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Event: 52

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Decreased, Calcium influx

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Decreased, Calcium influx

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Cellular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
neuron

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
calcium ion transport calcium ion decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Binding of antagonist to NMDARs impairs cognition KeyEvent Anna Price (send email) Open for citation & comment TFHA/WNT Endorsed
Binding of antagonist to NMDARs can lead to neuroinflammation and neurodegeneration KeyEvent Florianne Tschudi-Monnet (send email) Open for citation & comment TFHA/WNT Endorsed

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mice Mus sp. High NCBI
zebrafish Danio rerio High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

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Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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

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

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

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

No OECD method is available to measure intracellular Ca2+.

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

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

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

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

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

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

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

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

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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

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

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

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

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

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

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

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

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

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

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

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

Lewit-Bentley A, Réty S. (2000) EF-hand calcium-binding proteins. Curr Opin Struct Biol. 2000 Dec;10(6):637-43.

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

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

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

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