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Event: 52

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. 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. More help
Decreased, Calcium influx
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization
Cellular

Cell term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term
neuron

Organ term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  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 signaling 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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. 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 Agnes Aggy (send email) Open for citation & comment WPHA/WNT Endorsed
Binding of antagonist to NMDARs can lead to neuroinflammation and neurodegeneration KeyEvent Arthur Author (send email) Open for citation & comment WPHA/WNT Endorsed

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.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

An indication of the the relevant life stage(s) for this KE. More help

Sex Applicability

An indication of the the relevant sex for this KE. More help

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. 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

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.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). Do not provide detailed protocols. More help

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

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  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. 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.