals via glutamate receptoes. Synaptic transmission efficiency declines, reducing the ability of neurons to communicate effectively. Synaptic dysfunction, including reduced synaptic strength and plasticity, has been confirmed through electrophysiological studies and corroborated by literature focusing on the role of spines in neural networks.

KE6: Decrease of Neuronal Network Function

Network-level impairments are supported by experimental models and computational studies, as well as publications addressing the effects of synaptic disruptions on overall brain function.

Adverse Outcome (AO): Learning and Memory Impairment (AO 341)

The adverse outcome is the culmination of upstream events, supported by behavioral studies and widely recognized in reviews discussing neurotoxicity and cognitive decline.

Weight of Evidence Summary

Biological plausibility: In AOP 475, chemical stimulation of glutamate receptors, particularly NMDA receptors, results in excessive intracellular calcium influx. This calcium overload leads to a loss of drebrin from dendritic spines through several possible mechanisms, such as translocation via acto-myosin interaction from dendritic spines to dendritic shafts, degradation by calpain, the ubiquitin-proteasome system (UPS) and caspases, calcineurin-dependent dephosphorylation leading to drebrin destabilization, and supression of drebrin synathesis through inhibition of mRNA translation. Under normal physiological conditions, drebrin binds to actin within dendritic spines, stabilizing spine morphology. Temporary drebrin reduction occurs even during physiological NMDA receptor activation and calcium influx; however, drebrin returns to dendritic spines during long-term potentiation (LTP), stabilizing newly inserted receptors and slightly enlarging spine morphology. In contrast, prolonged drebrin loss during long-term depression (LTD) changed spine morphokogy and enhances endocytosis, reducing PSD95 and glutamate receptors. Pathological conditions exacerbate drebrin loss, which is implicated in memory impairment associated with Alzheimer's disease (AD).

Empirical Support: Drebrin reduction begins during mild cognitive impairment (MCI), an early stage of AD. Experimentally induced drebrin reduction via genetic manipulation or radiation exposure results in learning and memory deficits, which can be reversed upon restoration of drebrin levels. Furthermore, animal models of Alzheimer's disease also exhibit decreased drebrin levels.

Quantitative understanding:

Existing experimental studies have quantitatively demonstrated that prolonged or excessive activation of NMDA receptors leads to measurable increases in intracellular calcium levels, which correlate with significant reductions in drebrin levels in dendritic spines.　There is a quantitative relationship between the dose of glutamate applied to neuron cultures and the reduction of the number of drebrin clusters. Duration of the glutamate treatment showed shifts of the dose-response curve to the left.　Quantitative data from immunocytochemical assays using cultured neurons have also shown dose-dependent decreases in drebrin cluster density following exposure to NMDA receptor agonists or neurotoxicants. Additionally, quantitative correlations between the extent of drebrin loss and the severity of cognitive impairment have been reported in animal models and clinical studies, underscoring the potential to derive quantitative thresholds or benchmarks useful in chemical risk assessment.

Quantitative Consideration

Drebrin loss has dose-response relationships to concentration of glutamate, depending on the period of the trearment.

Cognitive impairment has dode-respose to drebrin level in the brain.

Considerations for Potential Applications of the AOP (optional)

AOP475 provides a framewirk useful in risk assessments of regulatory toxicology.

 KE 2078, drebrin loss, can be quantitatively measured using immunocytochemistry. Thus, targeted assays could be developed to evaluate chemicals' potential to disrupt synaptic function and cognitive performance. Quantitative measurement of the number of drebrin clusters, assessed by immunohistochemistry using neuronal cultures derived from frozen embryonic neurons, could be integrated into an IATA framework to provide a comprehensive neurotoxicity assessment, reducing reliance on animal testing.

References

1. Shirao T., Sekino Y. (Eds.) (2017) Drebrin. (Advances in Experimental Medicine and Biology, vol 1006) Tokyo, Springer
2. 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.
3. 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)
4. 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.
5. 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)
6. 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)
7. 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.
8. 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.
9. 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.
10. 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
11. 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.
12. 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.
13. 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

Domain of Applicability

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

Organ term

Domain of 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: 389: Increased, Intracellular Calcium overload

Short Name: Increased, Intracellular Calcium overload

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Please see KE Calcium influx, Decreasedin the AOP entitled Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.

Additional text, specific for the AOP “Acetylcholinesterase Inhibition leading to Neurodegeneration”:

Zebrafish have shown dysregulation in intracellular calcium ion levels following exposure to organophosphate compounds through similar mechanisms demonstrated in mammals (Faria et al. 2015).

Key Event Description

NMDAR agonist binding results in increased intracellular calcium, whereas NMDAR antagonist binding results in decreased intracellular calcium levels. For the relevant paragraphs below please see AOP entitled Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.

Biological state: KE Calcium influx, Decreased

Biological compartments: KE Calcium influx, Decreased

General role in biology: KE Calcium influx, Decreased

The text specific for the AOP "ionotropic glutamatergic receptors and cognition” and “Acetylcholinesterase inhibition leading to neurodegeneration”:

It is now well accepted that modest activation of NMDARs leading to modest increases in postsynaptic calcium are optimal for triggering LTD (Lledo et al. 1998; Bloodgood and Sabatin, 2007; Bloodgood et al. 2009), whereas much stronger activation of NMDARs leading to much larger increases in postsynaptic calcium are required to trigger LTP (Luscher and Malenka, 2012; Malenka 1994). Indeed, high-frequency stimulation causes a strong temporal summation of the excitatory postsynaptic potentials (EPSPs), and depolarization of the postsynaptic cell is sufficient to relieve the Mg2+ block of the NMDAR and allow a large amount of calcium to enter into the postsynaptic cells. Therefore, intra-cellular calcium is measured as a readout for evaluation NMDAR stimulation.

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?

Please see KE Calcium influx, Decreasedin the AOP entitled: Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.

References

Bloodgood BL, Sabatini BL., Nonlinear regulation of unitary synaptic signals by CaV2.3 voltage-sensitive calcium channels located in dendritic spines. Neuron, 2007, 53:249–260.

Bloodgood BL, Giessel AJ, Sabatini BL., Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines. PLoS Biol., 2009, 7: e1000190.

Faria, M., N. Garcia-Reyero, F. Padrós, P. J. Babin, D. Sebastián, J. Cachot, E. Prats, M. Arick Ii, E. Rial, A. Knoll-Gellida, G. Mathieu, F. Le Bihanic, B. L. Escalon, A. Zorzano, A. M. Soares and D. Raldúa (2015), "Zebrafish Models for Human Acute Organophosphorus Poisoning.” Sci Rep 5. DOI: 10.1038/srep15591.

Lledo PM, Zhang X, Sudhof TC, Malenka RC, Nicoll RA., Postsynaptic membrane fusion and long-term potentiation. Science, 1998, 279: 399–403.

Malenka RC. Synaptic plasticity in the hippocampus: LTP and LTD. Cell, 1994, 78: 535–538.

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.

Event: 2078: Loss of drebrin

Short Name: Loss of drebrin

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

The results can be applied when developmental neurotoxicity and neurotoxicity in the following way. 

1.Drebrin as a biomarker for Neurotoxicity: Drebrin localization in the dendritc spine of matured neuron is hightly sensitive to calcium in flux via NMDA receptors and calpain-mediated degradation. Its critical role in dendritic spine morphology and synaptic function makes it a potential biomarker for assessing neurotoxicity caused by chemical substances.

2.Evaluation of Effects on Synaptic Plasticity: The localization changes and degradation of drebrin can serve as indicators to assess the impact of chemicals on synaptic plasticity (e.g., LTP and LTD). 

3.Calcium-dependent Toxicity ;In cases where chemicals induce excitotoxicity through NMDA receptor-mediated calcium influx, drebrin degradation can clarify the specific pathways and extent of neurotoxic damage.

4.Evaluation of Risk mitigation strategied; Chemicals to prevent drebrin degradation can contribute to strategies to rescue the toxicity.

5.Evolutionary Conservation of Drebrin: Drebrin's evolutionary conservation allows for the development of neurotoxicity assessment systems that can be applied across various species, including non-human models, to support broader toxicological evaluations.

Example Scenarios for Application:

• Neurotoxicity screening for newly developed chemicals.
• Risk assessment of existing chemicals that may increase the likelihood of neurodegenerative diseases.
• Providing data for safety standards in industries such as pharmaceuticals, agriculture, and industrial chemicals.

Key Event Description

Glutamate induces drebrin exodus from dendritic spines to dendritic shafts, which is reported in matured cultured neuron of rodent and human iPS-derived neurons.

NMDA-induced excitotoxicity elicits the degradation of drebrin in matured cultured neurons of rodent hippocampus and cortex. This process is triggered by calcium influx and mediated by calpains.

Drebrin is an evolutionarily conserved actin-binding protein in dendritic spines.  Overexpression of drebrin A in neurons enlarges dendritic spines and decreases spine motility, whereas down-regulation of drebrin A in neurons decreases the density and width of dendritic spines and inhibits synaptic clustering of NMDARs.

Drebrin forms stable actin filaments and plays a pivotal role in dendritic spine morphogenesis (Hayashi and Shirao 1999; Takahashi et al., 2003; Takahashi et al., 2006).

During the initial stage of synaptic plasticity (either LTP or LTD), Ca²⁺ influx through the NMDA receptors arises and it brings out drebrin exodus from dendritic spines (Sekino et al., 2006).

Furthermore, prolonged NMDA-induced excitotoxicity induces calpain-mediated degradation of drebrin in vitro and in vivo.

How it is Measured or Detected

Loss of drebrin from dendritic spines can be detected by immunocytochemistry, ELISA or or Western blotting  in cultured human and rodent neurons and brain tissues (Counts et al., 2006; Ishizuka et al., 2014).

The twenty-one-day primary cultured neurons were prepared using frozen stock of dissociated hippocampal neurons (Koganezawa et al, 2023; Hanamura et al 2019). In brief, cells were cultured in multi well microplates with defined medium. 

For immunocytochemistry, the cultured neurons were incubated with chemicals for 1 hour. After fixation, cultured neurons were immunostained with anti-drebrin and anti-MAP2 antibodies. The cluster density of drebrin along the dendrites was automatically quantified using high content analysis instruments (Hanamura et al, 2019, Mitsuoka et al, 2019). 

For enzyme-linked immunoassay, after the incubation of chemicals the extracts of neurons were quantified using ELISA kit for drebrin (ALzMEd, Inc.),

References

Yamazaki H, Koganezawa N, Yokoo H, Sekino Y, Shirao T. Super-resolution imaging reveals the relationship between CaMKIIβ and drebrin within dendritic spines. Neurosci Res. 2024 Feb;199:30-35. doi: 10.1016/j.neures.2023.08.002. Epub 2023 Sep 1. PMID: 37659612.

Kajita Y, Kojima N, Shirao T. A lack of drebrin causes olfactory impairment. Brain Behav. 2024 Jan;14(1):e3354. doi: 10.1002/brb3.3354. PMID: 38376048; PMCID: PMC10757890.

Song L, Chen H, Qiao D, Zhang B, Guo F, Zhang Y, Wang C, Li S, Cui H. ZIP9 mediates the effects of DHT on learning, memory and hippocampal synaptic plasticity of male Tfm and APP/PS1 mice. Front Endocrinol (Lausanne). 2023 May 25;14:1139874. doi: 10.3389/fendo.2023.1139874. PMID: 37305050; PMCID: PMC10248430.

Lin W, Shiomoto S, Yamada S, Watanabe H, Kawashima Y, Eguchi Y, Muramatsu K, Sekino Y. Dendritic spine formation and synapse maturation in transcription factor-induced human iPSC-derived neurons. iScience. 2023 Feb 27;26(4):106285. doi: 10.1016/j.isci.2023.106285. Erratum in: iScience. 2023 Jul 20;26(8):107423. doi: 10.1016/j.isci.2023.107423. PMID: 37034988; PMCID: PMC10073939.

Koganezawa, N., Roppongi, R.T.,Sekino, Y., Tsutsui, I., Higa, A.,Shirao, T. Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons. J. Vis. Exp. (191), e64872, doi:10.3791/64872 (2023).

Mitsuoka T, Hanamura K, Koganezawa N, Kikura-Hanajiri R, Sekino Y, Shirao T. “Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay” J Pharmacol Toxicol Methods 2019 May 10:106583. doi: 10.1016/j.vascn.2019.106583.

Hanamura K, Koganezawa N, Kamiyama K, Tanaka N, Oka T, Yamamura M, Sekino Y, Shirao T. “High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons.” J Pharmacol Toxicol Methods. 2018 Sep - Oct;99:106607. doi: 10.1016/j.vascn.2019.106607.

Yamazaki H, Sasagawa Y, Yamamoto H, Bito H, Shirao T. CaMKIIβ is localized in dendritic spines as both drebrin-dependent and drebrin-independent pools. J Neurochem. 2018 Jul;146(2):145-159. doi: 10.1111/jnc.14449. Epub 2018 Jun 11. PMID: 29675826; PMCID: PMC6099455.

Cho C, MacDonald R, Shang J, Cho MJ, Chalifour LE, Paudel HK. Early growth response-1-mediated down-regulation of drebrin correlates with loss of dendritic spines. J Neurochem. 2017 Jul;142(1):56-73. doi: 10.1111/jnc.14031. Epub 2017 Apr 26. PMID: 28369888.

Liu Y, Xu YF, Zhang L, Huang L, Yu P, Zhu H, Deng W, Qin C. Effective expression of Drebrin in hippocampus improves cognitive function and alleviates lesions of Alzheimer's disease in APP (swe)/PS1 (ΔE9) mice. CNS Neurosci Ther. 2017 Jul;23(7):590-604. doi: 10.1111/cns.12706. Epub 2017 Jun 8. PMID: 28597477; PMCID: PMC6492767.

Sekino Y, Koganezawa N, Mizui T, Shirao T. Role of Drebrin in Synaptic Plasticity. Adv Exp Med Biol. 2017;1006:183-201. doi: 10.1007/978-4-431-56550-5_11. PMID: 28865021.

Puspitasari A, Koganezawa N, Ishizuka Y, Kojima N, Tanaka N, Nakano T, Shirao T. X Irradiation Induces Acute Cognitive Decline via Transient Synaptic Dysfunction. Radiat Res. 2016 Apr;185(4):423-30. doi: 10.1667/RR14236.1. Epub 2016 Mar 29. PMID: 27023259.

Chimura T, Launey T, Yoshida N (2015) Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo. PLoS ONE 10(4): e0125119. doi:10.1371/journal.pone.0125119

Jung G, Kim EJ, Cicvaric A, Sase S, Gröger M, Höger H, Sialana FJ, Berger J, Monje FJ, Lubec G. Drebrin depletion alters neurotransmitter receptor levels in protein complexes, dendritic spine morphogenesis and memory-related synaptic plasticity in the mouse hippocampus. J Neurochem. 2015 Jul;134(2):327-39. doi: 10.1111/jnc.13119. Epub 2015 Apr 29. PMID: 25865831.

Ishizuka Y, Shimizu H, Takagi E, Kato M, Yamagata H, Mikuni M, Shirao T. Histone deacetylase mediates the decrease in drebrin cluster density induced by amyloid beta oligomers. Neurochem Int. 2014 Oct;76:114-21. doi: 10.1016/j.neuint.2014.07.005. Epub 2014 Jul 21. PMID: 25058791.

Mizui T, Sekino Y, Yamazaki H, Ishizuka Y, Takahashi H, Kojima N, Kojima M, Shirao T. Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines. PLoS One. 2014 Jan 22;9(1):e85367. doi: 10.1371/journal.pone.0085367. PMID: 24465547; PMCID: PMC3899004.

Roppongi RT, Kojima N, Hanamura K, Yamazaki H, Shirao T. Selective reduction of drebrin and actin in dendritic spines of hippocampal neurons by activation of 5-HT(2A) receptors. Neurosci Lett. 2013 Jun 28;547:76-81. doi: 10.1016/j.neulet.2013.04.061. Epub 2013 May 14. PMID: 23684573.

Kojima N, Hanamura K, Yamazaki H, Ikeda T, Itohara S, Shirao T. Genetic disruption of the alternative splicing of drebrin gene impairs context-dependent fear learning in adulthood. Neuroscience. 2010 Jan 13;165(1):138-50. doi: 10.1016/j.neuroscience.2009.10.016. Epub 2009 Oct 24. PMID: 19837137.

Aoki C, Kojima N, Sabaliauskas N, Shah L, Ahmed TH, Oakford J, Ahmed T, Yamazaki H, Hanamura K, Shirao T. Drebrin a knockout eliminates the rapid form of homeostatic synaptic plasticity at excitatory synapses of intact adult cerebral cortex. J Comp Neurol. 2009 Nov 1;517(1):105-21. doi: 10.1002/cne.22137. PMID: 19711416; PMCID: PMC2839874.

Ivanov A, Esclapez M, Ferhat L. Role of drebrin A in dendritic spine plasticity and synaptic function: Implications in neurological disorders. Commun Integr Biol. 2009 May;2(3):268-70. doi: 10.4161/cib.2.3.8166. PMID: 19641748; PMCID: PMC2717538.

Takahashi H, Mizui T, Shirao T. Down-regulation of drebrin A expression suppresses synaptic targeting of NMDA receptors in developing hippocampal neurones. J Neurochem. 2006 Apr;97 Suppl 1:110-5. doi: 10.1111/j.1471-4159.2005.03536.x. PMID: 16635259.

Sekino Y, Tanaka S, Hanamura K, Yamazaki H, Sasagawa Y, Xue Y, Hayashi K, Shirao T. (2006)  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.Mar;31(3):493-504. doi: 10.1016/j.mcn.2005.11.003. Epub 2005 Dec 20. PMID: 16368245.

Kobayashi R, Sekino Y, Shirao T, Tanaka S, Ogura T, Inada K, Saji M. Antisense knockdown of drebrin A, a dendritic spine protein, causes stronger preference, impaired pre-pulse inhibition, and an increased sensitivity to psychostimulant. Neurosci Res. 2004 Jun;49(2):205-17. doi: 10.1016/j.neures.2004.02.014. PMID: 15140563.

Aoki C, Fujisawa S, Mahadomrongkul V, Shah PJ, Nader K, Erisir A. NMDA receptor blockade in intact adult cortex increases trafficking of NR2A subunits into spines, postsynaptic densities, and axon terminals. Brain Res. 2003 Feb 14;963(1-2):139-49. doi: 10.1016/s0006-8993(02)03962-8. PMID: 12560119.

Shim KS, Lubec G. Drebrin, a dendritic spine protein, is manifold decreased in brains of patients with Alzheimer's disease and Down syndrome. Neurosci Lett. 2002 May 24;324(3):209-12. doi: 10.1016/s0304-3940(02)00210-0. PMID: 12009525.

Tomidokoro Y, Harigaya Y, Matsubara E, Ikeda M, Kawarabayashi T, Shirao T, Ishiguro K, Okamoto K, Younkin SG, Shoji M. Brain Abeta amyloidosis in APPsw mice induces accumulation of presenilin-1 and tau. J Pathol. 2001 Aug;194(4):500-6. doi: 10.1002/path.897. PMID: 11523060.

Harigaya Y, Shoji M, Shirao T, Hirai S. Disappearance of actin-binding protein, drebrin, from hippocampal synapses in Alzheimer's disease. J Neurosci Res. 1996 Jan 1;43(1):87-92. doi: 10.1002/jnr.490430111. PMID: 8838578.

Event: 2242: Abnormality, dendritic spine morphology

Short Name: Dendritic spine abnormality

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Primary cultured neuron analysis

Synaptic protein expression analysis

Spine classification:

Electrophysiological analysis

Key Event Description

Abnormalities in dendritic spines have been implicated in a wide range of psychiatric and neurological disorders, with initial discoveries stemming from Golgi staining of postmortem brains. These spine pathologies have been observed across various conditions, including schizophrenia, autism spectrum disorders (ASD), Alzheimer's disease (AD), bipolar disorder, Down syndrome, and epilepsy.　Alzheimer's disease brains exhibit reduced synapse density and dendritic spine loss in the cortex and hippocampus, with greater spine loss associated with lower mental status　This synaptic pathology is considered one of the earliest features of AD, occurring prior to neuronal loss. The shared feature of spine pathology across these disorders suggests that dendritic spines may serve as a common substrate for many brain disorders involving deficits in information processing and neuronal connectivity.

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How it is Measured or Detected

Drebrin immunocytochemistry 

number of drebrin clustees 

References

Results from the National Center for Biotechnology Information (NCBI) at the U.S. National Library of Medicine (NLM).

Search: Dendritic Spine abnormality

Marin-Padilla M. Pyramidal cell abnormalities in the motor cortex of a child with Down's syndrome. A Golgi study. J Comp Neurol. 1976 May 1;167(1):63-81. doi: 10.1002/cne.901670105. PMID: 131810.

Gonatas NK, Moss A. Pathologic axons and synapses in human neuropsychiatric disorders. Hum Pathol. 1975 Sep;6(5):571-82. doi: 10.1016/s0046-8177(75)80042-6. PMID: 170188.

 Marin-Padilla M. Abnormal neuronal differentiation (functional maturation) in mental retardation. Birth Defects Orig Artic Ser. 1975;11(7):133-53. PMID: 764896.

Purpura DP. Normal and aberrant neuronal development in the cerebral cortex of human fetus and young infant. UCLA Forum Med Sci. 1975;(18):141-69. doi: 10.1016/b978-0-12-139050-1.50014-8. PMID: 128168.

Purpura DP. Dendritic spine "dysgenesis" and mental retardation. Science. 1974 Dec 20;186(4169):1126-8. doi: 10.1126/science.186.4169.1126. PMID: 4469701.

Search : decrease of drebrin

Xie MJ, Yagi H, Iguchi T, Yamazaki H, Hanamura K, Matsuzaki H, Shirao T, Sato M. Phldb2 is essential for regulating hippocampal dendritic spine morphology through drebrin in an adult-type isoform-specific manner. Neurosci Res. 2022 Dec;185:1-10. doi: 10.1016/j.neures.2022.09.010. Epub 2022 Sep 23. PMID: 36162735.

Event: 1944: Synaptic dysfunction

Short Name: Dysfunctional synapses

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Species applicability includes humans, using clinical assessments like neuroimaging, EEG, and cognitive tests; animal models (e.g., rodents) employing electrophysiological, behavioral, and biochemical assays; and cultured neuronal cells for molecular-level studies. Life-stage applicability covers developmental stages for neurodevelopmental disorders, adulthood for psychiatric and neurodegenerative diseases, and elderly populations for age-related cognitive decline. Experimental system applicability spans in vitro models for cellular mechanisms, ex vivo brain slices for electrophysiological studies, and in vivo animal models for behavioral and systemic analysis. Disease applicability involves neurological and psychiatric conditions such as Alzheimer's, Parkinson's, schizophrenia, depression, epilepsy, and autism spectrum disorders. Clearly defining these domains ensures accurate interpretation, facilitates translational research, and supports targeted therapeutic development for various neurological and psychiatric disorders.

Key Event Description

Synaptic dysfunction refers to the impairment or disruption of communication between neurons at synapses. It occurs when neurotransmitter release, receptor function, or synaptic structure is altered or damaged, leading to impaired signaling. Such dysfunction can contribute to various neurological and psychiatric conditions, including Alzheimer's disease, Parkinson's disease, depression, schizophrenia, and autism spectrum disorders. 

How it is Measured or Detected

• Electrophysiology (Patch Clamp, Field Potential Recording): Measures neurotransmition efficiency and receptor function by currents recordings. Evaluate synaptic plasticity, such as LTP and LTD
• Microdialysis or HPLC (High-Performance Liquid Chromatography): Quantifies neurotransmitter levels directly.
• Western Blot, ELISA: Evaluate proteins expression.
• Immunohistochemistry and imagings : Evaluate expression and locarization for pre- and post synaptic proteins, mitochondrial imaging, infkammation and immune responses using microscopies.
• Behavioral Tests (e.g., Morris Water Maze, Novel Object Recognition): Evaluate cognitive functions related to synaptic plasticity.
• Morphlogical changes: Electron Microscopy (EM): Visualize synaptic ultrastructure. Confocal or Two-photon Microscopy: Observe dendritic spine morphology and density changes. Golgi Staining: Analyze dendritic spine density and morphology.
• MRI, PET EEG

Event: 386: Decrease of neuronal network function

Short Name: Neuronal network function, Decreased

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

In vitro studies in brain slices applying electrophysiological techniques showed significant variability among species (immature rats, rabbits and kittens) related to synaptic latency, duration, amplitude and efficacy in spike initiation (reviewed in Erecinska et al., 2004).

Key Event Description

Biological state: There are striking differences in neuronal network formation and function among the developing and mature brain. The developing brain shows a slow maturation and a transient passage from spontaneous, long-duration action potentials to synaptically-triggered, short-duration action potentials.

Furthermore, at this precise developmental stage the neuronal network is characterised by "hyperexcitability”, which is related to the increased number of local circuit recurrent excitatory synapses and the lack of γ-amino-butyric acid A (GABAA)-mediated inhibitory function that appears much later. This “hyperexcitability” disappears with maturation when pairing of the pre- and postsynaptic partners occurs and synapses are formed generating population of postsynaptic potentials and population of spikes followed by developmental GABA switch. Glutamatergic neurotransmission is dominant at early stages of development and NMDA receptor-mediated synaptic currents are far more times longer than those in maturation, allowing more calcium to enter the neurons. The processes that are involved in increased calcium influx and the subsequent intracellular events seem to play a critical role in establishment of wiring of neural circuits and strengthening of synaptic connections during development (reviewed in Erecinska et al., 2004). Neurons that do not receive glutaminergic stimulation are undergoing developmental apoptosis.

During the neonatal period, the brain is subject to profound alterations in neuronal circuitry due to high levels of synaptogenesis and gliogenesis. For example, in neuroendocrine regions such as the preoptic area-anterior hypothalamus (POA-AH), the site of gonadotropin-releasing hormone (GnRH) system is developmentally regulated by glutamatergic neurons. The changes in the expression of the N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B system begin early in postnatal development, before the onset of puberty, thereby playing a role in establishing the appropriate environment for the subsequent maturation of GnRH neurons (Adams et al., 1999).

Biological compartments: Neural network formation and function happen in all brain regions but it appears to onset at different time points of development (reviewed in Erecinska et al., 2004). Glutamatergic neurotransmission in hippocampus is poorly developed at birth. Initially, NMDA receptors play important role but the vast majority of these premature glutamatergic synapses are “silent” possibly due to delayed development of hippocampal AMPA receptors. In contrast, in the cerebral cortex the maturation of excitatory glutamatergic neurotransmission happens much earlier. The “silent” synapses disappear by PND 7-8 in both brain regions mentioned above.

There is strong evidence suggesting that NMDA receptor subunit composition controls synaptogenesis and synapse stabilization (Gambrill and Barria, 2011). It is established fact that during early postnatal development in the rat hippocampus, synaptogenesis occurs in parallel with a developmental switch in the subunit composition of NMDA receptors from NR2B to NR2A. It is suggested that early expression of NR2A in organotypic hippocampal slices reduces the number of synapses and the volume and dynamics of spines. In contrast, overexpression of NR2B does not affect the normal number and growth of synapses. However, it does increase spine motility, adding and retracting spines at a higher rate. The C terminus of NR2B, and specifically its ability to bind CaMKII, is sufficient to allow proper synapse formation and maturation. Conversely, the C terminus of NR2A was sufficient to stop the development of synapse number and spine growth. These results indicate that the ratio of synaptic NR2B over NR2A controls spine motility and synaptogenesis, and suggest a structural role for the intracellular C terminus of NR2 in recruiting the signalling and scaffolding molecules necessary for proper synaptogenesis. Interestingly, it was found that genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent, the deletion of NR3A accelerates the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 sugesting that glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (Henson et al., 2012).

General role in biology: The development of neuronal networks can be distinguished into two phases: an early ‘establishment’ phase of neuronal connections, where activity-dependent and independent mechanisms could operate, and a later ‘maintenance’ phase, which appears to be controlled by neuronal activity (Yuste and Sur, 1999). These neuronal networks facilitate information flow that is necessary to produce complex behaviors, including learning and memory (Mayford et al., 2012).

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?

In vivo: The recording of brain activity by using electroencephalography (EEG), electrocorticography (ECoG) and local field potentials (LFP) assists towards the collection of signals generated by multiple neuronal cell networks. Advances in computer technology have allowed quantification of the EEG and expansion of quantitative EEG (qEEG) analysis providing a sensitive tool for time-course studies of different compounds acting on neuronal networks' function (Binienda et al., 2011). The number of excitatory or inhibitory synapses can be functionally studied at an electrophysiological level by examining the contribution of glutamatergic and GABAergic synaptic inputs. The number of them can be determined by variably clamping the membrane potential and recording excitatory and inhibitory postsynaptic currents (EPSCs or IPSCs) (Liu, 2004).

In vitro: Microelectrode array (MEA) recordings are also used to measure electrical activity in cultured neurons (Keefer et al., 2001, Gramowski et al., 2000; Gopal, 2003; Johnstone et al., 2010). MEAs can be applied in high throughput platforms to facilitate screening of numerous chemical compounds (McConnell et al., 2012). Using selective agonists and antagonists of different classes of receptors their response can be evaluated in a quantitative manner (Novellino et al., 2011; Hogberg et al., 2011).

Patch clamping technique can also be used to measure neuronal network activity.In some cases, if required, planar patch clamping technique can also be used to measure neuronal networks activity (e.g., Bosca et al., 2014).

References

Adams MM, Flagg RA, Gore AC., Perinatal changes in hypothalamic N-methyl-D-aspartate receptors and their relationship to gonadotropin-releasing hormone neurons. Endocrinology. 1999 May;140(5):2288-96.

Binienda ZK, Beaudoin MA, Thorn BT, Ali SF. (2011) Analysis of electrical brain waves in neurotoxicology: γ-hydroxybutyrate. Curr Neuropharmacol. 9: 236-239.

Bosca, A., M. Martina, and C. Py (2014) Planar patch clamp for neuronal networks--considerations and future perspectives. Methods Mol Biol, 2014. 1183: p. 93-113.

Erecinska M, Cherian S, Silver IA. (2004) Energy metabolism in mammalian brain during development. Prog Neurobiol. 73: 397-445.

Gambrill AC, Barria A. NMDA receptor subunit composition controls synaptogenesis and synapse stabilization. Proc Natl Acad Sci U S A. 2011:108(14):5855-60.

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.

Henson MA, Larsen RS, Lawson SN, Pérez-Otaño I, Nakanishi N, Lipton SA, Philpot BD. (2012) Genetic deletion of NR3A accelerates glutamatergic synapse maturation. PLoS One. 7(8).

Hogberg HT, Sobanski T, Novellino A, Whelan M, Weiss DG, Bal-Price AK. (2011) Application of micro-electrode arrays (MEAs) as an emerging technology for developmental neurotoxicity: evaluation of domoic acid-induced effects in primary cultures of rat cortical neurons. Neurotoxicology 32: 158-168.

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.

Liu G. (2004) Local structural balance and functional interaction of excitatory and inhibitory synapses in hippocampal dendrites. Nat Neurosci. 7: 373-379.

Mayford M, Siegelbaum SA, Kandel ER. (2012) Synapses and memory storage. Cold Spring Harb Perspect Biol. 4. pii: a005751.

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.

Novellino A, Scelfo B, Palosaari T, Price A, Sobanski T, Shafer TJ, Johnstone AF, Gross GW, Gramowski A, Schroeder O, Jügelt K, Chiappalone M, Benfenati F, Martinoia S, Tedesco MT, Defranchi E, D'Angelo P, Whelan M. (2011) Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals. Front Neuroeng. 4: 4.

Yuste R, Peinado A, Katz LC. (1992) Neuronal domains in developing neocortex. Science 257: 665-669.

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

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). 

Life stage applicability: This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). 

Sex applicability: This key event is not sex specific (Cekanaviciute et al., 2018), although sex-dependent cognitive outcomes have been recently ; Parihar et al., 2020). 

Evidence for perturbation by a prototypic stressor: Current literature provides ample evidence of impaired learning and memory being induced by ionizing radiation (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). 

Key Event Description

 (Adapted from KE: 341 - in blue) 

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 behavior. On the other hand, non-associative learning can be defined as an alteration in the behavioral 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 characterized 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 neural 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 behavioral 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, Hebb-Williams 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. 

RAM, Barnes, MWM, Hebb-Williams maze 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). The Hebb- Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004). 

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). 

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). 

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). 

Operant Responding. Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). 

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: 

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). 

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). 

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). 

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

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). 

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). 

Attentional set-shifting (ATSET) task. Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015). 

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 behavior. 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). 

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Appendix 2

List of Key Event Relationships in the AOP

10 µM