7440-61-1JFALSRSLKYAFGM-UHFFFAOYSA-NJFALSRSLKYAFGM-UHFFFAOYSA-N
UraniumUranium, isotope of mass 238
238U Element
UN 2979 (DOT)
Uranium I
DTXSID10425227440-43-9BDOSMKKIYDKNTQ-UHFFFAOYSA-NBDOSMKKIYDKNTQ-UHFFFAOYSA-N
CadmiumCadimium
CADMIUM BLUE
CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER
DTXSID102394014277-97-5VZFRNCSOCOPNDB-AOKDLOFSSA-NVZFRNCSOCOPNDB-AOKDLOFSSA-N
L-Domoic acid(2S,3S,4R,5'R)-2-Carboxy-4-(5'-carboxy-1'-methyl-1Z,3E-hexadienyl)-3-pyrrolidineacetic acid
DTXSID20274180GO:0005739mitochondrionGO:0017146NMDA selective glutamate receptor complexCHEBI:39124calcium ionGO:0008328ionotropic glutamate receptor complexCL:0000129microglial cellCL:0000127astrocyteGO:0007612learningGO:0007613memoryGO:0008219cell deathMP:0002229neurodegenerationGO:0004972NMDA glutamate receptor activityGO:0006816calcium ion transportGO:0099536synaptic signalingGO:0004970ionotropic glutamate receptor activityMP:0001847brain inflammation7functional change2decreased1increased11pathologicalUranium2021-08-05T14:28:502021-08-05T14:28:50Nanoparticles and Micrometer Particles2022-02-04T13:43:432022-02-04T13:43:43Cadmium2017-10-25T08:33:122017-10-25T08:33:12Sars-CoV-2<p>Virus from the coronaviridae family related to SARS-CoV, 229E, NL63, OC43, HKU1 and MERS.</p>
<p>Transmitted by aerosols</p>
2021-02-23T04:50:402022-09-09T05:09:36Chemical2017-02-07T13:22:422017-02-07T13:22:42SARS-CoV2020-03-01T10:42:462020-03-01T10:42:46Virus2018-05-29T07:10:012018-05-29T07:10:01Domoic acid2016-11-29T18:42:132016-11-29T18:42:13bacteria2021-02-23T05:15:412021-02-23T05:15:41WCS_9606human10090mouse10116ratWCS_7227fruit flyWCS_7955zebrafishWCS_160004gastropodsWikiUser_25human and other cells in culture10116Rattus norvegicusWCS_7227Drosophila melanogaster976298Primates sp. BOLD:AAA000110095mice9541Macaca fascicularisWikiUser_14MonkeyN/A, Mitochondrial dysfunction 1N/A, Mitochondrial dysfunction 1Cellular<p>Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.</p>
<p>Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006).</p>
<p>Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation.</p>
<p>A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM), are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (mPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010).</p>
<p><u>Metal-induced Mitochondrial Dysfunction</u><br />
Mitochondria are an important site of Ca2+ regulation and storage, taking up Ca2+ ions electrophoretically from the cytosol through a Ca2+ uniporter, which can then accumulate in the mitochondria (Roos et al., 2012; Orrenius et al., 2015). Similarities between calcium and metals, such as cadmium and lead, makes the entrance and accumulation of these metals into the mitochondria via calcium metals possible by mode of molecular mimicry (Mathews et al., 2013; Adiele et al., 2012). The outer mitochondrial membrane also contains the divalent metal transporter (DMT1), which allows for mitochondrial uptake of divalent metals such as Fe and Mn. When cells are under heavy metal-induced stress, DMT has been shown to be overexpressed in the mitochondrial membrane, making the mitochondria targets of metal toxicity and accumulation.</p>
<p>Heavy metal exposure in aerobic organisms increases ROS formation through redox cycling, where metals with different valence states (Fe, Cu, Cr, etc.) directly produce ROS as they are reduced by cellular antioxidants and then react with oxygen (Shaki et al., 2012; Shaki et al., 2013; Pourahmad et al., 2006; Santos et al., 2007). The production of highly reactive hydroxyl radicals under mitochondrial oxidative stress and in the presence of transition metals occurs via the Fenton reaction or Haber-Weiss reaction (Hancock et al., 2001; Valko et al., 2005; Adam-Vizi et al., 2010). Metals and ROS are capable of damaging mitochondrial DNA as well as mechanisms of DNA repair and proliferation arrest (Valko et al., 2005). Metals and ROS have the potential to directly damage mitochondrial membranes and structure by binding to and oxidizing membrane lipids and proteins. This structural damage can collapse the MMP and lead to the opening of the MPTP (Orrenius et al., 2015; Roos et al., 2012; Pourahmad et al., 2006). Uranium and mercury, for example, have both been shown to directly inhibit the mitochondrial electron transport chain and interfere with ATP production (Shaki et al., 2012; Roos et al., 2012). Furthermore, as previously mentioned, metals have been shown to inhibit ROS-detoxifying enzymes. By binding to these enzymes, metals can inhibit their antioxidant functions, and cause an accumulation of ROS and increased synthesis of more antioxidant enzymes in order to combat the oxidative stress (Blajszczak and Bonini, 2017).</p>
<p><strong>Summing up:</strong> Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.</p>
<p>Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail.</p>
<p>I. Mitochondrial dysfunction assays assessing a loss-of function.</p>
<p>1. Cellular oxygen consumption.</p>
<p>See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O<sub>2</sub> consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005).</p>
<p>2. Mitochondrial membrane potential (Δψm ).</p>
<p>The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. The classical, and still most quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode on suspensions of isolated mitochondria. The Δψm can also be measured in live cells by fluorimetric methods. These are based on dyes which accumulate in mitochochondria because of Δψm. Frequently used are tetramethylrhodamineethylester (TMRE), tetramethylrhodaminemethyl ester (TMRM) (Petronilli et al., 1999) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1). Mitochondria with intact membrane potential concentrate JC-1, so that it forms red fluorescent aggregates, whereas de-energized mitochondria cannot concentrate JC-1 and the dilute dye fluoresces green (Barrientos et al., 1999). Assays using TMRE or TMRM measure only at one wavelength (red fluorescence), and depending on the assay setup, de-energized mitochondria become either less fluorescent (loss of the dye) or more fluorescent (attenuated dye quenching).</p>
<p>3. Enzymatic activity of the electron transport system (ETS).</p>
<p>Determination of ETS activity can be dene following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990).</p>
<p>4. ATP content.</p>
<p>For the evaluation of ATP levels, various commercially-available ATP assay kits are offered based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005).</p>
<p><br />
II. Mitochondrial dysfunction assays assessing a gain-of function.</p>
<p><br />
1. Mitochondrial permeability transition pore opening (PTP).</p>
<p>The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999).</p>
<p>2. mtDNA damage as a biomarker of mitochondrial dysfunction.</p>
<p>Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b).</p>
<p>3. Generation of ROS and resultant oxidative stress.</p>
<p>a. General approach. Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine.</p>
<p>b. Measurement of the cellular glutathione (GSH) status. GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential). GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress.</p>
<p>c. Quantification of lipid peroxidation. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method.</p>
<p><br />
d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (<a class="external free" href="http://www.biotek.com/resources/articles/reactive-oxygen-species.html" rel="nofollow" target="_blank">http://www.biotek.com/resources/articles/reactive-oxygen-species.html</a>). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in the absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at site of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria.</p>
<p>e. Detection of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) production. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H<sub>2</sub>O<sub>2</sub> form increasing amounts of fluorescent product (Tarpley et al., 2004).</p>
<p>Summing up, mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (mPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion) (Promega assay, TB369; Radkowsky et al., 1986) • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent. All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE <a href="/wiki/index.php/Event:209" title="Event:209"> Oxidative Stress, Increase</a></p>
<table border="1" cellpadding="1" cellspacing="1">
<tbody>
<tr>
<td>
<p><strong>Assay Type & Measured Content</strong></p>
</td>
<td><strong>Description</strong></td>
<td><strong>Dose Range Studied</strong></td>
<td>
<p><strong>Assay Characteristics</strong></p>
<p><strong>(Length/Ease of use/Accuracy)</strong></p>
</td>
</tr>
<tr>
<td>
<p><strong>Rhodamine 123 Assay</strong></p>
<p>Measuring Mitochondrial membrane potential (MMP) and its collapse </p>
<p>(Shaki et al., 2012)</p>
</td>
<td>
<p>Mitochondrial uptake of cationic fluorescent dye, rhodamine 123, is used for estimation of mitochondrial membrane potential. The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively.</p>
</td>
<td>50, 100 and 500 μM of uranyl acetate</td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>TMRE fluorescence Assay</strong></p>
<p>Measuring Mitochondrial permeability transition pore (mPTP) opening</p>
<p>(Huser et al., 1998)</p>
</td>
<td>Laser scanning confocal microscopy in combination with the potentiometric fluorescence dye tetramethylrhodamine ethyl ester to monitor relative changes in membrane potential in single isolated cardiac mitochondria. The cationic dye distributes across the membrane in a voltage-dependent manner. Therefore, the large potential gradient across the inner mitochondrial membrane results in the accumulation of the fluorescent dye within the matrix compartment. Rapid depolarizations are caused by the opening of the transition pore.</td>
<td>1 µM cyclosporin A</td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>GSH / GSSG Determination Assay</strong></p>
<p>Measuring cellular glutathione (GSH) status; ratio of GSH/GSSG</p>
<p>(Owen & Butterfield, 2010; Shaki et al., 2013)</p>
</td>
<td>GSH and GSSG levels are determinted biochemically with DTNB (Ellman’s reagent). The developed yellow color was read at 412 nm on a spectrophotometer.</td>
<td>100 µM uranyl acetate</td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>TBARS Assay</strong></p>
<p>Quantification of lipid peroxidation</p>
<p>(Yuan et al., 2016)</p>
</td>
<td>MDA content, a product of lipid peroxidation, was measured using a thiobarbituric acid reactive substances (TBARS) assay. Briefly, the kidney cells were collected in 1 ml PBS buffer solution (pH 7.4) and sonicated. MDA reacts with thiobarbituric acid forming a colored product which can be measured at an absorbance of 532 nm.</td>
<td>200, 400, 800 µM uranyl acetate</td>
<td>
<p>Medium / medium</p>
<p>High accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Aequorin-based bioluminescence assay</strong></p>
<p>Increase in mitochondrial Ca<sup>2+</sup> influx</p>
<p>(Pozzan & Rudolf, 2009)</p>
</td>
<td>Together with GFP, the aequorin moiety acts as Ca<sup>2+</sup> sensor <em>in vivo</em>, which delivers emission energy to the GFP acceptor molecule in a BRET (Bioluminescence Resonance Energy Transfer) process; the Ca2+ can then be visualized with fluorescence microscopy.</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Western blot & immunostaining analyses</strong></p>
<p>Measuring cytochrome c release</p>
(Chen et al., 2000)</td>
<td>Examining the redistribution of Cyto c in cytosolic and mitochondrial cellular fractions. Cells are homogenized and centrifuged, then prepared for immunoblots. Cellular fractions were washed in PBS and lysed in 1% NP-40 buffer. Cellular proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes, probed using immunoblot analyses with antibodies specific to cyto c (6581A for Western and 65971A for immunostaining; Pharmingen)</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Quantikine Rat/Mouse Cytochrome c Immunoassay</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Shaki et al., 2012)</p>
</td>
<td>Cytochrome C release was measured a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing mono- clonal antibody specific for cytochrome c conjugated to horseradish peroxidase). After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm.</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Membrane potential and cell viability – Flow Cytometry</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Kruidering et al., 1997)</p>
</td>
<td>“Dc and viability were determined by analyzing the R123 and propidium iodide fluorescence intensity with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser, with the Lysis software program (Becton Dickinson). R123 is a cationic dye that accumulates in the negatively charged inner side of the mitochondria. When the potential drops, less R123 accumulates in the mitochondria, which results in a lower fluorescence signal. The potential was measured as follows: at the indicated times, a 500-ml sample of the cell suspension was taken and transferred to an Eppendorf minivial. To this sample, 100 ml of 6 mM R123 in buffer D was added. After incubation for 10 min at 37°C, the cell suspension was centrifuged for 5 min at 80 3 <em>g</em>. The cell pellet was resuspended in 200 ml of buffer D, containing 0.2 mM R123 and 10 mM propidium iodide, to prevent loss of R123 and to stain nonviable cells, respectively. The samples were transferred to FACScan tubes and analyzed immediately. Analysis was performed at a flow rate of<br />
60 ml/min. R123 fluorescence was detected by the FL1 detector with an emission detection limit below 560 nm. Propidium iodide fluorescence was detected by the FL3 detector, with emission detection above 620 nm. Per sample 3,000 to 5,000 cells were counted (Van de Water <em>et al.</em>, 1993)”</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
</tbody>
</table>
<p>Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010).</p>
CL:0000255eukaryotic cellNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHigh<p>Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal. 2005, 7(9-10):1140-1149.</p>
<p>Adam-Vizi, V., & Starkov, A. A. (2010). Calcium and mitochondrial reactive oxygen species generation: How to read the facts. Journal of Alzheimer's Disease : JAD, 20 Suppl 2, S413-S426. doi:10.3233/JAD-2010-100465</p>
<p>Adiele, R. C., Stevens, D., & Kamunde, C. (2012). Differential inhibition of electron transport chain enzyme complexes by cadmium and calcium in isolated rainbow trout (oncorhynchus mykiss) hepatic mitochondria. Toxicological Sciences, 127(1), 110-119. doi:10.1093/toxsci/kfs091</p>
<p>Bal-Price A. and Guy C. Brown. Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria. J. Neurochemistry, 2000, 75: 1455-1464.</p>
<p>Bal-Price A, Matthias A, Brown GC., Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J. Neurochem. 2002, 80: 73-80.</p>
<p>Barrientos A., and Moraes C.T. (1999) Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. Vol. 274, No. 23, pp. 16188–16197.</p>
<p>Belyaeva, E. A., Sokolova, T. V., Emelyanova, L. V., & Zakharova, I. O. (2012). Mitochondrial electron transport chain in heavy metal-induced neurotoxicity : Effects of cadmium , mercury , and copper. Thescientificworld, 2012, 1-14. doi:10.1100/2012/136063</p>
<p>Blajszczak, C., & Bonini, M. G. (2017). Mitochondria targeting by environmental stressors: Implications for redox cellular signaling. Toxicology, 391, 84-89. doi:10.1016/j.tox.2017.07.013</p>
<p>Brand MD, Nicholls DG. Assessing mitochondrial dysfunction in cells. Biochem J. 2011 Apr 15;435(2):297-312.</p>
<p>Braun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182.</p>
<p>Cammen M. Corwin, Susannah Christensen. John P. (1990) Electron transport system (ETS) activity as a measure of benthic macrofaunal metabolism MARINE ECOLOGY PROGRESS SERIES- (65) : 171-182.</p>
<p>Chen, Q., Gong, B., & Almasan, A. (2000). Distinct stages of cytochrome c release from mitochondria: Evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death and Differentiation, 7(2), 227-233. doi:10.1038/sj.cdd.4400629</p>
<p>Ciapaite, Lolita Van Eikenhorst, Gerco Bakker, Stephan J.L. Diamant, Michaela. Heine, Robert J Wagner, Marijke J. V. Westerhoff, Hans and Klaas Krab (2005) Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria Diabetes 54:4 944-951.</p>
<p>Correia SC, Santos RX, Perry G, Zhu X, Moreira PI, Smith MA. (2012). Mitochondrial importance in Alzheimer’s, Huntington’s and Parkinson’s diseases. Adv Exp Med Biol 724:205 – 221.</p>
<p>Cozzolino M, Ferri A, Valle C, Carri MT. (2013). Mitochondria and ALS: implications from novel genes and pathways. Mol Cell Neurosci 55:44 – 49.</p>
<p>Diepart, C, Verrax, J Calderon, PU, Feron, O., Jordan, BF, Gallez, B (2010) Comparison of methods for measuring oxygen consumption in tumor cells in vitroAnalytical Biochemistry 396 (2010) 250–256.</p>
<p>Farooqui T. and . Farooqui, A. A (2012) Oxidative stress in Vertebrates and Invertebrate: molecular aspects of cell signalling. Wiley-Blackwell,Chapter 27, pp:377- 385.</p>
<p>Fan LM, Li JM. Evaluation of methods of detecting cell reactive oxygen species production for drug screening and cell cycle studies. J Pharmacol Toxicol Methods. 2014 Jul-Aug;70(1):40-7.</p>
<p>Fiskum G. Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma. 2000 Oct;17(10):843-55.</p>
<p>Friberg H, Wieloch T. (2002). Mitochondrial permeability transition in acute neurodegeneration. Biochimie 84:241–250.</p>
<p>Fujikawa DG, The Role of Excitotoxic Programmed Necrosis in Acute Brain Injury. Computational and Structural Biotechnology Journal, 2015, 13: 212–221.</p>
<p>Graier WF, Frieden M, Malli R. (2007). Mitochondria and Ca2+ signaling: old guests, new functions. Pflugers Arch 455:375–396.</p>
<p>Green DR. (1998). Apoptotic pathways: the roads to ruin. Cell 94:695-698.</p>
<p>Grivennikova VG, Vinogradov AD. Generation of superoxide by the mitochondrial Complex I. Biochim Biophys Acta. 2006, 1757(5-6):553-61.</p>
<p>Hafner RP, Brown GC, Brand MD: Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the ‘top-down’ approach of metabolic control theory. Eur J Biochem188 :313 –319,1990.</p>
<p>Hancock, J. T., Desikan, R., & Neill, S. J. (2001). Role of reactive oxygen species in cell signalling pathways. Biochemical Society Transactions, 29(Pt 2), 345-350. doi:10.1042/0300-5127:0290345 [doi]</p>
<p>Hao, Y., Huang, J., Liu, C., Li, H., Liu, J., Zeng, Y., . . . Li, R. (2016). Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity. Scientific Reports, doi:10.1038/srep38942</p>
<p>Hao, Y., Ren, J., Liu, C., Li, H., Liu, J., Yang, Z., . . . Su, Y. (2014). Zinc protects human kidney cells from depleted uranium induced apoptosis. Basic & Clinical Pharmacology & Toxicology, 114, 271-280. doi:10.1111/bcpt.12167</p>
<p>Hinkle PC (1995) Measurement of ADP/O ratios. In Bioenergetics: A Practical Approach. Brown GC, Cooper CE, Eds. Oxford, U.K., IRL Press, p.5 –6.</p>
<p>Huerta-García, E., Perez-Arizti, J. A., Marquez-Ramirez, S. G., Delgado-Buenrostro, N. L., Chirino, Y. I., Iglesias, G. G., & Lopez-Marure, R. (2014). Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells. Free Radical Biology and Medicine, 73, 84-94. doi:10.1016/j.freeradbiomed.2014.04.026</p>
<p>Hüser, J., Rechenmacher, C. E., & Blatter, L. A. (1998). Imaging the permeability pore transition in single mitochondria. Biophysical Journal, 74(4), 2129-2137. doi:10.1016/S0006-3495(98)77920-2</p>
<p>Hynes, J.. Marroquin, L.D Ogurtsov, V.I. Christiansen, K.N. Stevens, G.J. Papkovsky, D.B. Will, Y. (2006)) Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes, Toxicol. Sci. 92 186–200.</p>
<p>James, P.E. Jackson, S.K.. Grinberg, O.Y Swartz, H.M. (1995) The effects of endotoxin on oxygen consumption of various cell types in vitro: an EPR oximetry study, Free Radic. Biol. Med. 18 (1995) 641–647.</p>
<p>Kang J, Pervaiz S. (2012). Mitochondria: Redox Metabolism and Dysfunction. Biochem Res Int 2012:896751.</p>
<p>Kann O, Kovács R. (2007). Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292:C641-576.</p>
<p>Karlsson, H. L., Gustafsson, J., Cronholm, P., & Möller, L. (2009). Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size. Toxicology Letters, 188(2), 112-118. doi:<a href="https://doi.org/10.1016/j.toxlet.2009.03.014" target="_blank">10.1016/j.toxlet.2009.03.014</a></p>
<p>Knott Andrew B., Guy Perkins, Robert Schwarzenbacher & Ella Bossy-Wetzel. Mitochondrial fragmentation in neurodegeneration. Nature Reviews Neuroscience, 2008, 229: 505-518.</p>
<p>Kruidering, M., Van De Water, B., De Heer, E., Mulder, G. J., & Nagelkerke, J. F. (1997). Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: Mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. The Journal of Pharmacology and Experimental Therapeutics, 280(2), 638-649.</p>
<p>Llaudet E, Hatz S, Droniou M, Dale N. Microelectrode biosensor for real-time measurement of ATP in biological tissue. Anal Chem. 2005, 77(10):3267-73.</p>
<p>Lee HC, Wei YH. (2012). Mitochondria and aging. Adv Exp Med Biol 942:311-327.</p>
<p>Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, et al. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem.2003;278:8516–8525.</p>
<p>Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 2006. 443:787-795.</p>
<p>Martin LJ. (2011). Mitochondrial pathobiology in ALS. J Bioenerg Biomembr 43:569 – 579.</p>
<p>Martinez-Cruz, Oliviert Sanchez-Paz, Arturo Garcia-Carreño, Fernando Jimenez-Gutierrez, Laura Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, <a class="internal mw-magiclink-isbn" href="/wiki/index.php/Special:BookSources/9789535100904">ISBN 978-953-51-0090-4</a>, Publisher InTech, 2012, 181-218.</p>
<p>Mathews, C. K., Holde, K. E. van, Appling, D. R., & Anthony-Cahill, S. J. (2013). Biochemistry (4th ed.). Toronto: Pearson.</p>
<p>McBride HM, Neuspiel M, Wasiak S. (2006). Mitochondria: more than just a powerhouse. Curr Biol 16:R551–560.</p>
<p>McCord, J.M. and I. Fidovich (1968) The Reduction of Cytochrome C by Milk Xanthine Oxidase. J. Biol. Chem. 243:5733-5760.</p>
<p>Mei Y, Thompson MD, Cohen RA, Tong X. (2013) Endoplasmic Reticulum Stress and Related Pathological Processes. J Pharmacol Biomed Anal.. 1:100-107.</p>
<p>Miccadei, S., & Floridi, A. (1993). Sites of inhibition of mitochondrial electron transport by cadmium. Elsevier Scientific Publishers Ireland Ltd., 89, 159-167.Xu, X. M., & Møller, S. G. (2010). ROS removal by DJ-1: Arabidopsis as a new model to understand Parkinson's Disease. Plant signaling & behavior, 5(8), 1034–1036. doi:10.4161/psb.5.8.12298</p>
<p>Modjtahedi N, Giordanetto F, Madeo F, Kroemer G. Apoptosis-inducing factor: vital and lethal. Trends Cell Biol. 2006 May;16(5):264-72.</p>
<p>Nunnari J, Suomalainen A. (2012). Mitochondria: in sickness and in health. Cell 148:1145–1159. Hajnóczky G, Csordás G, Das S, Garcia-Perez C, Saotome M, Sinha Roy S, Yi M. (2006). Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40:553-560.</p>
<p>Oliviert Martinez-Cruz, Arturo Sanchez-Paz, Fernando Garcia-Carreño, Laura Jimenez-Gutierrez, Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, <a href="/wiki/index.php/Special:BookSources/9789535100904">ISBN 978-953-51-0090-4</a>, Publisher InTech, 2012, 181-218.</p>
<p>Orrenius, S., Gogvadze, V., & Zhivotovsky, B. (2015). Calcium and mitochondria in the regulation of cell death. Biochemical and Biophysical Research Communications, 460(1), 72-81. doi:10.1016/j.bbrc.2015.01.137</p>
<p>Owen, J. B., & Butterfield, D. A. (2010). Measurement of oxidized/reduced glutathione ratio. Methods in Molecular Biology (Clifton, N.J.), 648, 269-277. doi:10.1007/978-1-60761-756-3_18 [doi]</p>
<p>Owens R.G. and King F.D. The measurement of respiratory lectron-transport system activity in marine zooplankton. Mar. Biol. 1975, 30:27-36.</p>
<p>Pan, Y., Leifer, A., Ruau, D., Neuss, S., Bonrnemann, J., Schmid, G., . . . Jahnen-Dechent, W. (2009). Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small, 5(8), 2067-2076. doi:10.1002/smll.200900466</p>
<p>Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F: Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 1999, 76:725-734.</p>
<p>Pourahmad, J., Ghashang, M., Ettehadi, H. A., & Ghalandari, R. (2006). A search for cellular and molecular mechanisms involved in depleted uranium (DU) toxicity. Environmental Toxicology, 21(4), 349-354. doi:10.1002/tox.20196</p>
<p>Pozzan, T., & Rudolf, R. (2009). Measurements of mitochondrial calcium in vivo. Biochimica Et Biophysica Acta (BBA) - Bioenergetics, 1787(11), 1317-1323. doi:<a href="https://doi.org/10.1016/j.bbabio.2008.11.012" target="_blank">https://doi.org/10.1016/j.bbabio.2008.11.012</a></p>
<p>Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI.</p>
<p>Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379.</p>
<p>Radkowsky, A.E. and E.M. Kosower (1986) Bimanes 17. (Haloalkyl)-1,5-diazabicyclo[3.3.O]octadienediones (halo-9,10- dioxabimanes): reactivity toward the tripeptide thiol, glutathione, J. Am. Chem. Soc 108:4527-4531.</p>
<p>Roos, D., Seeger, R., Puntel, R., & Vargas Barbosa, N. (2012). Role of calcium and mitochondria in MeHg-mediated cytotoxicity. Journal of Biomedicine and Biotechnology, 2012, 1-15. doi:10.1155/2012/248764</p>
<p>Ruch, W., P.H. Cooper, and M. Baggiollini (1983) Assay of H2O2 production by macrophages and neutrophils with Homovanillic acid and horseradish peroxidase. J. Immunol Methods 63:347-357.</p>
<p>Sanders LH, McCoy J, Hu X, Mastroberardino PG, Dickinson BC, Chang CJ, Chu CT, Van Houten B, Greenamyre JT. (2014a). Mitochondrial DNA damage: molecular marker of vulnerable nigral neurons in Parkinson's disease. Neurobiol Dis. 70:214-23.</p>
<p>Sanders LH, Howlett EH2, McCoy J, Greenamyre JT. (2014b) Mitochondrial DNA damage as a peripheral biomarker for mitochondrial toxin exposure in rats. Toxicol Sci. Dec;142(2):395-402.</p>
<p>Santos, N. A. G., Catão, C. S., Martins, N. M., Curti, C., Bianchi, M. L. P., & Santos, A. C. (2007). Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Archives of Toxicology, 81(7), 495-504. doi:10.1007/s00204-006-0173-2</p>
<p>Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015</p>
<p>Shaki, F., Hosseini, M., Ghazi-Khansari, M., & Pourahmad, J. (2013). Depleted uranium induces disruption of energy homeostasis and oxidative stress in isolated rat brain mitochondria. Metallomics, 5(6), 736-744. doi:10.1039/c3mt00019b</p>
<p>Single B, Leist M, Nicotera P. Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ. 1998 Dec;5(12):1001-3.</p>
<p>Tahira Farooqui and Akhlaq A. Farooqui. (2012) Oxidative stress in Vertebrates and Invertebrate: molecular aspects of cell signalling. Wiley-Blackwell,Chapter 27, pp:377- 385.</p>
<p>Tarpley, M.M., D.A. Wink, and M.B. Grisham (2004) Methods for detection of reactive Metabolites of Oxygen and Nitrogen: in vitro and in vivo considerations. Am . J. Physiol Regul Integr Comp Physiol. 286:R431-R444.</p>
<p>Valko, M., Morris, H., & Cronin, M. T. (2005). Metals, toxicity and oxidative stress. Current Medicinal Chemistry, 12(10), 1161-1208. doi:10.2174/0929867053764635 [doi]</p>
<p>von Heimburg, D. Hemmrich, K. Zachariah S.,. Staiger, H Pallua, N.(2005) Oxygen consumption in undifferentiated versus differentiated adipogenic mesenchymal precursor cells, Respir. Physiol. Neurobiol. 146 (2005) 107–116.</p>
<p>Waerzeggers, Yannic Monfared, Parisa Viel, Thomas Winkeler, Alexandra Jacobs, Andreas H. (2010) Mouse models in neurological disorders: Applications of non-invasive imaging, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1802, Issue 10, Pages 819-839.</p>
<p>Walker JE, Skehel JM, Buchanan SK. (1995) Structural analysis of NADH: ubiquinone oxidoreductase from bovine heart mitochondria. Methods Enzymol.;260:14–34.</p>
<p>Wang A, Costello S, Cockburn M, Zhang X, Bronstein J, Ritz B. (2011). Parkinson’s disease risk from ambient exposure to pesticides. Eur J Epidemiol 26:547-555.</p>
<p>Wang, L., Li, J., Li, J., & Liu, Z. (2009). Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol.Trace Elem.Res., 137, 69-78. doi:10.1007/s12011-009-8560-1</p>
<p>Wang Y., and Qin ZH., Molecular and cellular mechanisms of excitotoxic neuronal death, Apoptosis, 2010, 15:1382-1402.</p>
<p>Wieloch T. (2001). Mitochondrial Involvement in Acute Neurodegeneration 52:247–254.</p>
<p>Winklhofer, K. Haass,C (2010) Mitochondrial dysfunction in Parkinson's disease, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1802: 29-44.</p>
<p>Yuan, Y., Zheng, J., Zhao, T., Tang, X., & Hu, N. (2016). Uranium-induced rat kidney cell cytotoxicity is mediated by decreased endogenous hydrogen sulfide (H2S) generation involved in reduced Nrf2 levels. Toxicology Research, 5(2), 660-673. doi:10.1039/C5TX00432B</p>
<p>Zhang, H., Chang, Z., Mehmood, K., Abbas, R. Z., Nabi, F., Rehman, M. U., . . . Zhou, D. (2018). Nano copper induces apoptosis in PK-15 cells via a mitochondria-mediated pathway. Biological Trace Element Research, 181(1), 62-70. doi:10.1007/s12011-017-1024-0</p>
<p>Zhou, M., Z.Diwu, Panchuk-Voloshina, N. and R.P. Haughland (1997), A Stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: application in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem 253:162-168.</p>
2016-11-29T18:41:232024-03-14T11:12:18Impairment, Learning and memoryImpairment, Learning and memoryIndividual<p> </p>
<p>Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non-associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behaviour. On the other hand, non-associative learning can be defined as an alteration in the behavioural response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning.</p>
<p>The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterised by the non-conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer).</p>
<p>Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. For example, the hippocampus has been shown to be critical for spatial-temporal memory, visio-spatial memory, verbal and narrative memory, and episodic and autobiographical memory (Burgess et al., 2000; Vorhees and Williams, 2014). However, there is substantial evidence that fundamental learning and memory functions are not mediated by the hippocampus alone but require a network that includes, in addition to the hippocampus, anterior thalamic nuclei, mammillary bodies cortex, cerebellum and basal ganglia (Aggleton and Brown, 1999; Doya, 2000; Mitchell et al., 2002, Toscano and Guilarte, 2005; Gilbert et al., 2006, 2016). Thus, damage to variety of brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).While the prefrontal cortex and frontostriatal neuronal circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014).</p>
<p>For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioural tests described below.</p>
<p><strong>In laboratory animals:</strong> 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, <span style="color:#3498db">Hebb-Williams maze</span>, 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.</p>
<p>1) RAM, Barnes, MWM, <span style="color:#3498db">Hebb-Williams maze </span>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 <span style="color:#3498db">Hebb-Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004).</span></p>
<p>2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention – I have seen one of these objects before, but not this one (Cohen and Stackman, 2015).</p>
<p>3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009).</p>
<p>4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2001).</p>
<p><span style="color:#3498db">5) Operant Responding. Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). </span></p>
<p><strong>In humans:</strong> 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:</p>
<p>1) Rey Osterieth Complex Figure test (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006).</p>
<p>2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986).</p>
<p>3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994).</p>
<p>4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986).</p>
<p>5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011).</p>
<p>6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).</p>
<p><span style="color:#3498db">7) Attentional set-shifting (ATSET) task. Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015).</span></p>
<p>8. Comprehensive developmental inventory for infants and toddlers (CDIIT). The CDIIT was designed and standardized in 1996, and it measures the global, cognitive, language, motor, gross motor, fine motor, social, self-help and behavioral developmental status of children from 3 to 71 months old (Wang et al., 1998).</p>
<p><strong>In Honey Bees:</strong> 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)."</p>
<p>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).</p>
<p><span style="color:#3498db"><strong>Life stage applicability: </strong>This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). </span></p>
<p><span style="color:#3498db"><strong>Sex applicability:</strong> This key event is not sex specific (Cekanaviciute et al., 2018), although sex-dependent cognitive outcomes have been recently ; Parihar et al., 2020). </span></p>
<p><span style="color:#3498db"><strong>Evidence for perturbation by a prototypic stressor: </strong>Current literature provides ample evidence of impaired learning and memory being induced by ionizing radiation (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). </span></p>
HighMixedHighDuring brain developmentHighAdult, reproductively matureHighHighHighHighHighHigh<p>Aggleton JP, Brown MW. (1999) Episodic memory, amnesia, and the hippocampal-anterior thalamic axis. Behav Brain Sci. 22: 425-489.</p>
<p>Alexander RD (1990) Epigenetic rules and Darwinian algorithms: The adaptive study of learning and development. Ethology and Sociobiology 11:241-303.</p>
<p>Bellinger DC (2012) A strategy for comparing the contributions of environmental chemicals and other risk factors to neurodevelopment of children. Environ Health Perspect 120:501-507.</p>
<p>Burgess N (2002) The hippocampus, space, and viewpoints in episodic memory. Q J Exp Psychol A 55:1057-1080. Cohen, SJ and Stackman, RW. (2015). Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav. Brain Res. 285: 105-1176.</p>
<p><span style="color:#3498db">Cekanaviciute, E., S. Rosi and S. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, Multidisciplinary Digital Publishing Institute (MDPI) AG, Basel, https://doi.org/10.3390/ijms19113669. </span></p>
<p>Cohen, SJ and Stackman, RW. (2015). Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav. Brain Res. 285: 105-1176.</p>
<p>Curzon P, Rustay NR, Browman KE. Cued and Contextual Fear Conditioning for Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009.</p>
<p>D'Hooge R, De Deyn PP (2001) Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 36:60-90.</p>
<p>Doya K. (2000) Complementary roles of basal ganglia and cerebellum in learning and motor control. Curr Opin Neurobiol. 10: 732-739.</p>
<p>Eichenbaum H (2000) A cortical-hippocampal system for declarative memory. Nat Rev Neurosci 1:41-50.</p>
<p>Fivush R. The development of autobiographical memory. Annu Rev Psychol. 2011;62:559-82.</p>
<p>Gilbert ME, Sanchez-Huerta K, Wood C (2016) Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.</p>
<p>Gilbert ME, Rovet J, Chen Z, Koibuchi N. (2012) Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology 33: 842-52.</p>
<p>Gilbert ME, Sui L (2006) Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.</p>
<p>Guirfa, M., Sandoz, J.C., 2012. Invertebrate learning and memory: fifty years of olfactory conditioning of the proboscis extension response in honeybees. Learn. Mem. 19 (2),<br />
54–66.</p>
<p>Herold, C, Lässer, MM, Schmid, LA, Seidl, U, Kong, L, Fellhauer, I, Thomann,PA, Essig, M and Schröder, J. (2015). Neuropsychology, Autobiographical Memory, and Hippocampal Volume in “Younger” and “Older” Patients with Chronic Schizophrenia. Front. Psychiatry, 6: 53.</p>
<p><span style="color:#3498db">Hladik, D. and S. Tapio. (2016), "Effects of ionizing radiation on the mammalian brain", Mutation Research/Reviews in Mutation Research, Vol. 770, Elsevier B. b., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.003. </span></p>
<p><span style="color:#3498db">Heisler, J. M. et al. (2015), "The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice", Journal of Visualized Experiments, 96, JoVe, Cambridge, https://doi.org/10.3791/51944. Heisler, J. M. et al. (2015), "The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice", Journal of Visualized Experiments, 96, JoVe, Cambridge, https://doi.org/10.3791/51944. </span></p>
<p>LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.<em> </em>STOTEN. 584-585, 751-775.</p>
<p>Lezak MD (1984) Neuropsychological assessment in behavioral toxicology--developing techniques and interpretative issues. Scand J Work Environ Health 10 Suppl 1:25-29.</p>
<p>Lezak MD (1994) Domains of behavior from a neuropsychological perspective: the whole story. Nebr Symp Motiv 41:23-55.</p>
<p>Makris SL, Raffaele K, Allen S, Bowers WJ, Hass U, Alleva E, Calamandrei G, Sheets L, Amcoff P, Delrue N, Crofton KM.(2009) A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect. Jan;117(1):17-25.</p>
<p>Menzel, R., 2012. The honeybee as a model for understanding the basis of cognition. Nat. Rev. Neurosci. 13 (11), 758–768.</p>
<p>Mitchell AS, Dalrymple-Alford JC, Christie MA. (2002) Spatial working memory and the brainstem cholinergic innervation to the anterior thalamus. J Neurosci. 22: 1922-1928.</p>
<p>OECD. 2007. OECD guidelines for the testing of chemicals/ section 4: Health effects. Test no. 426: Developmental neurotoxicity study. www.Oecd.Org/dataoecd/20/52/37622194.Pdf [accessed may 21, 2012].</p>
<p>OECD (2008) Nr 43 GUIDANCE DOCUMENT ON MAMMALIAN REPRODUCTIVE TOXICITY TESTING AND ASSESSMENT. ENV/JM/MONO(2008)16</p>
<p>Ono T. (2009) Learning and Memory. Encyclopedia of neuroscience. M D. Binder, N. Hirokawa and U. Windhorst (Eds). Springer-Verlag GmbH Berlin Heidelberg. pp 2129-2137.</p>
<p><span style="color:#3498db">Parihar, V. K. et al. (2020), "Sex-Specific Cognitive Deficits Following Space Radiation Exposure", Frontiers in Behavioral Neuroscience, Vol. 14, https://doi.org/10.3389/fnbeh.2020.535885. </span></p>
<p><span style="color:#3498db">Pritchett, K. and G. Mulder. (2004), "Hebb-Williams mazes.", Contemporary topics in laboratory animal science, Vol. 43/5, http://www.ncbi.nlm.nih.gov/pubmed/15461441. </span></p>
<p>Puig, M.V., Antzoulatos, E.G., Miller, E.K., 2014. Prefrontal dopamine in associative learning and memory. Neuroscience 282, 217–229.</p>
<p><span style="color:#3498db">Rabin, B. M. et al. (2002), "Effects of Exposure to 56Fe Particles or Protons on Fixed-ratio Operant Responding in Rats", Journal of Radiation Research, Vol. 43/S, https://doi.org/10.1269/jrr.43.S225. </span></p>
<p>Roberts AC, Bill BR, Glanzman DL. (2013) Learning and memory in zebrafish larvae. Front Neural Circuits 7: 126.</p>
<p>Rohlman DS, Lucchini R, Anger WK, Bellinger DC, van Thriel C. (2008) Neurobehavioral testing in human risk assessment. Neurotoxicology. 29: 556-567.</p>
<p>Shin, MS, Park, SY, Park, SR, Oeol, SH and Kwon, JS. (2006). Clinical and empirical applications of the Rey-Osterieth complex figure test. Nature Protocols, 1: 892-899.</p>
<p>Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372-376.</p>
<p>Stanton ME, Spear LP (1990) Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol Teratol 12:261-267.</p>
<p>Talley, JL. (1986). Memory in learning disabled children: Digit span and eh Rey Auditory verbal learning test. Archives of Clinical Neuropsychology, Elseiver.</p>
<div>
<div>T.M. Wang, C.W. Su, H.F. Liao, L.Y. Lin, K.S. Chou, S.H. Lin The standardization of the comprehensive developmental inventory for infants and toddlers Psychol. Test., 45 (1998), pp. 19-46</div>
<div> </div>
<div>Toscano CD, Guilarte TR. (2005) Lead neurotoxicity: From exposure to molecular effects. Brain Res Rev. 49: 529-554.</div>
</div>
<p>U.S.EPA. 1998. Health effects guidelines OPPTS 870.6300 developmental neurotoxicity study. EPA Document 712-C-98-239.Office of Prevention Pesticides and Toxic Substances.</p>
<p>Vorhees CV, Williams MT (2014) Assessing spatial learning and memory in rodents. ILAR J 55:310-332.</p>
<div>
<div>Willoughby KA, McAndrews MP, Rovet JF. Accuracy of episodic autobiographical memory in children with early thyroid hormone deficiency using a staged event. Dev Cogn Neurosci. 2014 Jul;9:1-11.</div>
</div>
<p> </p>
2016-11-29T18:41:242023-06-26T12:44:45Cell injury/deathCell injury/deathCellular<p style="text-align:justify">Two types of cell death can be distinguished by morphological features, although it is likely that these are two ends of a spectrum with possible intermediate forms. Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of the cell into discrete bodies with intact plasma membranes. These are rapidly phagocytosed by neighbouring cells. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. In contrast, necrotic cell death is characterized by cell swelling and lysis. This is usually a consequence of profound loss of mitochondrial function and resultant ATP depletion, leading to loss of ion homeostasis, including volume regulation, and increased intracellular Ca2+. The latter activates a number of nonspecific hydrolases (i.e., proteases, nucleases, and phospholipases) as well as calcium dependent kinases. Activation of calpain I, the Ca2+-dependent cysteine protease cleaves the death-promoting Bcl-2 family members Bid and Bax which translocate to mitochondrial membranes, resulting in release of truncated apoptosis-inducing factor (tAIF), cytochrome c and endonuclease in the case of Bid and cytocrome c in the case of Bax. tAIF translocates to cell nuclei, and together with cyclophilin A and phosphorylated histone H2AX (γH2AX) is responsible for DNA cleavage, a feature of programmed necrosis. Activated calpain I has also been shown to cleave the plasma membrane Na+–Ca2+ exchanger, which leads to build-up of intracellular Ca2+, which is the source of additional increased intracellular Ca2+. Cytochrome c in cellular apoptosis is a component of the apoptosome.</p>
<p style="text-align:justify">DNA damage activates nuclear poly(ADP-ribose) polymerase-1(PARP-1), a DNA repair enzyme. PARP-1 forms poly(ADP-ribose) polymers, to repair DNA, but when DNA damage is extensive, PAR accumulates, exits cell nuclei and travels to mitochondrial membranes, where it, like calpain I, is involved in AIF release from mitochondria. A fundamental distinction between necrosis and apoptosis is the loss of plasma membrane integrity; this is integral to the former but not the latter. As a consequence, lytic release of cellular constituents promotes a local inflammatory reaction, whereas the rapid removal of apoptotic bodies minimizes such a reaction. The distinction between the two modes of death is easily accomplished in vitro but not in vivo. Thus, although claims that certain drugs induce apoptosis have been made, these are relatively unconvincing. DNA fragmentation can occur in necrosis, leading to positive TUNEL staining <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">(<span style="font-size:16px">see explanation below</span>)</span></span>. Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis.</p>
<p style="text-align:justify">Two alternative pathways - either extrinsic (receptor-mediated) or intrinsic (mitochondria-mediated) - lead to apoptotic cell death. The initiation of cell death begins either at the plasma membrane with the binding of TNF or FasL to their cognate receptors or within the cell. The latter is due to the occurrence of intracellular stress in the form of biochemical events such as oxidative stress, redox changes, covalent binding, lipid peroxidation, and consequent functional effects on mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, or DNA. The intrinsic mitochondrial pathway involves the initiator, caspase-9, which, when activated, forms an “apoptosome” in the cytosol, together with cytochrome c, which translocates from mitochondria, Apaf-1 and dATP. The apoptosome activates caspase-3, the central effector caspase, which in turn activates downstream factors that are responsible for the apoptotic death of a cell (Fujikawa, 2015). Intracellular stress either directly affects mitochondria or can lead to effects on other organelles, which then send signals to the mitochondria to recruit participation in the death process (Fujikawa, 2015; Malhi et al., 2010).<sup> </sup>Constitutively expressed nitric oxide synthase (nNOS) is a Ca2+-dependent cytosolic enzyme that forms nitric oxide (NO) from L-arginine, and NO reacts with the free radical such as superoxide (O2−) to form the very toxic free radical peroxynitrite (ONOO−). Free radicals such as ONOO−, O2 − and hydroxyl radical (OH−) damage cellular membranes and intracellular proteins, enzymes and DNA (Fujikawa, 2015; Malhi et al., 2010; Kaplowitz, 2002; Kroemer et al., 2009). </p>
<p> </p>
<p><strong>Necrosis:</strong></p>
<p style="text-align:justify">Lactate dehydrogenase (LDH) is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor. The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample (Chan et al., 2013). </p>
<p style="text-align:justify">The MTT assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes include XTT, MTS and the WSTs. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light (Berridgeet al.,2005).</p>
<p style="text-align:justify">Propidium iodide (PI) is an intercalating agent and a fluorescent molecule used to stain necrotic cells. It is cell membrane impermeant so it stains only those cells where the cell membrane is destroyed. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm (Moore et al.,1998)</p>
<p style="text-align:justify">Alamar Blue (resazurin) is a fluorescent dye. The oxidized blue non fluorescent Alamar blue is reduced to a pink fluorescent dye in the medium by cell activity (O'Brien et al., 2000) (12).</p>
<p style="text-align:justify">Neutral red uptake, which is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in lysosomes (Repetto et al., 2008)(13). <span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Moreover, quantification of ATP, signaling the presence of metabolically active cells, can be performed (CellTiter-Glo; Promega).</span></span></p>
<p style="text-align:justify">ATP assay: Quantification of ATP, signaling the presence of metabolically active cells (CellTiter-Glo; Promega).</p>
<p style="text-align:justify"><br />
<strong>Apoptosis:</strong></p>
<p style="text-align:justify">TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage.</p>
<p style="text-align:justify">Caspase activity assays measured by fluorescence. During apoptosis, mainly caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. Antibodies to the 85 kDa fragment of cleaved PARP or to caspase-3 both serve as markers for apoptotic cells that can be monitored using immunofluorescence (Li, Peng et al., 2004).</p>
<p style="text-align:justify">Hoechst 33342 staining: Hoechst dyes are cell-permeable and bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. The stained, condensed or fragmented DNA is a marker of apoptosis (Loo, 2002; Kubbies and Rabinovitch, 1983). </p>
<p style="text-align:justify">Acridine Orange/Ethidium Bromide staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis.</p>
<p>Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).<sup> </sup></p>
CL:0000255eukaryotic cellNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHighHigh<ul>
<li>Fujikawa, D.G. (2015), The role of excitotoxic programmed necrosis in acute brain injury, Comput Struct Biotechnol J, vol. 13, pp. 212-221.</li>
<li>Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.</li>
<li>Kaplowitz, N. (2002), Biochemical and Cellular Mechanisms of Toxic Liver Injury, Semin Liver Dis, vol. 22, no. 2,<span style="color:#000000"> </span><a class="external free" href="http://www.medscape.com/viewarticle/433631" rel="nofollow" target="_blank"><span style="color:#000000">http://www.medscape.com/viewarticle/433631</span></a><span style="color:#000000"> </span>(accessed on 20 January 2016).</li>
<li>Kroemer, G. et al., (2009), Classification of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death Differ, vol. 16, no. 1, pp. 3-11.</li>
<li>Chan, F.K., K. Moriwaki and M.J. De Rosa (2013), Detection of necrosis by release of lactate dehydrogenase (LDH) activity, Methods Mol Biol, vol. 979, pp. 65–70.</li>
<li>Berridge, M.V., P.M. Herst and A.S. Tan (2005), Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, vol. 11, pp 127-152.</li>
<li>Moore, A, et al.(1998), Simultaneous measurement of cell cycle and apoptotic cell death,Methods Cell Biol, vol. 57, pp. 265–278.</li>
<li>Li, Peng et al. (2004), Mitochondrial activation of apoptosis, Cell, vol. 116, no. 2 Suppl,pp. S57-59, 2 p following S59.</li>
<li>Loo, D.T. (2002), TUNEL Assay an overview of techniques, Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage, chapter 2, Didenko VV (ed.), Humana Press Inc.</li>
<li>Kubbies, M. and P.S. Rabinovitch (1983), Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes, Cytometry, vol. 3, no. 4, pp. 276–281.</li>
<li>Fink, S.L. and B.T. Cookson (2005), Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells, Infect Immun, vol. 73, no. 4, pp.1907-1916.</li>
<li>O'Brien J, Wilson I, Orton T, Pognan F. 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European journal of biochemistry / FEBS 267(17): 5421-5426.</li>
<li>Repetto G, del Peso A, Zurita JL. 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols 3(7): 1125-1131.</li>
</ul>
2016-11-29T18:41:222022-07-15T09:46:25N/A, NeurodegenerationN/A, NeurodegenerationTissue<p style="margin-left:7.0pt">The term neurodegeneration is a combination of two words - "neuro," referring to nerve cells and "degeneration," referring to progressive damage. The term "neurodegeneration" can be applied to several conditions that result in the loss of nerve structure and function, and neuronal loss by necrosis and/or apoptosis</p>
<p>Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of “neurodegenerative diseases" including Huntington's, Alzheimer’s and Parkinson’s disease. All of these conditions lead to progressive brain damage and neurodegeneration.</p>
<p>Alzheimer's disease is characterised by loss of neurons and synapses in the cerebral cortex and certain subcortical regions, with gross atrophy of the affected regions; symptoms include memory loss.</p>
<p>Parkinson's disease (PD) results from the death of dopaminergic neurons in the midbrain substantia nigra pars compacta; symptoms include bradykinesia, rigidity, and resting tremor.</p>
<p>Several observations suggest correlative links between environmental exposure and neurodegenerative diseases, but only few suggest causative links:</p>
<p>Only an extremely small proportion (less than 5%) of neurodegenerative diseases are caused by genetic mutations (Narayan and Dragounov, 2017). The remainders are thought to be caused by the following:</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->A build up of toxic proteins in the brain (Evin et al., 2006)</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->A loss of mitochondrial function that leads to the oxidative stress and creation of neurotoxic molecules that trigger cell death (apoptotic, necrotic or autophagy) (Cobley et al., 2018)</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->Changes in the levels and activities of neurotrophic factors (Kazim and Iqbal, 2016; Machado et al., 2016; Rodriguez et al., 2014)</p>
<p style="margin-left:68.05pt"><!--[if !supportLists]--><span style="font-family:symbol">· </span><!--[endif]-->Variations in the activity of neural networks (Greicius and Kimmel, 2012)</p>
<p><strong>Protein aggregation</strong>: the correlation between neurodegenerative disease and protein aggregation in the brain has long been recognised, but a causal relationship has not been unequivocally established (Lansbury et al., 2006; Kumar et al., 2016). The dynamic nature of protein aggregation mean that, despite progress in understanding its mechanisms, its relationship to disease is difficult to determine in the laboratory.</p>
<p>Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered pre-symptomatically, drastically reduce the incidence of these diseases.</p>
<p><strong>Loss of mitochondrial function</strong>: many lines of evidence suggest that mitochondria have a central role in neurodegenerative diseases (Lin and Beal, 2006). Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Dysfunction of mitochondria induces oxidative stress, production of free radicals, calcium overload, and mutations in mitochondrial DNA that contribute to neurodegenerative diseases. In all major examples of these diseases there is strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis. Moreover, an impressive number of disease- specific proteins interact with mitochondria. Thus, therapies targeting basic mitochondrial processes, such as energy metabolism or free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise.</p>
<p style="margin-left:7.0pt"><strong>Decreased level of neurotrophic factors</strong>: decreased levels and activities of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), have been described in a number of neurodegenerative disorders, including Huntington's disease, Alzheimer disease and Parkinson disease (Zuccato and Cattaneo, 2009). These studies have led to the development of experimental strategies aimed at increasing BDNF levels in the brains of animals that have been genetically altered to mimic the aforementioned human diseases, with a view to ultimately influencing the clinical treatment of these conditions. Therefore BDNF treatment is being considered as a beneficial and feasible therapeutic approach in the clinic.</p>
<p style="margin-left:7.0pt"><strong>Variations in the activity of neural networks</strong>: Patients with various neurodegenerative disorders show remarkable fluctuations in neurological functions, even during the same day (Palop et al., 2006). These fluctuations cannot be caused by sudden loss or gain of nerve cells. Instead, it is likely that they reflect variations in the activity of neural networks and, perhaps, chronic intoxication by abnormal proteins that the brain is only temporarily able to overcome.</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">Neurodegeneration in relation to COVID19 </span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">SARS-CoV-2 patients present elevated plasma levels of neurofilament light chain protein (NfL), which is a well-known biochemical indicator of neuronal injury (Kanberg et al., 2020). Postmortem brain autopsies demonstrate virus invasion to different brain regions, including the hypothalamus and olfactory bulb, accompanied by neural death and demyelination (Archie and Cucullo 2020; Heneka et al. 2020).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Autopsy results of patients with SARS showed ischemic neuronal damage and demyelination; viral RNA was detected in brain tissue, particularly accumulating in and around the hippocampus (Gu et al. 2005).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Brain magnetic resonance imaging (MRI) investigations in SARS-CoV-2 patients show multifocal hyperintense white matter lesions and cortical signal abnormalities (particularly in the medial temporal lobe) on fluid-attenuated inversion recovery (FLAIR), along with intracerebral hemorrhagic and microhemorrhagic lesions, and leptomeningeal enhancement (Kandemirli et al. 2020; Kremer et al. 2020; Mohammadi et al., 2020).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Moreover, eight COVID-19 patients with signs of encephalopathy had anti–SARS-CoV-2 antibodies in their CSF, and 4 patients had CSF positive for 14-3-3-protein suggesting ongoing neurodegeneration (Alexopoulos et al. 2020).</span></span></span></p>
<p>The assays for measurements of necrotic or apoptotic cell death are described in the Key Event: Cell injury/Cell death</p>
<p>Recent neuropathological studies have shown that Fluoro-Jade, an anionic fluorescent dye, is a good marker of degenerating neurons. Fluoro-Jade and Fluoro-Jade B were found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death (Schmued et al., 2005). More recently, Fluoro-Jade C was shown to be highly resistant to fading and compatible with virtually all histological processing and staining protocols (Schmued et al., 2005). In addition, Fluoro-Jade C is a good tool for detecting acutely and chronically degenerating neurons (Ehara and Ueda, 2009).</p>
<p>The necrotic and apoptotic cell death pathways are quite well conserved throughout taxa (Blackstone and Green, 1999, Aravind et al., 2001). It has been widely suggested that apoptosis is also conserved in metazoans, although despite conservation of Bcl-2 proteins, APAF-1, and caspases there is no biochemical evidence of the existence of the mitochondrial pathway in either C. elegans or Drosophila apoptosis (Baum et al., 2007; Blackstone and Green, 1999).</p>
UBERON:0000955brainHighMixedHighDuring brain development, adulthood and agingHighHighModerate<p>Aravind, L., Dixit, V. M., and Koonin, E. V. (2001). Apoptotic Molecular Machinery: Vastly Increased Complexity in Vertebrates Revealed by Genome Comparisons. Science 291, 1279-1284.</p>
<p>Baum, J. S., Arama, E., Steller, H., and McCall, K. (2007). The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis. Cell Death Differ 14, 1508-1517.</p>
<p>Blackstone, N. W., and Green, D. R. (1999). The evolution of a mechanism of cell suicide. Bioessays 21, 84-88.</p>
<p>Cobley JN, Fiorello ML, Bailey DM (2018) 13 reasons why the brain is susceptible to oxidative stress. Redox Biol 15: 490-503</p>
<p>Ehara A, Ueda S. 2009. Application of Fluoro-Jade C in acute and chronic neurodegeneration models: utilities and staining differences. Acta histochemica et cytochemica 42(6): 171-179.</p>
<p>Evin G, Sernee MF, Masters CL (2006) Inhibition of gamma-secretase as a therapeutic intervention for Alzheimer's disease: prospects, limitations and strategies. CNS Drugs 20: 351-72</p>
<p>Greicius MD, Kimmel DL (2012) Neuroimaging insights into network-based neurodegeneration. Curr Opin Neurol 25: 727-34</p>
<p>Kazim SF, Iqbal K (2016) Neurotrophic factor small-molecule mimetics mediated neuroregeneration and synaptic repair: emerging therapeutic modality for Alzheimer's disease. Mol Neurodegener 11: 50</p>
<p>Kumar V, Sami N, Kashav T, Islam A, Ahmad F, Hassan MI (2016) Protein aggregation and neurodegenerative diseases: From theory to therapy. Eur J Med Chem 124: 1105-1120</p>
<p>Lansbury1 PT & Lashuel HA (2006) A century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 443, 774-779.</p>
<p>Lin1 MT & Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787-795</p>
<p>Machado V, Zoller T, Attaai A, Spittau B (2016) Microglia-Mediated Neuroinflammation and Neurotrophic Factor-Induced Protection in the MPTP Mouse Model of Parkinson's Disease-Lessons from Transgenic Mice. Int J Mol Sci 17</p>
<p>Narayan P, Dragunow M (2017) Alzheimer's Disease and Histone Code Alterations. Adv Exp Med Biol 978: 321-336</p>
<p>Palop JJ, Chin1 J & Mucke L, Review Article A network dysfunction perspective on neurodegenerative diseases. 2006, Nature 443, 768-773</p>
<p>Rodrigues TM, Jeronimo-Santos A, Outeiro TF, Sebastiao AM, Diogenes MJ (2014) Challenges and promises in the development of neurotrophic factor-based therapies for Parkinson's disease. Drugs Aging 31: 239-61</p>
<p>Schmued LC, Stowers CC, Scallet AC, Xu L. 2005. Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res 1035(1): 24-31.</p>
<p>Zuccato C & Cattaneo E, Brain-derived neurotrophic factor in neurodegenerative diseases.2009, Nature Reviews Neurology 5, 311-3</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">COVID19-related references relevant to KE Neurodegeneration:</span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Alexopoulos et al. Anti-SARS-CoV-2 antibodies in the CSF, blood-brain barrier dysfunction, and neurological outcome: Studies in 8 stuporous and comatose patients. Neurol Neuroimmunol Neuroinflamm. 2020 Sep 25;7(6):e893.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Archie SR, Cucullo L. Cerebrovascular and neurological dysfunction under the threat of COVID-19: is there a comorbid role for smoking and vaping? Int J Mol Sci. 2020 21(11):3916 12. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gu J et al. Multiple organ infection and the pathogenesis of SARS. J Exp Med. 2005;202:415–424.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Heneka MT, et al. Immediate and long-term consequences of COVID-19 infections for the development of neurological disease. Alzheimers Res Ther. 2020 12(1):1–3.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Kandemirli SG, et al. Brain MRI findings in patients in the intensive care unit with COVID-19 infection. Radiology. 2020 Oct;297(1):E232-E235.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Kanberg N, et al. Neurochemical evidence of astrocytic and neuronal injury commonly found in COVID-19. Neurology. 2020 Sep 22;95(12):e1754-e1759.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Kremer S, et al. Brain MRI findings in severe COVID-19: a retrospective observational study. Radiology. 2020 Nov;297(2):E242-E251.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Mohammadi S. et al. Understanding the Immunologic Characteristics of Neurologic Manifestations of SARS-CoV-2 and Potential Immunological Mechanisms. Mol Neurobiol. 2020 Dec;57(12):5263-5275.</span></span></span></p>
2016-11-29T18:41:242021-02-23T05:07:07Overactivation, NMDARsOveractivation, NMDARsCellular<p><strong>Biological state:</strong> Please see MIE <a href="/wiki/index.php/Event:201" title="Event:201"> NMDARs, Binding of antagonist</a></p>
<p><strong>Biological compartments:</strong> Please see MIE <a href="/wiki/index.php/Event:201" title="Event:201"> NMDARs, Binding of antagonist</a></p>
<p><strong>General role in biology:</strong> Please see MIE <a href="/wiki/index.php/Event:201" title="Event:201"> NMDARs, Binding of antagonist</a></p>
<p>The above chapters belong to the AOP entitled: <em>Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities</em> since the general characteristic of the NMDA receptor biology is the same for both AOPs.</p>
<p>Additional text, specific for this AOP:</p>
<p>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 (<em>Calcium-dependent processes are describe in Key Event Calcium influx, increased</em>). Postsynaptic Ca2+ signals of different amplitudes and durations are able to induce either LPT or LTD.</p>
<p>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).</p>
<p>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).</p>
<p>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.</p>
<p>Additional text, specific for the AOP “Acetylcholinesterase inhibition leading to neurodegeneration”:</p>
<p> 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).</p>
<p><!--[endif]----><!--[endif]----></p>
<p><em>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? </em></p>
<p>No OECD methods are available to measure the activation state of NMDA receptors.</p>
<p>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).</p>
<p>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.</p>
<p>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).</p>
<p>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).</p>
<p>Neuronal network function can be also measured using optical detection of neuronal spikes both in vivo and in vitro (Wilt et al., 2013).</p>
<p>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.</p>
<p>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).</p>
<p>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).</p>
CL:0000540neuronHighHighHighHigh<p><br />
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: <a class="external free" href="http://www.ncbi.nlm.nih.gov/books/NBK5274/" rel="nofollow" target="_blank">http://www.ncbi.nlm.nih.gov/books/NBK5274/</a>.</p>
<p>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.</p>
<p>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.</p>
<p>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”, <em>NeuroToxicology</em> <strong>44</strong>: 17-26. DOI: 10.1016/j.neuro.2014.04.006.</p>
<p>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.</p>
<p>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”, <em>Bulletin of Environmental Contamination and Toxicology</em> <strong>96</strong>(6): 707-713. DOI: 10.1007/s00128-016-1798-3.</p>
<p>Gopal K., Neurotoxic effects of mercury on auditory cortex networks growing on microelectrode arrays: a preliminary analysis. Neurotoxicol Teratol., 2003, 25: 69-76.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Malenka RC, Bear MF., LTP and LTD: An embarrassment of riches. Neuron, 2004, 44: 5–21.</p>
<p>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.</p>
<p>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.</p>
<p>McDonough, J. H., Jr. and T. M. Shih (1997), "Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology”, <em>Neurosci Biobehav Rev</em> <strong>21</strong>(5): 559-579.</p>
<p>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.</p>
<p>Ogasawara H, Doi T, Kawato M. Systems biology perspectives on cerebellar long-term depression. Neurosignals, 2008, 16 (4): 300–17.</p>
<p>Ogdon D, Stanfield P., Patch clamp techniques for single channel and whole-cell recording. Chapter 4, pages 53-78, (<a class="external free" href="http://www.utdallas.edu/~tres/microelectrode/microelectrodes_ch04.pdf" rel="nofollow" target="_blank">http://www.utdallas.edu/~tres/microelectrode/microelectrodes_ch04.pdf</a>).</p>
<p>Paradiso MA, Bear MF, Connors BW., Neuroscience: exploring the brain. 2007, Hagerstwon, MD: Lippincott Williams & Wilkins. p. 718. <a class="internal mw-magiclink-isbn" href="/wiki/index.php/Special:BookSources/0781760038">ISBN 0-7817-6003-8</a>.</p>
<p>Purves D., Neuroscience (4th ed.). Sunderland, Mass: Sinauer., 2008, pp. 197–200. <a class="internal mw-magiclink-isbn" href="/wiki/index.php/Special:BookSources/0878936971">ISBN 0-87893-697-1</a>.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
2016-11-29T18:41:242023-01-04T18:39:50Increased, Intracellular Calcium overloadIncreased, Intracellular Calcium overloadCellular<p>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 <em>Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.</em></p>
<p><strong>Biological state:</strong> KE <a href="/wiki/index.php/Event:52" title="Event:52"> Calcium influx, Decreased</a></p>
<p><strong>Biological compartments:</strong> KE <a href="/wiki/index.php/Event:52" title="Event:52"> Calcium influx, Decreased</a></p>
<p><strong>General role in biology:</strong> KE <a href="/wiki/index.php/Event:52" title="Event:52"> Calcium influx, Decreased</a></p>
<p><br />
The text specific for the AOP "ionotropic glutamatergic receptors and cognition” and “Acetylcholinesterase inhibition leading to neurodegeneration”:</p>
<p>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.</p>
<p><em>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? </em></p>
<p>Please see KE <a href="/wiki/index.php/Event:52" title="Event:52"> Calcium influx, Decreasedin</a> the AOP entitled: <em>Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.</em></p>
<p>Please see KE <a href="/wiki/index.php/Event:52" title="Event:52"> Calcium influx, Decreasedin</a> the AOP entitled <em>Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.</em></p>
<p> </p>
<p>Additional text, specific for the AOP “Acetylcholinesterase Inhibition leading to Neurodegeneration”:</p>
<p>Zebrafish have shown dysregulation in intracellular calcium ion levels following exposure to organophosphate compounds through similar mechanisms demonstrated in mammals <!--[if supportFields]><span
style='font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ascii-theme-font:
minor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";
color:black;mso-themecolor:text1'><span style='mso-element:field-begin'></span><span
style='mso-spacerun:yes'> </span>ADDIN EN.CITE
<EndNote><Cite><Author>Faria</Author><Year>2015</Year><RecNum>288</RecNum><DisplayText>(Faria
et al.
2015)</DisplayText><record><rec-number>288</rec-number><foreign-keys><key
app="EN" db-id="92990sdd8px5aie0tw7559riap0ffvxf0x5a"
timestamp="1569977726">288</key></foreign-keys><ref-type
name="Journal Article">17</ref-type><contributors><authors><author>Faria,
M.</author><author>Garcia-Reyero,
N.</author><author>Padrós, F.</author><author>Babin, P.
J.</author><author>Sebastián,
D.</author><author>Cachot, J.</author><author>Prats,
E.</author><author>Arick Ii, M.</author><author>Rial,
E.</author><author>Knoll-Gellida,
A.</author><author>Mathieu, G.</author><author>Le
Bihanic, F.</author><author>Escalon, B.
L.</author><author>Zorzano, A.</author><author>Soares,
A. M.</author><author>Raldúa,
D.</author></authors></contributors><titles><title>Zebrafish
Models for Human Acute Organophosphorus
Poisoning</title><secondary-title>Sci
Rep</secondary-title></titles><periodical><full-title>Sci
Rep</full-title></periodical><pages>15591</pages><volume>5</volume><edition>2015/10/22</edition><keywords><keyword>Acetylcholinesterase</keyword><keyword>Animals</keyword><keyword>Chemical
Terrorism</keyword><keyword>Chlorpyrifos</keyword><keyword>Disease
Models, Animal</keyword><keyword>Humans</keyword><keyword>Organophosphate
Poisoning</keyword><keyword>Small Molecule
Libraries</keyword><keyword>Zebrafish</keyword></keywords><dates><year>2015</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>2045-2322</isbn><accession-num>26489395</accession-num><urls><related-urls><url>https://www.ncbi.nlm.nih.gov/pubmed/26489395</url><url>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614985/pdf/srep15591.pdf</url></related-urls></urls><custom2>PMC4614985</custom2><electronic-resource-num>10.1038/srep15591</electronic-resource-num><language>eng</language></record></Cite></EndNote><span
style='mso-element:field-separator'></span></span><![endif]-->(Faria et al. 2015)<!--[if supportFields]><span style='font-size:11.0pt;
font-family:"Calibri",sans-serif;mso-ascii-theme-font:minor-latin;mso-hansi-theme-font:
minor-latin;mso-bidi-font-family:"Times New Roman";color:black;mso-themecolor:
text1'><span style='mso-element:field-end'></span></span><![endif]-->.</p>
CL:0000255eukaryotic cellHigh<p>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.</p>
<p>Bloodgood BL, Giessel AJ, Sabatini BL., Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines. PLoS Biol., 2009, 7: e1000190.</p>
<p>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.” <em>Sci Rep</em> <strong>5</strong>. DOI: 10.1038/srep15591.</p>
<p>Lledo PM, Zhang X, Sudhof TC, Malenka RC, Nicoll RA., Postsynaptic membrane fusion and long-term potentiation. Science, 1998, 279: 399–403.</p>
<p>Malenka RC. Synaptic plasticity in the hippocampus: LTP and LTD. Cell, 1994, 78: 535–538.</p>
<p>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.</p>
2016-11-29T18:41:242020-06-26T04:45:09Decreased, Neuronal network function in adult brainDecreased, Neuronal network function in adult brainOrgan<p><b>Biological state:</b>
</p><p>In the brain, neurons never work alone. They create a network where the activity of one cell directly influences many others. Each neuron is a specialized cell and when activated, it fires an electrochemical signal along the axon. A neuron fires only if the total signal received at the cell body from the dendrites exceeds a certain level (the firing threshold). The strength of the signal received by a neuron (and therefore its chances of firing) critically depends on the efficacy of the synapses. Each synapse actually contains a synaptic cleft with neurotransmitter that transmits a signal across the gap. During synaptic transmission neurotransmitters are released by a presynaptic neuron and bind to and activate the receptors of the postsynaptic neuron in response to a threshold of action potential. Synaptic transmission relies on: the availability of the neurotransmitter; the release of the neurotransmitter by exocytosis; the binding of the postsynaptic receptor by the neurotransmitter; the functional response of the postsynaptic cell; and the subsequent removal or deactivation of the neurotransmitter.
Neurons form complex networks of synapses through which action potentials travel. When the nerve impulse arrives at the synapse, it may cause the release of neurotransmitters, which influence another (postsynaptic) neuron. The postsynaptic neurons receive inputs from many additional neurons, both excitatory and inhibitory. The excitatory and inhibitory influences are summed (neural summation) resulting in inhibition or "firing" (i.e., generate an action potential) if the threshold potential has been reached. The voltage at which an action potential is triggered happens if enough voltage-dependent sodium channels are activated and the net inward sodium current exceeds all outward currents (Kolb and Whishaw, 2003). Therefore, at the beginning of the action potential, the Na+ channels open and Na+ moves into the axon, causing depolarization. Re-polarization occurs when the K+ channels open and K+ moves out of the axon. This creates a change in polarity between the outside of the cell and the inside. The impulse travels down from the axon hillock in one direction only, to the axon terminal. Here, the neurotransmitter is released releasing neurotransmitter at the synaptic cleft to pass along information to another adjacent neuron. Excitatory inputs bring a neuron closer to a firing threshold, while inhibitory inputs bring the neuron farther from threshold. An action potential is an "all-or-none" event; neurons whose membranes have not reached threshold will not fire, while those that do, will fire.
One of the most influential researchers into neurological systems (Donald Hebb) postulated that learning consisted principally in altering the "strength" of synaptic networking. Recent research in cognitive science, in particular in the area of non-conscious information processing, have further demonstrated the enormous capacity of the human mind to learn simple input-output co-variations from extremely complex stimuli. Consequently, the neurodegeneration and cell death disrupt the natural rhythms of brain network communication. Cognitive disorders are primarily associated with dysfunction of the neurons of the prefrontal cortex, hippocampus and with changes mainly in NMDARs function (Wang et al, 2015).
</p><p><b>Biological compartments:</b>
</p><p>The interface through which neurons interact with their neighbours usually consists of several axon terminals connected via synapses to dendrites on other neurons. If the hippocampal or cortical neurons are damaged or killed by the over-activation of receptors for the excitatory neurotransmitter glutamate, such as the NMDA, kainate and AMPA receptors, the neuronal networking and number of synapses are decreased. Indeed, it has been proved that lesions of the hippocampus in humans prevent the acquisition of new episodic memories suggesting that hippocampus-dependent memory is mediated, at least in part, by hippocampal synaptic plasticity that is a prominent feature of hippocampal synapses of the neuronal network (Neves et al., 2008). Since the finding that the hippocampus plays a pivotal role in long-term memory consolidation (dogma, well established fact in the literature, described in the text books; e.g. Andersen et al., 2007; Byrne, 2008; Eichenbaum, 2002), many proposals have been made regarding its specific role. A prominent view of the mechanisms underlying consolidation of episodic memories involves fast formation (e.g., via Hebbian mechanisms) of strong associations between hippocampal sparse patterns of activity and distributed neocortical representations.
Recent research on the primate prefrontal cortex discovered that the pyramidal cell circuits that generate the persistent firing underlying spatial working memory communicate through synapses on spines containing NMDARs with NR2B subunits (GluN2B) in the post-synaptic density. This contrasts with synapses in the hippocampus and primary visual cortex, where GluN2B receptors are both synaptic and extrasynaptic. Cholinergic stimulation of nicotinic α7 receptors within the glutamate synapse is necessary for NMDAR actions (Wang and Arnsten, 2015).
</p><p><b>General role in biology:</b>
</p><p><b>Glutamatergic neurotransmission (NMDA, AMPA and KA receptors)</b>
</p><p>The network of glutamatergic neurons is heavily involved in long-term synaptic plasticity, the main process linked to learning and memory. At the same time over-activation of these neurons (excitotoxicity) leads to neuronal cell death that can be mediated by increased levels of extracellular glutamate or a molecule that behaves as its analogue. Glutamate acts at a variety of ionotropic receptors, including AMPARs, kainate receptors, and NMDARs. The NMDARs have been of particular interest due to their unique properties. They require neuronal depolarization to relieve their Mg++ block, and are permeable to Ca++ that can initiate second-messenger signalling events, such as mediating neuroplasticity or negative feedback through Ca++-sensitive K+ channels. There have been extensive studies on the glutamate NMDAR and AMPAR mechanisms underlying long-term synaptic plasticity in the primary visual cortex and in CA1 neurons of the hippocampus (Liu et al., 2004; Cho et al., 2009; Lüscher and Malenka, 2012).
Neuronal network function and long-term plasticity is also regulated by the levels of AMPAR expression as the number of AMPARs inserted into the post-synaptic density can mediate the degree of spine depolarization and thus the NMDAR opening. Synaptic plasticity in the mature visual cortex appears to be governed by GluN2A subunits, which have faster kinetics than GluN2B. GluN2B receptors are expressed in synapses early in development, but many move to extra-synaptic locations in the mature visual cortex and hippocampus (Goebel-Goody et al., 2009). The actions of NMDARs on the dorsolateral prefrontal cortex neuronal circuitry network underlying spatial working memory in primates and it mechanism is described in detail by Wang and Arnsten (2015). In the hippocampus, there is some evidence that long-term potentiation (LTP) is mediated by synaptic GluN2A, while long-term depression is mediated by extrasynaptic GluN2B receptors (Liu et al., 2004).
Kainate receptors (KARs) also play an important role in neuronal network function. They play a major function in the pre-synaptic terminal, in particular in the hippocampus. Activation of kainate receptors in have been shown to regulate glutamate release (Jane et al., 2009) and to both depress and factilitate transmission in different synapses. Pre-synaptic kainate receptors in the hippocampus facilitate AMPA and NMDA receptor-mediated transmission at mossy fibre-CA3 synapses (Lauri et al, 2005). Activation of post-synaptic KARs facilitates activation of NMDARs as it has been described in the context of DomA exposure.
</p><p><b>Role of other neurotransmitters</b>
</p><p>It is important to stress that other classical neurotransmitter systems also play an important role in learning and memory processes (Blokland 1996). The role of the most critical neurotransmitters has been evaluated in a meta-analysis based on studies of four behavioral tasks relevant for evaluation of rat cognitive functions such as Morris water maze, radial maze, passive avoidance, and spontaneous alternation (Myhrer, 2003). Calculation of impact factors (percentage of significant effects of chemical agents like agonists, antagonists, neurotoxins) showed that glutamate was ranking highest (93), followed by GABA (81), dopamine (81), acetylcholine (81), serotonin (55), and norepinephrine (48).
</p><p><b>GABA-ergic</b> receptors: indeed, presynaptic GABA B receptors mediate GABA-dependent inhibition of glutamate release, impacting plasticity of hippocampal synapses and hippocampus-dependent memory function (Vigot et al., 2006). A critical link between GABABR heterodimer conformational dynamics and local regulation of release probability at hippocampal synapses has been recently proved (Laviv et al., 2010).
</p><p><b>5-Hydroxytrytamine</b> (serotonin) type 3A receptors (5-HT3ARs), as the only ligand-gated ion channels in the serotonin receptor family, are known to regulate neuronal excitation and release of GABA in hippocampal interneurons, playing also an important role in glutamatergic synaptic plasticity. Deletion of the 5-HT3AR gene in transgenic mice abolished NMDAR-dependent long-term depression (LTD) induced by low-frequency stimulation (LFS) in hippocampal CA1 synapses in slices. In addition, 5-HT3ARs disruption inhibited AMPARs internalization, without altering basal surface levels of AMPARs. These observations revealed an important role of 5-HT3ARs in NMDAR-dependent long-term depression, which is critical for learning behaviours (Yu et al., 2014).
</p><p><b>The cholinergic hypothesis</b> claims that the decline in cognitive functions in dementia is predominantly related to a decrease in cholinergic neurotransmission. This hypothesis has led to great interest in the putative involvement of the cholinergic neurotransmission in learning and memory processes (Blokland 1996; Bracco et al., 2014).
</p><p><b>Dopamine</b> plays diverse roles in human behaviour and cognition but it is mainly involved in motivation, decision-making, reward processing, attention, working memory and learning (Steinberg and Janak, 2012; Labudda et al., 2010).
</p><p><b>Noradrenaline</b> is associated with memory processing as it induces lasting changes in the brain that could sustain memories over time (Gazarini et al., 2013). As confirmed later on its neurotransmission indeed strengthens memory-related synaptic plasticity such as long-term potentiation, allowing memories to be formed and maintained in a more intense and enduring manner, a notion particularly valid for those with emotional content (Joëls et al. 2011). Like other types of memory, an emotional memory has to be consolidated to allow its later retrieval. Accumulating evidence has indicated that noradrenaline acts during these gradual stages to fine-tune the strength and/or persistence of a memory (Guzmán-Ramos et al. 2012; Gazarini et al., 2013).
</p><p><em>
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?
</em>
</p><p>Neuronal network activity is fundamental to brain function and now can be measured using in vitro and in vivo techniques such as:
</p><p>1. Two-photon imaging of cell populations in vivo that are labelled with fluorescent calcium indicators. Two-photon imaging relies on fluorescence excitation and, in general, necessitates staining of cells with fluorescent dyes. Various staining methods have been developed for in vivo calcium measurements. Single cells can be filled with membrane-impermeable calcium indicators via intracellular recording electrodes or by single-cell electroporation. The basic aspects of in vivo calcium imaging and recent developments that allow evaluation of the neural circuits activity are described by Göbel et al., (2007a).
With new imaging technology, scientists are now better able to visualize neural circuits connecting brain regions in humans. Advances in genetic engineering, microscopy, and computing are enabling scientists to begin to map the connections between individual nerve cells.
</p><p>2. Optical detection of neuronal spikes both in vivo and in vitro.
Assuming action potential (AP) as the only trigger of calcium influx, spike patterns are directly reflected in the trains of calcium transients. Each fluorescence trace is the convolution of the spike train with the single AP-evoked calcium transient plus added noise. The temporal resolution will be limited by the acquisition rate of the network scanning approach. In addition, the signal-to-noise ratio of fluorescence signals will be a decisive factor for the accuracy of the reconstruction.
</p><p>3. Microelectrode array (MEA) recordings in primary cultures. Glutamate analogues effects on neuronal network activity can be assessed (Lantz et al., 2014) and neuronal spontaneous activity evaluation is already used for screening purposes (Valdivia et al., 2014).
</p><p>4. To understand the function of a neural circuit, it is important to discriminate its sub-network components. This is possible through counterstaining of specific neuronal and glial cell types, especially in bulk loaded tissue where markers need not be calcium sensitive. In addition, transgenic mice with fluorescent protein expression in specific neuronal subsets, allow separation of functional signals into different neuronal subtypes (Göbel et al., 2007b).
</p><p>5. Combined positron emission tomography (PET) and magnetic resonance imaging (MRI) is a new tool to study functional processes in the brain, including the response to a stimulus simultaneously using PET. Functional MRI (fMRI), is used to assesses at the same time fast vascular and oxygenation changes during activation. These results demonstrate the feasibility of combined PET-MRI for the simultaneous study of the brain at activation and rest, revealing comprehensive and complementary information to further decode brain function and brain networks (Wehrl et al., 2013).
</p><p>6. Seed-based correlative analysis of [18F]fluorodeoxyglucose (FDG)-PET (FDG-PET) differences in images (resting state minus activation) is suitable to identify cerebral networks in rats. Using awake and freely moving animals enables functional network analysis of complex behavioral paradigms (Rohleder et al., 2015).
</p><p>Although most experiments at present are carried out in anesthetized animals, several approaches for imaging in awake behaving animals have been devised that ultimately aim at directly correlating neuronal network dynamics with behaviour (Dombeck et al., 2007, Arenkiel et al., 2007). Finally, through expression of light-activated channel proteins, it might become possible in the future to not only read-out but also control neuronal networks in vivo (Garaschuk et al., 2006)since with the development of X-ray, CT, and MRI, deep neural networks involved in learning and memory processes can be studied in vivo (Cheng et al., .2014).
</p><p>7. NMDAR overactivation-induced LTD that decrease number of spine density can be measured in vitro using GFP technology and by cofilin-F-actin quantification (Calabrese et al., 2014).
</p><p>Current behavioural tests used for evaluating neural network function:
</p><p>1. The Morris water maze: this test is developed to measure spatial orientation in rats
The rat has to swim around the pool to search for a platform onto which he can escape from the water. In one condition, the platform is visible, rising 1 cm above the water surface. In a Second condition the rat has to learn to find the hidden platform provided it remains in the fixed position relative to distal room cues.
</p><p>2. Radial maze: In the T-maze version of working memory, the animal has to remember only a single item for each trial. In the radial arm version of the working memory procedure rats have to learn multiple items.
</p><p>3. Passive avoidance: fear-motivated avoidance tests are usually based on electric current as source of punishment.
</p><p>4. Spontaneous alternation: spontaneous alternation is spatial alternation and represents a tendency to avoid stimulus re-exposure during exploratory behaviour. T-maze (simple or multiple), Y-maze, and radial maze are used to quantify an innate, unlearned response in rats.
</p><p>These four behavioural tests are described in detail in the review by Myhrer (Myhrer et al., 2003).
</p><p>The ability to process complex spatiotemporal information through neuronal networking is a fundamental process underlying the behaviour of all higher organisms.
The most studied are the neuronal networks of rodents (e.g Reig et al., 2015) and primates (e.g. Wang and Arnsten, 2015) and extremely large amount of the published data exist to support this topic.
Invertebrates hold neural circuitries in various degrees of complexity and there are studies describing how neurons are organized into functional networks to generate behaviour. (Wong and Wong, 2004; Marder, 1994).
</p>UBERON:0000955brain<p><br />
Andersen Per, Richard Morris, David Amaral, Tim Bliss and John O'Keefe, eds., The Hippocampus Book. Oxford University Press. ISBN, 2007, 978-0-19-510027-3.
</p><p>Arenkiel BR, Peca J, Davison IG, Feliciano C, Deisseroth K, Augustine GJ, Ehlers MD, Feng G. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron, 2007, 54: 205–218.
</p><p>Blokland A, Acetylcholine: a neurotransmitter for learning and memory? Brain Research Reviews, 1996, 21: 285-300.
</p><p>Bracco L, Bessi V, Padiglioni S, Marini S1, Pepeu G., Do cholinesterase inhibitors act primarily on attention deficit? A naturalistic study in Alzheimer's disease patients. J Alzheimers Dis. 2014, 40(3):737-42.
</p><p>Byrne John H, ed., Learning and Memory: A comprehensive reference. Elsevier. 2008, <a href="/wiki/index.php/Special:BookSources/9780123705099" class="internal mw-magiclink-isbn">ISBN 978-0-12-370509-9</a>.
</p><p>Calabrese B., Saffin JM, Halpain SH. Activity-Dependent Dendritic Spine Shrinkage and Growth Involve Downregulation of Cofilin via Distinct Mechanisms, 2014 DOI: 10.1371/journal.pone.0094787
</p><p>Cheng Da, Haixian Zhang, Yongsheng Sang. Brain CT Image Classification with Deep Neural Networks,Chapter from Proceedings of the 18th Asia Pacific Symposium on Intelligent and Evolutionary Systems, 2014, Volume 1 of the series Proceedings in Adaptation, Learning and Optimization pp 653-662.
</p><p>Cho KK, Khibnik L, Philpot BD, Bear MF., The ratio of NR2A/B NMDA receptor subunits determines the qualities of ocular dominance plasticity in visual cortex. Proc Natl Acad Sci USA 2009, 106: 5377–5382.
</p><p>Dombeck DA, Khabbaz AN, Collman F, Tank DW., Imaging large scale neural activity with cellular resolution in awake mobile mice. Neuron, 2007, 56: 43–57.
</p><p>Eichenbaum Howard, The Cognitive Neuroscience of Memory. Oxford University Press US. 2002, <a href="/wiki/index.php/Special:BookSources/9780195141757" class="internal mw-magiclink-isbn">ISBN 978-0-19-514175-7</a>.
</p><p>Gazarini L, Cristina A. Jark Stern, Antônio P., Carobrez and Leandro J. Bertoglio. 2013 Enhanced noradrenergic activity potentiates fear memory consolidation and reconsolidation by differentially recruiting α1- and β-adrenergic receptorsCurrent Issue Learning Memory, 2013, 20: 210-219.
</p><p>Göbel W, Fritjof Helmchen, In Vivo Calcium Imaging of Neural Network Function, Physiology, 2007a, 22: 358-365.
</p><p>Göbel W, Fritjof Helmchen, New Angles on Neuronal Dendrites In Vivo, Journal of Neurophysiology, 2007b, 98: 3770-3779.
</p><p>Goebel-Goody SM, Davies KD, Alvestad Linger RM, Freund RK, Browning MD, Phospho-regulation of synaptic and extrasynaptic N-methyl-d-aspartate receptors in adult hippocampal slices. Neuroscience 2009, 158: 1446–1459.
</p><p>Garaschuk O, Milos RI, Grienberger C, Marandi N, Adelsberger H, Konnerth A., Optical monitoring of brain function in vivo: from neurons to networks. Pflügers Arch., 2006, 453: 385–396.
</p><p>Guzmán-Ramos K, Osorio-Gómez D, Moreno-Castilla P, Bermúdez-Rattoni F., Post-acquisition release of glutamate and norepinephrine in the amygdala is involved in taste-aversion memory consolidation. Learn Mem., 2012, 19: 231–238.
</p><p>Jane DE, Lodge D, Collingridge GL. Kainate receptors: pharmacology, function and therapeutic potential. Neuropharmacology. 2009;56(1):90-113.
</p><p>Joëls M, Fernandez G, Roozendaal B., Stress and emotional memory: A matter of timing. Trends Cogn Sci., 2011, 15: 280–288.
</p><p>Kolb, Bryan; Whishaw, Ian Q., Fundamentals of Human Neuropsychology (5th ed.). 2003, Worth. pp. 102–104. <a href="/wiki/index.php/Special:BookSources/9780716753001" class="internal mw-magiclink-isbn">ISBN 978-0-7167-5300-1</a>.
</p><p>Labudda K, Brand M, Mertens M, Ollech I, Markowitsch HJ and Woermann FG., Decision making under risk condition in patients with Parkinson’s disease: A behavioural and fMRI study. Behavioral Neurology. 2010, 23(3): 131-143.
</p><p>Lantz SR1, Mack CM2, Wallace K2, Key EF3, Shafer TJ2, Casida JE3. 2014, Glufosinate binds N-methyl-D-aspartate receptors and increases neuronal network activity in vitro, Neurotoxicology. 2014, 45:38-47.
</p><p>Lauri S.E., Mikael Segerstråle, Aino Vesikansa, Francois Maingret, Christophe Mulle, Graham L. Collingridge, John T. R. Isaac, Tomi Taira. Endogenous Activation of Kainate Receptors Regulates Glutamate Release and Network Activity in developing Hippocampus. The Journal of Neuroscience, 2005, 25(18): 4473-4484.
</p><p>Laviv Tal, Inbal Riven, Iftach Dolev, Irena Vertkin, Bartosz Balana, Paul A. Slesinger, Inna Slutsky, Basal GABA Regulates GABABR Conformation and Release Probability at Single Hippocampal Synapses. Neuron, 2010, 67: 253–267.
</p><p>Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, et al., Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 2004, 304: 1021–1024.
</p><p>Lüscher C., Malenka RC. NMDA receptor-dependent longterm potentiation and long-term depression (LTP/LTD). Cold Spring Harb Perspect Biol 2012, 4: pii: a005710.
</p><p>Marder E., Invertebrate neurobiology. Polymorphic neural networks. Curr Biol. 1994, 4: 752-4.
</p><p>Myhrer T., Neurotransmitter systems involved in learning and memory in the rat: a meta-analysis based on studies of four behavioral tasks. Brain Res Brain Res Rev. 2003, 41(2-3):268-87.
</p><p>Neves G., Sam F. Cooke & Tim V. P. Bliss, Synaptic plasticity, memory and the hippocampus: a neural network approach to causality. Nature Reviews Neuroscience, 2008, 9: 65-75.
</p><p>Reig R, Zerlaut Y, Vergara R, Destexhe A, Sanchez-Vives MV., Gain modulation of synaptic inputs by network state in auditory cortex in vivo. J Neurosci. 2015, 35(6) :2689-702.
</p><p>Rohleder Cathrin, F. Leweke, Bernd Neumaier, Alexander Drzezga, Heike Endepols Characterization of functional neural networks using [18F]fluorodeoxyglucose (FDG)-PET in awake rats. J Nucl Med May 1, 2015, 56 no. supplement 3 1542.
</p><p>Steinberg EE and Janak PH., Establishing causality for dopamine in neural function and behavior with optogenetics. Brain Research, 2012, 9: 52-63.
</p><p>Valdivia P., Matt Martin, William R. LeFew, James Ross, Keith A. Houck, Timothy J. Shafer, Multi-well microelectrode array recordings detect neuroactivity of ToxCast compounds, NeuroToxicology, 2014, 44: 204–217.
</p><p>Vigot, R., Barbieri, S., Brauner-Osborne, H., Turecek, R., Shigemoto, R., Zhang, Y.P., Lujan, R., Jacobson, L.H., Biermann, B., Fritschy, J.M., et al., Differential compartmentalization and distinct functions of GABAB receptor variants. Neuron, 2006, 50: 589–601.
</p><p>Wang M., Amy F T Arnsten, Contribution of NMDA receptors to dorsolateral prefrontal cortical networks in primates, Neurosci Bull April 1, 2015, 31(2): 191–197
</p><p>Wehrl Hans F, Mosaddek Hossain, Konrad Lankes, Chih-Chieh Liu, Ilja Bezrukov, Petros Martirosian, Fritz Schick, Gerald Reischl, Bernd J Pichler, Simultaneous PET-MRI reveals brain function in activated and resting state on metabolic, hemodynamic and multiple temporal scales. Nature Medicine, 2013, 19: 1184–1189.
</p><p>Wong Y.H. and Wong J.T.Y, Invertebrate Neural Networks, Neuro-Signals, 2004, 13, No. 1-2.
</p><p>Yu Y, Cao DQ, Xu HY, Sun M, Huang ZL, Yung WH, Lu N, Huang Y6., 5-HT3A receptors are required in long-term depression and AMPA receptor internalization. Neuroscience. 2014, 278:105-12.
</p>2016-11-29T18:41:252017-09-16T10:15:07Binding of agonist, Ionotropic glutamate receptorsBinding of agonist, Ionotropic glutamate receptorsMolecular<p>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.
</p><p><br />
<b>Biological state:</b>
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.
</p><p><br />
<b>Biological compartments:</b>
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).
</p><p><br />
<b>General role in biology:</b>
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:
</p><p>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).
</p><p>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).
</p><p>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).
</p><p><em>
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?
</em>
</p><p>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; <a rel="nofollow" target="_blank" class="external free" href="http://pdsp.med.unc.edu/UNC-CH%20Protocol%20Book.pdf">http://pdsp.med.unc.edu/UNC-CH%20Protocol%20Book.pdf</a>; <a rel="nofollow" target="_blank" class="external free" href="http://www.currentprotocols.com/WileyCDA/CPUnit/refId-ph0120.html">http://www.currentprotocols.com/WileyCDA/CPUnit/refId-ph0120.html</a>). 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).
</p><p>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)
</p><hr /> The major determinants for ligand e.g. for both co-agonist glycine binding and L-glutamate binding are well conserved between species from lower organism to mammals (reviewed in Xia and Chiang, 2009). PCR analysis, cloning and subsequent sequencing of the seal lion NMDA receptors showed 80% homology to those from rats, but more than 95% homologus to those from dogs (Gill et al., 2010).CL:0000540neuronHighHighHighHighHigh<p><br />
(for Abstract and MIE)
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>Daoudal G, Debanne D, Long-term plasticity of intrinsic excitability: learning rules and mechanisms. Learn Mem., 2003, 10(6):456-65.
</p><p>Dingledine R, Borges K, Bowie D, Traynelis SF., The glutamate receptor ion channels. Pharmacol Rev., 1999, 51: 7–61.
</p><p>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.
</p><p>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.
</p><p>Glanzman DL., Associative learning: Hebbian flies. Curr Biol., 2005, 7: 15(11):R416-9.
</p><p>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.
</p><p>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.
</p><p>Lefebvre KA, Robertson A., Domoic acid and human exposure risks: a review.Toxicon. 2010, 56: 218-30.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>Neville KR, Lytton WW. Potentiation of Ca2+ influx through NMDA channels by action potentials: a computer model. Neuroreport., 1999, 10(17): 3711-6.
</p><p>Pulido OM., Domoic acid toxicologic pathology: a review. Mar Drugs., 2008, 6: 180-219.
</p><p><br />
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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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.
</p><p>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: <a rel="nofollow" target="_blank" class="external free" href="http://www.ncbi.nlm.nih.gov/books/NBK5286/">http://www.ncbi.nlm.nih.gov/books/NBK5286/</a>
</p><p>Retrieved from <a rel="nofollow" target="_blank" class="external free" href="https://aopkb.org/aopwiki/index.php/?oldid=27027">https://aopkb.org/aopwiki/index.php/?oldid=27027</a>
</p>2016-11-29T18:41:272017-09-16T10:15:07NeuroinflammationNeuroinflammationTissue<p>Neuroinflammation or brain inflammation differs from peripheral inflammation in that the vascular response and the role of peripheral bone marrow-derived cells are less conspicuous. The most easily detectable feature of neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signalling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001), as well as in the production of reactive oxygen (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signalling and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004).</p>
<p>Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006 ; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells scan the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defence), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005; Moehle and West, 2015): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been descibed recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1alpha), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.</p>
<p> </p>
<p><strong>Neuroinflammation and Brain development</strong></p>
<p>During brain development, microglia are known to play a critical role as shapers of neural circuits, by providing trophic factors and by remodeling and pruning synapses (Rajendran and Paolicelli, 2018). In addition to playing a role in synaptic management, microglia are important for the pruning of dying neurons and in the clearance of debris (<a href="#_ENREF_43" title="Harry, 2013 #5042">Harry, 2013</a>). Microglia seem to affect also processes associated with neuronal proliferation and differentiation (Harry and Kraft, 2012). Similarly to microglia, astrocytes have instructive roles in neurogenesis, gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis, and synaptic pruning (Reemst et al., 2016).</p>
<p>The development-dependent reactivity of microglial cells and astrocytes is not well known. Ischemic insult appears to elicit similar microglial reactivity both during brain development and in adulthood (<a href="#_ENREF_3" title="Baburamani, 2014 #6737">Baburamani et al, 2014</a>; <a href="#_ENREF_54" title="Leonardo, 2009 #6879">Leonardo & Pennypacker, 2009</a>). In contrast, treatment with lead acetate was previously shown to result in a more pronounced microglial and astrocyte reactivity in immature 3D rat brain cell cultures as compared to mature ones (<a href="#_ENREF_101" title="Zurich, 2002 #3368">Zurich et al, 2002</a>). Astrocyte reactivity was also more pronounced in immature 3D rat brain cell cultures following paraquat exposure, whereas development-dependent differences in the phenotype of reactive microglia were observed (Sandström et al., 2017). This suggests that neuroinflammation is occurring during brain development and may express a different phenotype than in adulthood, and that dysfunction of microglia and astrocyte during brain development could contribute to neurodevelopmental disorders and potentially to late-onset neuropathology (Reemst et al., 2016).</p>
<p> </p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Neuroinflammation in relation to COVID19</strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">SARS-CoV-2 patients with moderate and severe COVID-19 presented an elevated plasma levels of glial fibrillary acidic protein (GFAP), which is known as biochemical indicator of glial activation (Kanberg et al., 2020).</span></span></p>
<p>Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:</p>
<ul>
<li>Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.</li>
<li>The most frequently used astrocyte marker is GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflammatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for staining of astrocytes (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.</li>
<li>All immunocytochemical methods can also be applied to cell culture models.</li>
<li>In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).</li>
<li>Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of activation markers. This can for instance be done by PCR quantification of inflammatory factors, by measurement of the respective mediators, e.g. by ELISA-related immuno-quantification. Such markers include:</li>
<li>Pro- and anti-inflammatory cytokine expression (IL-1β; TNF-α, Il-6, IL-4); or expression of immunostimmulatory proteins (e.g. MHC-II)</li>
<li>Itgam, CD86 expression as markers of M1 microglial phenotype</li>
<li>Arg1, MRC1, as markers of M2 microglial phenotype</li>
</ul>
<p>For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</p>
<p> </p>
<p><strong>Regulatory example using the KE</strong></p>
<p>Measurement of glial fibrillary acidic protein (GFAP) in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67). It has been used on rare occasions for other toxicant evaluations.</p>
<p>Neuroinflammation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure, <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">or SARS-CoV-2 and other coronavirus infection. </span>Some references (non-exhaustive list) are given below for illustration:</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Human: Vennetti et al., 2006</span></p>
<p>Monkey (Macaca fascicularis): Charleston et al., 1994, 1996</p>
<p>Rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002</p>
<p>Mouse: Liu et al., 2012</p>
<p>Zebrafish: Xu et al., 2014.</p>
UBERON:0000955brainHighMixedHighDuring brain development, adulthood and agingHighHighModerateLowModerate<p><span style="font-size:12px">Aguzzi, A., Barres, B.A., Bennett, M.L., 2013. Microglia: scapegoat, saboteur, or something else? Science 339(6116), 156-161.</span></p>
<p><span style="font-size:12px">Aloisi, F., 2001. Immune function of microglia. Glia 36, 165-179.</span></p>
<p><span style="font-size:12px">Aschner M (1998) Immune and inflammatory responses in the CNS: modulation by astrocytes. ToxicolLett 103: 283-287</span></p>
<p><span style="font-size:12px">Banati, R. B. (2002). "Visualising microglial activation <em>in vivo</em>." Glia 40: 206-217. </span></p>
<p><span style="font-size:12px">Baburamani AA, Supramaniam VG, Hagberg H, Mallard C (2014) Microglia toxicity in preterm brain injury. <em>Reprod Toxicol</em> <strong>48:</strong> 106-112</span></p>
<p><span style="font-size:12px">Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</span></p>
<p><span style="font-size:12px">Carson, M.J., Thrash, J.C., Walter, B., 2006. The cellular response in neuroinflammation: The role of leukocytes, microglia and astrocytes in neuronal death and survival. Clin Neurosci Res 6(5), 237-245.</span></p>
<p><span style="font-size:12px">Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.</span></p>
<p><span style="font-size:12px">Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.</span></p>
<p><span style="font-size:12px">Claycomb, K.I., Johnson, K.M., Winokur, P.N., Sacino, A.V., Crocker, S.J., 2013. Astrocyte regulation of CNS inflammation and remyelination. Brain Sci 3(3), 1109-1127.</span></p>
<p><span style="font-size:12px">Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</span></p>
<p><span style="font-size:12px">Eng LF, Ghirnikar RS, Lee YL (2000) Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000). NeurochemRes 25: 1439-1451</span></p>
<p><span style="font-size:12px">Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</span></p>
<p><span style="font-size:12px">Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93.</span></p>
<p><span style="font-size:12px">Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907.</span></p>
<p><span style="font-size:12px">Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.</span></p>
<p><span style="font-size:12px">Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34.</span></p>
<p><span style="font-size:12px">Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35</span></p>
<p><span style="font-size:12px">Graeber MB, Streit WJ (1990) Microglia: immune network in the CNS. Brain Pathol 1: 2-5</span></p>
<p><span style="font-size:12px">Harry GJ and Kraft AD (2012) Microglia in the developing brain: apotential target with lifetime effects. <a href="https://www.ncbi.nlm.nih.gov/pubmed/22322212" title="Neurotoxicology.">Neurotoxicology.</a> 33(2):191-206.</span></p>
<p><span style="font-size:12px">Harry GJ (2013) Microglia during development and aging. <em>Pharmacology & therapeutics</em> <strong>139:</strong> 313-326</span></p>
<p><span style="font-size:12px">Kanberg N, et al. Neurochemical evidence of astrocytic and neuronal injury commonly found in COVID-19. Neurology. 2020 Sep 22;95(12):e1754-e1759</span></p>
<p><span style="font-size:12px">Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444</span></p>
<p><span style="font-size:12px">Kraft AD, Harry GJ., Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. International Journal of Environmental research and Public Health., 2011, 8(7): 2980-3018.</span></p>
<p><span style="font-size:12px">Kreutzberg GW (1995) Microglia, the first line of defence in brain pathologies. Arzneimttelforsch 45: 357-360</span></p>
<p><span style="font-size:12px">Kreutzberg GW (1996) Microglia : a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318</span></p>
<p><span style="font-size:12px">Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42</span></p>
<p><span style="font-size:12px">Leonardo CC, Pennypacker KR (2009) Neuroinflammation and MMPs: potential therapeutic targets in neonatal hypoxic-ischemic injury. <em>J Neuroinflammation</em> <strong>6:</strong> 13</span></p>
<p><span style="font-size:12px">Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638): 481-487.</span></p>
<p><span style="font-size:12px">Little AR, Miller DB, Li S, Kashon ML, O'Callaghan JP. 2012. Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis. Neurotoxicol Teratol 34(1): 72-82.</span></p>
<p><span style="font-size:12px">Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.</span></p>
<p><span style="font-size:12px">Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and <em>in vivo</em> conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87.</span></p>
<p><span style="font-size:12px">Maresz K, Ponomarev ED, Barteneva N, Tan Y, Mann MK, Dittel BN (2008) IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages. J Reprod Immunol 78: 140-148</span></p>
<p><span style="font-size:12px">Moehle MS, West AB (2015) M1 and M2 immune activation in Parkinson's Disease: Foe and ally? Neuroscience 302:59-73 doi:10.1016/j.neuroscience.2014.11.018</span></p>
<p><span style="font-size:12px">Monnet-Tschudi F, Zurich MG, Honegger P (2007) Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26: 339-346</span></p>
<p><span style="font-size:12px">Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19. </span></p>
<p><span style="font-size:12px">Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958-969</span></p>
<p><span style="font-size:12px">Nakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc & Haematol Disorders 4: 65-84.</span></p>
<p><span style="font-size:12px">Perego C, Fumagalli S, De Simoni MG (2011) Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation 8: 174</span></p>
<p><span style="font-size:12px">Ponomarev ED, Maresz K, Tan Y, Dittel BN (2007) CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci 27: 10714-10721</span></p>
<p><span style="font-size:12px">Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81: 374-389</span></p>
<p><span style="font-size:12px"><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Rajendran%20L%5BAuthor%5D&cauthor=true&cauthor_uid=29563239"><span style="color:#000000">Rajendran L</span></a><span style="color:#000000"><sup>1</sup>, </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Paolicelli%20RC%5BAuthor%5D&cauthor=true&cauthor_uid=29563239"><span style="color:#000000">Paolicelli RC</span></a><span style="color:#000000"> (2018). Microglia-Mediated Synapse Loss in Alzheimer's Disease. </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/29563239" title="The Journal of neuroscience : the official journal of the Society for Neuroscience."><span style="color:#000000">J Neurosci.</span></a><span style="color:#000000"> 38:2911-2919.</span></span></p>
<p><span style="font-size:12px">Ransohoff RM. 2016. A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19(8): 987-991.</span></p>
<p><span style="font-size:12px"><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Reemst%20K%5BAuthor%5D&cauthor=true&cauthor_uid=27877121"><span style="color:#000000">Reemst K</span></a><span style="color:#000000">, </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Noctor%20SC%5BAuthor%5D&cauthor=true&cauthor_uid=27877121"><span style="color:#000000">Noctor SC</span></a><span style="color:#000000">, </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Lucassen%20PJ%5BAuthor%5D&cauthor=true&cauthor_uid=27877121"><span style="color:#000000">Lucassen PJ</span></a><span style="color:#000000">, </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Hol%20EM%5BAuthor%5D&cauthor=true&cauthor_uid=27877121"><span style="color:#000000">Hol EM</span></a><span style="color:#000000">. (</span>2016) The Indispensable Roles of Microglia and Astrocytes during Brain Development. <a href="https://www.ncbi.nlm.nih.gov/pubmed/27877121" title="Frontiers in human neuroscience.">Front Hum Neurosci.</a> 10:566. DOI:10.3389/fnhum.2016.00566</span></p>
<p><span style="font-size:12px">Rivest, S., 2009. Regulation of innate immune responses in the brain. Nat Rev Immunol 9(6), 429-439.</span></p>
<p><span style="font-size:12px">Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001</span></p>
<p><span style="font-size:12px">Sandstrom J, Broyer A, Zoia D, et al. (2017a) Potential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures. Neurotoxicology 60:116-124 doi:10.1016/j.neuro.2017.04.010</span></p>
<p><span style="font-size:12px">Sandstrom J, Eggermann E, Charvet I, et al. (2017b) Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing. Toxicol In Vitro 38:124-135 doi:10.1016/j.tiv.2016.10.001</span></p>
<p><span style="font-size:12px">Streit, W.J., Walter, S.A., Pennell, N.A., 1999. Reactive microgliosis. Progress in Neurobiol. 57, 563-581.</span></p>
<p><span style="font-size:12px">Struzynska L, Dabrowska-Bouta B, Koza K, Sulkowski G (2007) Inflammation-Like Glial Response in Lead-Exposed Immature Rat Brain. Toxicol Sc 95:156-162</span></p>
<p><span style="font-size:12px">Venneti S, Lopresti BJ, Wiley CA. 2006. The peripheral benzodiazepine receptor (Translocator protein 18kDa) in microglia: from pathology to imaging. Prog Neurobiol 80(6): 308-322.</span></p>
<p><span style="font-size:12px">von Tobel, J. S., P. Antinori, et al. (2014). "Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype." Neurotoxicology 44C: 61-70.</span></p>
<p><span style="font-size:12px">Xu DP, Zhang K, Zhang ZJ, Sun YW, Guo BJ, Wang YQ, et al. 2014. A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-kappaB and the PKCalpha/PI3-K/Akt pathways. Neurochem Int 78: 76-85.</span></p>
<p><span style="font-size:12px">Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate <em>in vitro</em>: Implication of glial reactions. J Neurosc Res 70: 108-116.</span></p>
2016-11-29T18:41:232022-07-15T09:54:27ce45a297-1f86-4c40-aae0-f997d0a93d88d58bcf47-999b-4505-b1c5-696e1ad94129<p>The NMDA receptor is distinct from the other glutamate receptors in two ways: first, it is both ligand-gated and voltage-dependent; second, it requires co-activation by two ligands: glutamate and either glycine or D-serine. Following membrane depolarization, the co-agonists, L-glutamate and glycine must bind to their respective sites on the receptor to open the channel. On activation, the NMDA receptor allows the influx of extracellular calcium ions into the postsynaptic neuron and neurotransmission occurs (reviewed in Higley and Sabatini, 2012). Calcium flux through NMDA receptors is also thought to be critical in synaptic plasticity, a cellular mechanism for learning and memory. Indeed, NMDA receptor–dependent synaptic potentiation (LTP) and depression (LTD) are two forms of activity-dependent long-term changes in synaptic efficacy that are believed to represent cellular correlates of learning and memory processes. The best characterized form of NMDA receptor-dependent LTP and LTD occurs between CA3 and CA1 pyramidal neurons of the hippocampus (Luscher and Malenka, 2012). It is now well established that modest activation of NMDARs leads to modest increases in postsynaptic calcium, triggering LTD, whereas much stronger activation of NMDARs leading to much larger increases in postsynaptic calcium are required to trigger LTP (Luscher and Malenka, 2012). The high-frequency stimulation causes a strong temporal summation of the excitatory postsynaptic potentials, 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 post-synaptic cells.</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt"><span style="background-color:white"><span style="font-size:10.5pt">There is structural and functional mechanistic understanding supporting this relationship between NMDAR overactivation and increased intracellular calcium.</span></span></span></span></p>
<p>The relationship between the upstream and downstream key event is plausible as the expression of the functional NMDA receptors is commonly carried out or assessed by Ca2+ imaging method. Calcium imaging techniques have been extensively utilized in the literature to investigate the potential interactions between NMDA-evoked Ca2+ influx and NMDA receptor activation. Approximately 15% of the current through NMDA receptors is mediated by Ca2+ under physiological conditions (Higley and Sabatini, 2012).</p>
<p>It has been shown that less than five and, occasionally, only a single NMDA receptor opens under physiological conditions, causing a total Ca2+ influx of about 6000 ions into a spine head reaching a concentration of ∼10 µm (Higley and Sabatini, 2012). However, the majority of the ions are rapidly eliminated by binding to Ca2+ proteins, reaching ∼1 µM of free Ca2+ concentration (Higley and Sabatini, 2012).</p>
<p>It has been shown that in rat primary forebrain cultures the intracellular Ca2+ increases after activation of the NMDA receptor through administration of NMDA but this increase in Ca2+ is blocked when the cells are cultured under Ca2+ free conditions, demonstrating that the NMDA-evoked increase in intracellular Ca2+ derives from extracellular and not intracellular sources (Liu et al., 2013).</p>
<p>Indirect mechanism of domoic acid (DA) induced overactivation of NMDARs that result in Ca2+ overload: depolarization of the pre-synaptic cell activates the release of endogenous Ca2+ which mobilizes vesicles containing GLU to the membrane surface. Glutamate (GLU) is then released into the synaptic cleft by exocytosis where it is able to interact with cell surface receptors. Exogenous DA can interact within the synaptic cleft with each of the three ionotropic receptor subtypes including the kainate, AMPA, and NMDA receptors on cell membranes. Activation of the kainate and AMPA receptors results in release of Ca2+ via coupled ion channels, into the post-synaptic cell. DA is also able to bind to NMDA receptors that are linked to both Ca2+ and NA/K+ ion channels and results in a cellular influx of both Na+ and Ca2+. Unlike GLU, DA induces prolonged receptor activation causing a constant influx of cations into the cell and the appropriate chemical cues for desensitization are blocked. The excess intracellular Ca2+ causes disruption of cellular function, cell swelling and ultimately cell death (Lefebvre and Robertson,2010).</p>
<p>Glufosinate (GLF) is the methylphosphinate analog of glutamate that directly can activate NMDARs (Lantz et al., 2014, Matsumura et al., 2001, Faro et al., 2013) (as described in KE: <em>NMDARs, Binding of agonist</em>). It is well established in the existing literature that activation of NMDARs leads to the intra-cellular Ca2+ overload and based on this assumption it can be suggested that an exposure to GLF leads to increased intra-cellular calcium levels.</p>
<p><em>Include consideration of temporal concordance here </em></p>
<p><strong>Domoic acid (DomA)</strong></p>
<ul>
<li>Treatment of mouse cerebellar granule neurons (CGNs) with 1 or 10 µM DomA causes increase of intracellular Ca2+ by approximately 5 or 8 fold compared to controls, respectively (Giordano et al., 2006). Interestingly, when the cells are exposed simultaneously to DomA and the NMDA receptor antagonist MK-801, the Ca2+ levels measured are close to control levels, indicating that the Ca2+ elevation evoked by DomA involves activation of NMDA receptors (Giordano et al., 2006).</li>
</ul>
<ul>
<li>The same research group has performed a time course study by applying a high and a low DomA concentration and using CGNs from Gclm (+/+) and Gclm (−/−) mice lacking glutathione (Giordano et al., 2007). The low DomA dose (0.1μM) causes a small and delayed increase in intracellular Ca2+ concentration with a full recovery by 20 min. When the experiment is performed in the absence of extracellular calcium, this increase of intracellular Ca2+ levels in the presence of DomA is abolished, indicating that this change in homeostasis of Ca2+ is due to ion entry from outside the cell. However, this recording of intracellular Ca2+ is antagonised only by NBQX (AMPA receptor antagonist), but not by MK-801 (NMDA receptor antagonist). On the other hand, the higher DomA concentration (10μM) causes a rapid and robust increase in intracellular Ca2+, which lasts even after 25 min. This effect is antagonized by both NBQX and MK-801, suggesting that not only AMPA but also NMDA receptors are involved in Ca2+ elevation evoked by DomA at high doses (Giordano et al., 2007).</li>
</ul>
<ul>
<li>In an earlier study, the time course and concentration dependence of the increase in intracellular Ca2+ stimulated by DomA has been examined in 10-13 day-in-culture CGNs (Berman et al., 2002). DomA produces a rapid and concentration-dependent increase in intracellular Ca2+, showing the maximal increase at 10 μM DomA (Berman et al., 2002). At this concentration, fluo-3 fluorescence that is used to measure Ca2+ elevates rapidly during the first 40 s of exposure, increases more slowly before peaking at 3.5 min, after which the signal diminishes steadily over the 30 min course of the experiment to 55% of peak values. The EC50 for DomA-induced increase in intracellular Ca2+ is 0.61 μM. In the same study, the NMDA receptor antagonist MK-801 significantly reduced both peak and final plateau of intracellular Ca2+ by 30 and 70%, respectively (Berman et al., 2002).</li>
</ul>
<ul>
<li>These three studies (Giordano et al., 2006; 2007; Berman et al., 2002) do not provide a simultaneous measurement of NMDA receptor activation by DomA and intracellular Ca2+ levels. However, they do provide indirect evidence of NMDA receptor activation involvement in increased intracellular Ca2+ concentrations induced by DomA as they have used known antagonists of the NMDA receptors that reverses the situation in both KEs (blocking upstream KE will block downstream KE).</li>
</ul>
<ul>
<li>In an in vivo study it was indirectly shown that the microinjection to adult male Sprague Dawley rats of 10 μM DomA increased intracellular Ca2+ levels. A significant upregulation of phosphorylated calcium-calmodulin-dependent kinase II (CaMKII) and phosphorylated cAMP response element binding protein (CREB) levels was recorded, possibly due to increased intracellular Ca2+ levels induced by DomA (Qiu and Currás-Collazo, 2006).</li>
</ul>
<p>In CGNs, the co-treatment with 10 µM DomA and the kainate/AMPA receptor antagonist NBQX maintains Ca2+ levels near to control levels, suggesting that the Ca2+ elevation evoked by DomA is mediated by the activation of both AMPA/kainate and of NMDA receptors (Giordano et al., 2006).</p>
<p>The voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine (5 μM) and NBQX (10 μM), a competitive AMPA/kainate receptor antagonist reduces the peak and final intracellular Ca2+ concentration in CGNs (Berman et al., 2002), strengthening the view that the increase of Ca2+ influx is not only mediated by NMDA receptors but also by AMPA/kainate receptors and VSCCs.</p>
<p> </p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Table 1: Summary of available data describing responses of intracellular calcium to NMDA receptor activation. DA, DomA = Domoic Acid. Glu = Glutamate. NMDA = N-methyl-D-aspartate. The following are NMDA receptor (NMDAR) antagonists: D-AP5 = D-2-amino-5-phosphonopentanoate. MK-801 = Dizocilpine.</span></span></p>
<table border="1" cellspacing="0" style="border-collapse:collapse; font-size:75%">
<tbody>
<tr>
<td>
<p><strong>Stressor</strong></p>
</td>
<td>
<p><strong>Experimental Model</strong></p>
</td>
<td>
<p><strong>Tested concentrations</strong></p>
</td>
<td>
<p><strong>Exposure route</strong></p>
</td>
<td>
<p><strong>Exposure duration</strong></p>
</td>
<td>
<p><strong>Overactivation of NMDAR (KE up) (measurements, quantitative if available)</strong></p>
</td>
<td>
<p><strong>Increased intracelllular Ca 2+ levels (KE down) (measurements, quantitative if available)</strong></p>
</td>
<td>
<p><strong>References</strong></p>
</td>
<td>
<p><strong>Temporal Relationship</strong></p>
</td>
<td>
<p><strong>Dose-response relationship</strong></p>
</td>
<td>
<p><strong>Incidence</strong></p>
</td>
<td>
<p><strong>Comments</strong></p>
</td>
</tr>
<tr>
<td>
<p>DomA</p>
</td>
<td>
<p>Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice</p>
</td>
<td>
<p>0.01 to 10 µM</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Time course (15 to 120 min)</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>5 and 8 fold increase of [Ca2+]i compared to controls.</p>
</td>
<td>
<p>Giordano et al., 2006</p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p>The cells were exposed simultaneously to DA and the NMDA receptor antagonist MK-801 and the Ca2+ levels were found to be close to control levels, indicating that the Ca2+ elevation evoked by DA involves activation of NMDA receptors.</p>
</td>
</tr>
<tr>
<td>
<p>DomA</p>
</td>
<td>
<p>CGNs from Gclm (+/+) and Gclm (−/−) mice</p>
</td>
<td>
<p>0.01 to 10 µM</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Time course (0 to 25 min)</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>0.1μM domoic acid caused a small and delayed increase (4 fold) in [Ca2+]i, with a full recovery by 20 min.In contrast, the higher concentration of domoic acid (10μM) caused a rapid and robust increase (8 fold) in [Ca2+]i, which was still elevated after 25 min.</p>
<p>0.1μM DA increases [Ca2+]M by about 3 fold, with a delay of about 15 min. In contrast, no changes in [Ca2+]M were observed following 10μM of DA.</p>
</td>
<td>
<p>Giordano et al., 2007</p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p>At the low concentration (0.1μM), the recording of intracellular Ca2+ was antagonized only by NBQX (AMPA receptor antagonist), but not by MK-801 (NMDA receptor antagonist). On the other hand, the higher DA concentration (10μM) caused a rapid and robust increase in intracellular Ca2+ . This effect was antagonized by both NBQX and MK-801, suggesting the importance of NMDA receptors in Ca2+ elevation evoked by DA but only at high doses</p>
</td>
</tr>
<tr>
<td>
<p>DomA</p>
</td>
<td>
<p>10-13 DIV CGNs obtained from 8-day-old Sprague–Dawley rats</p>
</td>
<td>
<p>0.1 to 30 µM</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Time course (0 to 45 min)</p>
</td>
<td>
<p> </p>
</td>
<td>
<p>EC50 for DA-induced increase in intracellular Ca2+ was 0.61 μM</p>
</td>
<td>
<p>Berman et al., 2002</p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p>The NMDA receptor antagonist MK-801 significantly reduced both peak and final plateau of intracellular Ca2+ by 30 and 70%, respectively</p>
</td>
</tr>
<tr>
<td>
<p>DomA</p>
</td>
<td>
<p>Adult male Sprague Dawley rats</p>
</td>
<td>
<pre>
10 µM
</pre>
</td>
<td>
<p>Brain microinjection</p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Increased phosphorylated CaMKII and phosphorylated CREB levels</p>
</td>
<td>
<p>Qiu and Currás-Collazo, 2006</p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Glutamate, NMDA</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Mouse cortical astrocytes</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Glutamate: 100 µM<br />
NMDA: 20 µM</span></span></td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Brief (1s application)</span></span></td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Increased intracellular Ca2+ measured through Fluo-3 Flourescence</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Palygin et al., 2011</span></span></td>
<td> </td>
<td> </td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Provides time-series data of intracellular calcium measured through fluoresence given an application of Glu, NMDA, or Glu + D-AP5 in mouse cortical astrocytes. Cells were additionally exposed to D-AP5, an NMDA antagonist, and showed reduced fluorscence changes. (added by DS for AOP 281)</span></span></td>
</tr>
<tr>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Glutamate</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Cultured rat hippocampal neurons</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">500 µM</span></span></td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Time course (0 to 45 minutes)</span></span></td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Increased intracellular Ca2+ measured through Fura-2 Flourescence</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Michaels and Rothman, 1990</span></span></td>
<td> </td>
<td> </td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Provides time-series data of intracellular calcium measured through fluoresence, as well as directly providing calculated intracellular calcium concentrations in response to high concentrations of applied Glu, both alone and with antagonists. (added by DS for AOP 281)</span></span></td>
</tr>
<tr>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">NMDA, Glutamate</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Neocortical neurons of Swiss-Webster mice</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Glutamate: 300 µM<br />
NMDA: 300 µM</span></span></td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Time course (0 to 20 minutes) and (0 to 2 minutes)</span></span></td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Increased intracellular Ca2+ measured through Fura-2/AM, Fura-2/K+, Fura-2/dextran, BTC</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Hyrc et al., 1997</span></span></td>
<td> </td>
<td> </td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Provides time-series data of intracellular calcium measured through a variety of fluorsence calcium indicators given an application of the selective agonist NMDA. </span></span><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">(added by DS for AOP 281)</span></span></td>
</tr>
<tr>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Glutamate</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Computational model (CA1 pyramidal neuron)</span></span></td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Models the concentration of Ca2+ in spine(s) of neuron</span></span></td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Hu et al., 2018</span></span></td>
<td> </td>
<td> </td>
<td> </td>
<td><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">Developed a computational model of a glutamatergic spine which models intracellular calcium dynamics and sources of calcium influx including activation of NMDA receptors. </span></span><span style="font-size:8.0pt"><span style="font-family:"Arial",sans-serif">(added by DS for AOP 281)</span></span></td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>Glufosinate (GLF)</strong></p>
<p>There are no data showing that an exposure to GLF causes an increase in intra-cellular calcium. Such assumption can be proposed based on a fact that GLF directly activates NMDR as described in the MIE and other relevant KEs of this AOP.</p>
<p>A case of a 59-yr-old woman who ingested a herbicide containing glufosinate was suffering from severe intoxication, however, she did not develop convulsions, which experimentally occurs in rats treated with GLF (Koyama et al., 1994) and is described in other human cases (Watanabe and Sano 1998).</p>
<p><em>Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? </em></p>
<p>The experiments describing semi-quantitative effects for these KERs are described in the table above.</p>
HighUnspecificHighAll life stagesLowHighHigh<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11.0pt"><span style="font-family:"Helvetica Neue""><span style="color:black">NMDARs have been shown to regulate calcium ion flow in a variety of species including zebrafish and rats </span></span></span><span style="font-size:11.0pt"><span style="font-family:"Helvetica Neue""><span style="color:black">(Horzmann and Freeman, 2016, el Nasr et al., 1990)</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Helvetica Neue""><span style="color:black">.</span></span></span></span></span></p>
<p><br />
Berman FW, LePage KT, Murray TF., Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca(2+) influx pathway. Brain Res., 2002, 924: 20-29.</p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">el Nasr, M. S., Peruche, B., Roβberg, C., Mennel, H.-D. & Krieglstein, J. 1990. Neuroprotective effect of memantine demonstrated in vivo and in vitro. <em>European Journal of Pharmacology,</em> 185<strong>,</strong> 19-24. DOI: <a href="https://doi.org/10.1016/0014-2999(90)90206-L" style="color:blue; text-decoration:underline">https://doi.org/10.1016/0014-2999(90)90206-L</a>.</span></span></p>
<p>Faro LR, Ferreira Nunes BV, Alfonso M, Ferreira VM, Durán R., Role of glutamate receptors and nitric oxide on the effects of glufosinate ammonium, an organophosphate pesticide, on in vivo dopamine release in rat striatum. Toxicology., 2013, Sep 15, 311: 154-61.</p>
<p>Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG., Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol. 2006., 70: 2116-2126.</p>
<p>Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG., Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.</p>
<p>Higley MJ, Sabatini BL., Calcium signalling in dendritic spines. Cold Spring Harb Perspect Biol., 2012, 4: a005686.</p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Horzmann, K. A. & Freeman, J. L. 2016. Zebrafish get connected: investigating neurotransmission targets and alterations in chemical toxicity. <em>Toxics,</em> 4. </span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Hu, E., Mergenthal, A., Bingham, C. S., Song, D., Bouteiller, J. M. & Berger, T. W. 2018. A Glutamatergic Spine Model to Enable Multi-Scale Modeling of Nonlinear Calcium Dynamics. <em>Front Comput Neurosci,</em> 12<strong>,</strong> 58. DOI: 10.3389/fncom.2018.00058.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Hyrc, K., Handran, S. D., Rothman, S. M. & Goldberg, M. P. 1997. Ionized intracellular calcium concentration predicts excitotoxic neuronal death: observations with low-affinity fluorescent calcium indicators. <em>J Neurosci,</em> 17<strong>,</strong> 6669-77. DOI: 10.1523/jneurosci.17-17-06669.1997.</span></span></p>
<p>Koyama K, Andou Y, Saruki K, Matsuo H., Delayed and severe toxicities of a herbicide containing glufosinate and a surfactant. Vet Hum Toxicol., 1994, 36: 17-8.</p>
<p>Lantz Stephen R , Cina M. Mack , Kathleen Wallace, Ellen F. Key , Timothy J. Shafer , John E. Casida., Glufosinate binds N-methyl-D aspartate receptors and increases neuronal network activity in vitro. NeuroToxicology, 2014, 45: 38–47.</p>
<p>Lefebvre KA, Robertson A. Domoic acid and human exposure risks: a review. Toxicon. 2010 Aug 15;56(2):218-30.</p>
<p>Liu F, Patterson TA, Sadovova N, Zhang X, Liu S, Zou X, Hanig JP, Paule MG, Slikker W Jr, Wang C., Ketamine-induced neuronal damage and altered N-methyl-D-aspartate receptor function in rat primary forebrain culture. Toxicol Sci., 2013, 131: 548-557.</p>
<p>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.</p>
<p>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(1-2): 123-5.</p>
<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Michaels, R. L. and Rothman, S. M. 1990. Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. <em>J Neurosci,</em> 10<strong>,</strong> 283-92. DOI: 10.1523/jneurosci.10-01-00283.1990.</span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Palygin, O., Lalo, U. & Pankratov, Y. 2011. Distinct pharmacological and functional properties of NMDA receptors in mouse cortical astrocytes. <em>British Journal of Pharmacology,</em> 163<strong>,</strong> 1755-1766. DOI: 10.1111/j.1476-5381.2011.01374.x.</span></span></p>
<p>Qiu S, Currás-Collazo MC., Histopathological and molecular changes produced by hippocampal microinjection of domoic acid. Neurotoxicol Teratol., 2006, 28: 354-362.</p>
<p>Watanabe T, Sano T., Neurological effects of glufosinate poisoning with a brief review. Hum Exp Toxicol. 1998, 17: 35-9.</p>
2016-11-29T18:41:332023-09-10T20:11:554f8c9fd6-ed55-4019-8287-e45ac29ddde8920190dd-6d6a-43ab-86a2-21af66b6c4c0<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Cell death of neurons directly causes neurodegeneration characterized by abnormal neuronal <span style="color:black">loss </span><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue""><span style="color:black">(Przedborski et al., 2003)</span></span></span><span style="color:black">. While </span>the upstream event is unspecific as to the type of cell affected, neurodegeneration is caused by cell death in neurons specifically.</span></span></p>
<p>There is well established mechanistic understanding supporting the relationship between these two KEs.</p>
<p>Neurodegeneration in the strict sense of the word, is referring to any pathological condition primarily affecting brain cell populations (Przedborski et al., 2003). At the histopathological level, neurodegenerative conditions are described by neuronal death and reactive gliosis (Przedborski et al., 2003).</p>
<p><em>Include consideration of temporal concordance here</em></p>
<p><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue""><span style="color:black">Evidence applicable to domoic acid (DomA):</span></span></span></p>
<ul>
<li>
<p><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Acute brain damage induced by DomA is characterized by neurodegenerative changes consisting of neuronal shrinkage, vacuolization of the cytoplasm, cell drop out, edema, microvacuolation of the neuropil and hydropic cytoplasmic swelling of resident astrocytes (reviewed in Pulido et al., 2008). These histopathological changes can be identified within structures of the limbic system, in hippocampus, in the CA3, CA4 or hilus of the dentate gyrus (DG) (reviewed in Pulido et al., 2008). Other brain areas known to be affected by DomA include: the olfactory bulb, the piriform and entorhinal cortices, the lateral septum, the subiculum, the arcuate nucleus and several amygdaloid nuclei. The area postrema is another target for DomA toxicity as it has been identified in both rodents and non-human primates, providing a possible explanation of emetic symptoms (nausea, retching, and/or vomiting) induced by DomA. There has been an effort to map and create a 3-D reconstruction of DomA-induced neurodegeneration in the mouse brain demonstrating that the affected areas include the olfactory bulb, septal areas and the limbic system (Colman et al., 2005; Barlow et al., 2004).</span></span></span></span></big></p>
</li>
<li>
<p><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Female Sprague-Dawley rats dosed once intraperitoneally (i.p.) with 0, 1, 2, 4, or 7.5 DomA mg /kg of body weight were euthanized after 24 h and their nervous system was examined for microscopic alterations revealing neuronal degeneration and vacuolation of the neurophil in the limbic and the olfactory systems (Tryphonas et al., 1990).</span></span></span></span></big></p>
</li>
<li>
<p><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">The mean of TUNEL positive cells in the hippocampus was increased (6-fold) in mice injected i.p.</span></span><span style="font-size:8.0pt"> </span><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">at a dose of 2 DomA mg/kg once a day for 3 weeks (Lu et al., 2012). However, the same treatment protocol did not cause any neurodegeneration (Lu et al., 2012). In contrast, when the same treatment was prolonged for one more week (total 4 weeks), the mean values of NeuN-positive cells in the hippocampal CA1 sections of DomA-treated cells decreased by 3 fold compared to controls (Lu et al., 2012). This study showed that the incidence of upstream KE (cell death) was higher than the incidence of downstream KE (neurodegeneration) and that upstream KE (cell death) preceded downstream KE (neurodegeneration).</span></span></span></span></big></p>
</li>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">The bcl-2 and bax mRNA levels in the hippocampus were significantly increased at 16 h and gradually decreased at 24 h following the administration of DomA (0.75 mg/kg body weight) in adult rats. In situ hybridization analysis revealed complete loss of bcl-2, bax, and caspase-3 mRNA at 24 h after DA administration in the region of the hippocampus, whereas neurodegeneration by Nissl staining was detected at the same time point but was more pronounced after 5 days (Ananth et al., 2001). This study showed that both KEs occurred after exposure to the same dose of DomA and that the upstream KE (cell death) occurred earlier than the downstream KE (neurodegeneration).</span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Adult rats received i.p. injections with DomA 1.0 mg/kg/h until animals exhibited first motor seizures. After a week of recovery, aggressive behaviors and motor seizures of the animals had been monitored for 3h twice a week. After 12 weeks, animals were euthanized and brains were examined for indications of cell loss by using thionine (Nissl) staining, which highlights the cell bodies of all living neurons. In piriform cortex a reduced cell density was noted in the medial layer 3 (1.3-1.8 fold decrease compared to controls), an area that shows also prominent amino cupric staining (stain that assesses neuronal damage) (Tiedeken and Ramsdell, 2013a). The same research group reported that by following the above experimental procedure but sacrificing the rats 7 days after DomA-induced seizures intense and widespread silver reaction product in the olfactory bulb occurred, whereas minor or no evident damage was found in the hippocampus (Tiedeken et al., 2013b).</span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Intraperitoneal injection of DomA 0.5 mg/kg to adult C57BL/6 male mice resulted in loss of 32% and 30% of Nissl-stained neurons in hilus and CA1 pyramidal layer of the hippocampus, respectively, compared to control mice 7 d after the administration (Antequera et al., 2012).</span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">The severity and extent of hippocampal neuronal degeneration varied significantly depending on the dose of DomA (1 μM to 1 mM) that was tested after microinjection to adult male Sprague Dawley rats (Qiu and Currás-Collazo, 2006). In rats dosed with 1 mM DomA and sacrificed after 24 h, histopathological analysis using toluidine blue staining revealed extensive neuronal damage throughout the ipsilateral hippocampal structure. Shrunken, disorganized and densely stained neurons of irregular shape were identified throughout CA1, CA2, CA3 pyramidal layer as well as the dentate gyrus hilus and granule cells layer. For the 100 μM group animals, CA1 neuronal changes were less prominent, whereas 10 μM and 1 μM DomA did not produced any resolvable histopathological changes (Qiu and Currás-Collazo, 2006).</span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Adult male rats treated with 2 mg/kg DomA i.p. were sacrificed after 3 d and showed that the silver stain used to assess neurodegeneration clearly distinguished treated from control animals, whereas a number of other markers failed to do so (Scallet et al., 2005). The same results were found after even longer exposure times (7 d) to DomA (Appel et al., 1997).</span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Male Wistar rats were given a single i.v. injection of DA (0.75 mg/kg) in the right external jugular vein and brain sections were stained with Nissl stain at 5 d after DomA administration. Histopathological analysis revealed a large number of darkly stained shrunken neurons in the hippocampus (Ananth et al., 2003). However, complete absence of hippocampal neurons was observed in CA1 and CA3 regions in DomA treated animals at 3 months after DomA administration (Ananth et al., 2003).</span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">In 2-3 week old hippocampal slice cultures, derived from 7 day old rat pups, DomA (0.1-100 µM) was added to the culture medium and neurodegeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was measured. The CA1 region appeared to be most sensitive to DomA, with an EC50 value of 6 µM DomA after estimating the PI-uptake at 72 h (Jakobsen et al., 2002).</span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Cynomolgus monkeys were given i.v. a range of DomA doses from 0.25 to 4.0 mg/kg. Silver staining of brain sections revealed that doses in the range of 0.5-1.0 mg/kg produced a small area of silver grains restricted to axons of the hippocampal CA2 stratum lucidum, whereas higher concentrations produced degenerating axons and cell bodies (Slikker et al., 1998). The same research group treated i.v. adult monkeys with DomA at one of a range of doses from 0.25 to 4 mg/kg. After a week, silver staining demonstrated degenerating axons and cell bodies that was mild and restricted to CA2 stratum lucidum at a lower doses (0.5 to 1.0 DomA mg/kg). Doses of more than 1.0 mg/kg caused widespread damage to pyramidal neurons and axon terminals of CA4, CA3, CA2, CA1, and subiculum subfields of the hippocampus. However, when DomA was orally administered to cynomolgus monkeys at doses of 0.5 mg/kg for 15 days and then at 0.75 mg/kg for another 15 days no histopathoogical changes in the brain were detected (Truelove et al., 1997). </span></span></span></span></big></li>
</ul>
<ul>
<li><big><span style="font-size:12pt"><span style="font-size:10.5pt">In humans, autopsy of individuals intoxicated by DomA revealed brain damage characterized by neuronal necrosis and in the hippocampus and the amygdaloid nucleus (Pulido, 2008). The thalamus and subfrontal cortex were damaged only in some patients suffering from Amnesic Shellfish Poisoning (ASP). The detailed examination of one patient intoxicated by DomA revealed complete neuronal loss in the CA1, CA3 and CA4 regions, whereas moderate loss was seen in the CA2 region (Cendes et al., 1995). Non-severe neuronal loss was detected in amygdale, overlying cortex, the dorsal and ventral septal nuclei, the secondary olfactory areas, and the nucleus accumbens (Cendes et al., 1995).</span></span></big></li>
</ul>
<p> </p>
<table border="1" cellspacing="0" style="border-collapse:collapse; font-size:75%">
<tbody>
<tr>
<td style="background-color:#f0f0f0"><strong>Stressor</strong></td>
<td style="background-color:#f0f0f0"><strong>Experimental Model</strong></td>
<td style="background-color:#f0f0f0"><strong>Tested concentrations</strong></td>
<td style="background-color:#f0f0f0"><strong>Exposure route</strong></td>
<td style="background-color:#f0f0f0"><strong>Exposure duration</strong></td>
<td style="background-color:#f0f0f0"><strong>Cell death (KE up) (measurements, quantitative if available)</strong></td>
<td style="background-color:#f0f0f0"><strong>Neurodegeneration (KE down) (measurements, quantitative if available)</strong></td>
<td style="background-color:#f0f0f0"><strong>References</strong></td>
<td style="background-color:#f0f0f0"><strong>Temporal Relationship</strong></td>
<td style="background-color:#f0f0f0"><strong>Dose-response relationship</strong></td>
<td style="background-color:#f0f0f0"><strong>Incidence</strong></td>
<td style="background-color:#f0f0f0"><strong>Comments</strong></td>
</tr>
<tr>
<td>DomA</td>
<td>Female Sprague-Dawley rats</td>
<td>0, 1, 2, 4, or 7.5 DomA mg /kg</td>
<td>intraperitoneally (i.p.)</td>
<td>Euthanized after 24 h</td>
<td> </td>
<td>Neuronal degeneration and vacuolation of the neuropil in the limbic and the olfactory systems</td>
<td>Tryphonas et al., 1990</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>16-month-old male ICR mice</td>
<td>2 mg/kg</td>
<td>Intraperitoneally (i.p.)</td>
<td>Once a day for 3 or 4 weeks</td>
<td>The mean of TUNEL positive cells in the hippocampus was increased (6 fold). The levels of bcl-2, procaspase-3 and procaspase-12 were significantly decreased and the activation of caspase-3 and caspase-12 in the mouse hippocampus were increased.</td>
<td>The mean OD of NeuN immunoreactivity in the hippocampus of mice decreased (3 fold) indicating significant neuron loss by apoptosis, which is one of the pathological hallmarks of neurodegeneration</td>
<td>Lu et al., 2012</td>
<td>Upstream KE (cell death) precedes downstream KE (neurodegeneration)</td>
<td>Same dose</td>
<td>Incidence of upstream KE (cell death) is higher than the incidence of downsteam KE (neurodegeneration)</td>
<td>Mice treated with DomA once a day for 3 weeks showed that apoptosis was increased. However, the same treatment protocol did not cause any neurodegeneration. In contrast, when the same treatment was prolonged for one more week (total 4 weeks) induced marked neuron loss.</td>
</tr>
<tr>
<td>DomA</td>
<td>Adult rats</td>
<td>0.75 mg/kg</td>
<td>intravenously (i.v.)</td>
<td>Euthanized after 2, 5, 14, or 21 days</td>
<td>The bcl-2 and bax mRNA levels in the hippocampus were significantly increased at 16 h and gradually decreased at 24 h following the administration of DomA. In situ hybridization analysis revealed complete loss of bcl-2, bax, and caspase-3 mRNA at 24 h after DomA administration in the region of hippocampus.</td>
<td>Neurodegeneration by Nissl staining was detected at the same time point but was reported to be more pronounced after 5 days</td>
<td>Ananth et al., 2001</td>
<td>Upstream KE (cell death) occurs earlier that downstream KE (neurodegeneration).</td>
<td>Same dose</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>Adult rats</td>
<td>1.0 mg/kg/h until animals exhibited first motor seizures</td>
<td>i.p.</td>
<td>Euthanized after 12 weeks</td>
<td> </td>
<td>In piriform cortex a reduced cell density was noted in the medial layer 3 (1.3-1.8 fold decrease compared to controls), an area that showed also prominent amino cupric staining (stain that assesses neuronal damage).</td>
<td>Tiedeken and Ramsdell, 2013a</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>Adult rats</td>
<td>1.0 mg/kg/h until animals exhibited first motor seizures</td>
<td>i.p.</td>
<td>Euthanized after 1 week</td>
<td> </td>
<td>Intense and widespread silver reaction product in the olfactory bulb, whereas minor or no evident damage was found in hippocampus.</td>
<td>Tiedeken et al., 2013b</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>Adult C57BL/6 male mice</td>
<td>0.5 mg/kg</td>
<td>i.p.</td>
<td>Euthanized after 1 week</td>
<td> </td>
<td>DomA treatment resulted in the loss of 32% and 30% of Nissl-stained neurons in hilus and CA1 pyramidal layer of the hippocampus, respectively, compared to control mice.</td>
<td>Antequera et al., 2012</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>Adult male Sprague Dawley rats</td>
<td>1 μM to 1 mM</td>
<td>microinjection</td>
<td>Euthanized after 24 h</td>
<td> </td>
<td>In rats dosed with 1 mM DomA and sacrificed after 24 h, histopathological analysis using toluidine blue staining revealed extensive neuronal damage throughout the ipsilateral hippocampal structure. Shrunken, disorganized and densely stained neurons of irregular shape were identified throughout CA1, CA2, CA3 pyramidal layer as well as the dentate gyrus hilus and granule cells layer. For the 100 μM group animals, CA1 neuronal changes were less prominent, whereas 10 μM and 1 μM DomA did not produce resolvable histopathological changes.</td>
<td>Qiu and Currás-Collazo, 2006</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>Adult male rats</td>
<td>2 mg/kg</td>
<td>i.p.</td>
<td>Euthanized after 3 or 7 days</td>
<td> </td>
<td>DA treatment for 3 d showed that the silver stain that was used to assess neurodegeneration clearly distinguished treated from control animals , the same was true for longer exposure time (7 d).</td>
<td>Scallet et al., 2005, Appel et al., 1997</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>Male Wistar rats</td>
<td>0.75 mg/kg</td>
<td>i.v.</td>
<td>Euthanized after 5 days or 3 months</td>
<td> </td>
<td>Histopathological analysis revealed a large number of darkly stained shrunken neurons in the hippocampus However, complete absence of hippocampal neurons was observed in CA1 and CA3 regions in DA treated animals at 3 months after DomA administration.</td>
<td>Ananth et al., 2003</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>2-3 week old rat hippocampal slice cultures, derived from 7 day old rat pups</td>
<td>0.1-100 µM</td>
<td> </td>
<td>72 h</td>
<td> </td>
<td>DomA induced neurodegeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields. The CA1 region appeared to be most sensitive to DomA, with an EC50 value of 6 µM DomA, estimated from the PI-uptake at 72 h .</td>
<td>Jakobsen et al., 2002</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>DomA</td>
<td>Cynomolgus monkeys</td>
<td>0.25 to 4.0 mg/kg</td>
<td>i.v.</td>
<td>Euthanized after 1 week</td>
<td> </td>
<td>Silver staining of brain sections revealed that doses in the range of 0.5-1.0 mg/kg produce a small area of silver grains restricted to axons of the hippocampal CA2 stratum lucidum, whereas higher concentrations revealed degenerating axons and cell bodies. After a week, silver staining demonstrated degenerating axons and cell bodies that was mild and restricted to CA2 stratum lucidum at the lower doses (0.5 to 1.0 DomA mg/kg). Doses of more than 1.0 mg/kg caused widespread damage to pyramidal neurons and axon terminals of CA4, CA3, CA2, CA1, and subiculum subfields of the hippocampus.</td>
<td>Slikker et al., 1998, Truelove et al., 1997</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
</tr>
</tbody>
</table>
<p>Gap of knowledge: there are no studies showing that glufosinate (GLF)-induced cell death leads to neurodegeneration.</p>
<h4><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue""><span style="color:black">Evidence applicable to acetylcholinesterase inhibition:</span></span></span></span></span></h4>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Experiments using the nerve agent soman, an acetylcholinesterase inhibitor, showed major changes in various areas of the brain including the cerebral cortex, piriform cortex, amygdala, hippocampus, thalamus and striatum from neuronal lesions </span></span><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">(Acon-Chen et al., 2016)</span></span><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">.</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue""><span style="color:black">There are various methods to categorize neurodegeneration from cell death, and there are “different clinical pictures” depending on the area or areas of the brain affected (Przedborski et al., 2003).</span></span></span></span></span></p>
<h4><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue""><span style="color:black">Domoic acid considerations:</span></span></span></span></span></h4>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Zebrafish were exposed for 36-weeks to DomA and showed no excitotoxic neuronal death and no histopathological lesions in glutamate-rich brain areas (Hiolski et al., 2014). </span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">Administration of DomA (9.0 mg DomA kg(-1) bw, i.p.) to seabream (<em>Sparus aurata</em>) lead to measurement of 0.61, 0.96, and 0.36 mg DomA kg(-1) of brain tissue at 1, 2 and 4 hours. At this dose but also at lower concentrations (0.45 and 0.9 mg DomA kg(-1) bw) no major permanent brain damage was detected (Nogueira et al., 2010). Leopard sharks possess the molecular target for DomA but it has been shown to be resistant to doses of DomA that can cause neurotoxicity to other vertebrates, suggesting the presence of some protective mechanism (Schaffer et al., 2006).</span></span></span></span></p>
<p><span style="font-size:10.5pt"><span style="font-family:"Helvetica Neue"">All these reports suggest species specific susceptibility to DomA toxicity.</span></span></p>
<p><em>Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? </em></p>
<p>The experiments describing semi quantitative effects for this KERs are described in the table above.</p>
HighUnspecificHighAdultHighHighHighLow<p><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#333333">Neurodegeneration from cell death is widely accepted, neurodegenerative models have used various species including mice and zebrafish for different neurodegenerative diseases </span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#333333">(Dawson et al., 2018)</span></span></span></p>
<h4>Information Specific to DomA</h4>
<p>There is an overall agreement regarding the histopathology of the brain lesions related to acute DomA neurotoxicity across certain species. Data derived from humans, rodents, non-human primates and sea lions suggest that common neurodegeneration features in selected brain areas are found despite the fact that study design, estimated exposure, processing of samples and history of event may differ (Pulido, 2008).</p>
<p>Furthermore, the distribution of brain damage by DomA has also been established by magnetic resonance imaging microscopy (MRM) for both human and rat, demonstrating similar distribution as that described by histopathological studies (Pulido, 2008).</p>
<p>It is important to notice that human sensitivity to DomA exposure is well documented in the published literature and seems to be much higher than in other species (Lefebvre and Robertson 210; Barlow et al., 2004). In 1987 in Canada, more than 200 people became acutely ill after ingesting mussels contaminated with DomA. The outbreak resulted in 20 hospitalizations and four deaths. Clinical effects observed included gastrointestinal symptoms and neurotoxic effects such as hallucinations, memory loss and coma. For this reason, the condition was termed amnesic shellfish poisoning (Barlow et al., 2004). The neurotoxic properties of DomA result in neuronal degeneration and necrosis in specific regions of the hippocampus (Teitelbaum et al., 1990).</p>
<p><br />
<span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Acon-Chen, C., Koenig, J. A., Smith, G. R., Truitt, A. R., Thomas, T. P. & Shih, T. M. 2016. Evaluation of acetylcholine, seizure activity and neuropathology following high-dose nerve agent exposure and delayed neuroprotective treatment drugs in freely moving rats. <em>Toxicology Mechanisms and Methods,</em> 26<strong>,</strong> 378-388. DOI: 10.1080/15376516.2016.1197992.</span></span>Ananth C, Thameem DS, Gopalakrishnakone P, Kaur C., Domoic acid-induced neuronal damage in the rat hippocampus: changes in apoptosis related genes (bcl-2, bax, caspase-3) and microglial response. J Neurosci Res., 2001, 66: 177-190.</p>
<p>Ananth C, Gopalakrishnakone P, Kaur C., Induction of inducible nitric oxide synthase expression in activated microglia following domoic acid (DA)-induced neurotoxicity in the rat hippocampus. Neurosci Lett., 2003, 338: 49-52.</p>
<p>Antequera D, Bolos M, Spuch C, Pascual C, Ferrer I, Fernandez-Bachiller MI, Rodríguez-Franco MI, Carro E., Effects of a tacrine-8-hydroxyquinoline hybrid (IQM-622) on Aβ accumulation and cell death: involvement in hippocampal neuronal loss in Alzheimer's disease. Neurobiol Dis., 2012, 46: 682-691.</p>
<p>Appel NM, Rapoport SI, O'Callaghan JP., Sequelae of parenteral domoic acid administration in rats: comparison of effects on different anatomical markers in brain. Synapse, 1997, 25: 350-358.</p>
<p>Barlow Jeffery B, T, Moizer K, Paul S, and Boyle C., Amnesic shellfish poison. Food Chem Toxicol., 42: 545-557.</p>
<p>Cendes F, Andermann F, Carpenter S, Zatorre RJ, Cashman NR., Temporal lobe epilepsy caused by domoic acid intoxication: evidence for glutamate receptor-mediated excitotoxicity in humans. Ann Neurol., 1995, 37: 123-6.</p>
<p>Colman JR, Nowocin KJ, Switzer RC, Trusk TC, Ramsdell JS., Mapping and reconstruction of domoic acid-induced neurodegeneration in the mouse brain. Neurotoxicol Teratol., 2005, 27: 753-767.</p>
<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Dawson, T. M., Golde, T. E. & Lagier-Tourenne, C. 2018. Animal models of neurodegenerative diseases. <em>Nature Neuroscience,</em> 21<strong>,</strong> 1370-1379. DOI: 10.1038/s41593-018-0236-8.</span></span></p>
<p>Hiolski EM, Kendrick PS, Frame ER, Myers MS, Bammler TK, Beyer RP, Farin FM, Wilkerson HW, Smith DR, Marcinek DJ, Lefebvre KA., Chronic low-level domoic acid exposure alters gene transcription and impairs mitochondrial function in the CNS. Aquat Toxicol., 2014, 155: 151-159.</p>
<p>Jakobsen B, Tasker A, Zimmer J., Domoic acid neurotoxicity in hippocampal slice cultures. Amino Acids, 2002, 23: 37-44.</p>
<p>Lefebvre Kathi A. and Robertson Alison, Domoic acid and human exposure risks: A review, Toxicon, 2010, 56: 218–230.</p>
<p>Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF., Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52: 646-659.</p>
<p>Nogueira I, Lobo-da-Cunha A, Afonso A, Rivera S, Azevedo J, Monteiro R, Cervantes R, Gago-Martinez A, Vasconcelos V., Toxic effects of domoic acid in the seabream Sparus aurata. Mar Drugs, 2010, 8: 2721-2732.</p>
<p>Przedborski S, Vila M, Jackson-Lewis V., Neurodegeneration: What is it and where are we? J Clin Invest., 2003, 111: 3-10.</p>
<p>Pulido OM., Domoic acid toxicologic pathology: a review. Mar Drugs, 2008, 6: 180-219.</p>
<p>Qiu S, Currás-Collazo MC., Histopathological and molecular changes produced by hippocampal microinjection of domoic acid. Neurotoxicol Teratol., 2006, 28: 354-362.</p>
<p>Scallet AC, Schmued LC., Johannessen JN. Neurohistochemical biomarkers of the marine neurotoxicant, domoic acid. Neurotoxicol Teratol., 2005, 27: 745-752.</p>
<p>Schaffer P, Reeves C, Casper DR, Davis CR., Absence of neurotoxic effects in leopard sharks, Triakis semifasciata, following domoic acid exposure. Toxicon., 2006, 47: 747-752.</p>
<p>Slikker W Jr, Scallet AC, Gaylor DW., Biologically-based dose-response model for neurotoxicity risk assessment. Toxicol Lett., 1998, 102-103: 429-433.</p>
<p>Teitelbaum JS, Zatorre RJ, Carpenter S, Gendron D, Evans AC, Gjedde A, and Cashman NR., Neurologic sequelae of domoic acid intoxication due to the ingestion of contaminated mussels. N Engl J Med., 1990, 322: 1781-1787.</p>
<p>Tiedeken JA, Muha N, Ramsdell JS., A cupric silver histochemical analysis of domoic acid damage to olfactory pathways following status epilepticus in a rat model for chronic recurrent spontaneous seizures and aggressive behavior. Toxicol Pathol., 2013a, 41: 454-69.</p>
<p>Tiedeken JA, Ramsdell JS., Persistent neurological damage associated with spontaneous recurrent seizures and atypical aggressive behavior of domoic acid epileptic disease. Toxicol Sci., 2013b, 133: 133-43.</p>
<p>Truelove J, Mueller R, Pulido O, Martin L, Fernie S, Iverson F., 30-day oral toxicity study of domoic acid in cynomolgus monkeys: lack of overt toxicity at doses approaching the acute toxic dose. Nat Toxins., 1997, 5: 111-114.</p>
<p>Tryphonas L, Truelove J, Nera E, Iverson F., Acute neurotoxicity of domoic acid in the rat. Toxicol Pathol., 1990, 18: 1-9.</p>
2016-11-29T18:41:332023-09-10T19:25:358367d882-24f4-4763-a5d9-999a7d5b4bfc920190dd-6d6a-43ab-86a2-21af66b6c4c0<p>It is well accepted that chronic neuroinflammation is involved in the pathogenesis of neurodegenerative diseases (McNaull et al., 2010; Tansey and Goldberg, 2009; Thundyil and Lim, 2015 ). Chronic neuroinflammation can cause secondary damage (Kraft and Harry, 2011). The mechanisms by which neuroinflammation (i.e. activated microglia and astrocytes) can kill neurons and induce/exacerbate the neurodegenerative process has been suggested to include the release of nitric oxide that causes inhibition of neuronal respiration, ROS and RNS production, and rapid glutamate release resulting in excitotoxic death of neurons (Brown & Bal-Price, 2003; Kraft & Harry, 2011; Taetzsch & Block, 2013). Glial reactivity is also associated with excessive production and release of pro-inflammatory cytokines that not only affect neurons, but also have detrimental feedback effects on microglia (Heneka et al., 2014). For example, sustained exposure to bacterial lipopolysaccharide (LPS) or to other pro-inflammatory mediators was shown to restrict microglial phagocytosis of misfiled and aggregated proteins (Sheng et al., 2003). Systemic immune challenge during pregnancy leading to microglial activation caused increased deposition of amyloid plaques and tau hyperphosphorylation in aged mice (Krstic et al., 2012, 2013), suggesting that neuroinflammation is involved in the amyloid plaques and neurofibrillary tangles formation. There is further evidence that the formation of neurofibrillary tangles is caused by microglial cell-driven neuroinflammation, since LPS-induced systemic inflammation increased tau pathology (Kitazawa et al., 2005).</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">Sars-CoV-2 specific evidence:</span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Studies on post-mortem cases indicate that lymphocytes and monocytes infiltrate in brain vessel walls, exacerbating the neuronal degeneration and demyelination process (Wu et al., 2020)</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">The aberrant immune response characterized by a surge in cytokine levels (e.g., IL-6) derived by SARS-CoV-2 accelerates the process of neurodegeneration that may contribute to the development of neurodegenerative diseases (Debnath et al. 2020).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">SARS-CoV-2 can infect human brain organoids resulting in unique metabolic changes and the death of infected and neighbouring neurons. This phenotype is accompanied by impaired synaptogenesis (Song et al., 2020) (Mesci et al, 2020). </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Moreover, it is hypothesized that an autoimmune reaction mediated by the cross-reaction between viral particles and myelin basic protein may provide the driving force for neural demyelination, as part of the neurodegenerative process. This hypothesis is supported by the fact that the genome of other coronaviruses like CoV-OC43 and CoV-229E, as well as their antibodies, has been isolated from the CNS of Multiple Sclerosis (MS) patients, and coronavirus-like particles have been found in perivascular cuffs of human MS brain (Montalvan et al. 2020). In fact, the virus might lie dormant in astrocytes and oligodendrocytes and trigger the autoimmunity mediated by molecular mimicry (Mohammadi et al., 2020).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Neurons are the target cells undergoing degeneration during infection, in part due to apoptosis (de Assis et al. 2020). Intracerebral inoculation with CoV-OC43 in susceptible mice led to an acute encephalitis, with neuronal cell death by necrosis and apoptosis (Jacomy H, et al. 2006).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">SARS-CoV infection causes neuronal death (even in the absence of encephalitis) in mice transgenic for human ACE2. Death of the animal likely results from dysfunction and/or death of infected neurons, especially those located in cardiorespiratory centres in the medulla. The absence of the host cell receptor prevents severe murine brain disease (Netland J, et al. 2008).</span></span></span></p>
<p>Neuroinflammation is a component of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Neumann, 2001) which may play a secondary or an active primary role in the disease process (Hirsch and Hunot, 2009). McNaull and coworkers (McNaull et al., 2010) suggested that early developmental onset of brain inflammation could be linked with late onset of Alzheimer’s disease. A recent paper by Krstic and coworkers (2012) showed that a systemic immune challenge during late gestation predispose mice to develop Alzheimer’s like pathology when aging, suggesting a causal link between systemic inflammation, neuroinflammation, and the onset of Alzheimer’s disease. Regarding toxicant-induced neuroinflammation, microglial/astrocyte activation and chronic neuron damage may continue for years after initial exposure (Taetsch and Block, 2013), suggesting that chronical neuroinflammation and neurodegeneration have a slow long-term temporal evolution. Ongoing neuroinflammation can be visualized in patients using the positron emission tomography (PET) ligand [11C] (R)-PK11195 (Cagnin et al., 2001). Recent genome-wide association study (GWAS) analyses of sporadic Alzheimer's disease revealed a set of genes that point to a pathogenic role of neuroinflammation in Alzheimer's disease (for review, see Heneka et al., 2014). High levels of pro-inflammatory cytokines produced by activated microglia and astrocytes are detected in the brain of Alzheimer's subjects and animal models (McGeer and McGeer, 1998; Janelsins et al., 2005). </p>
<p><em>Include consideration of temporal concordance here </em></p>
<p>Pb</p>
<p>Rats treated from gestation day 5 till postnatal day 180 with a mixture of Pb/Cd/As showed in early adulthood increased levels of IL-1b, IL-6 and TNF-a in hippocampus and frontal cortex associated with increased Ab levels, where Pb applied alone triggered maximal Ab induction (Ashok et al., 2015). Similarly, monkeys exposed during infancy to Pb (from birth to 400 days to 1 mg Pb /kg/day) showed in aging (23 y old) an overexpression of APP and Abeta (Bihaqi et al., 2011), and of Tau mRNA and protein (Bihaqi and Zawa, 2013). Similar observations were made in old rats (18-20 months) when exposed to Pb (0.2% in drinking water) from postnatal day 1 to 20 (Basha et al., 2005; Zawia and Basha, 2005; Bihaqi et al., 2014). This was associated with cognitive impairment, observed only if animals were exposed when young (Bihaqi et al., 2014). Perinatal exposure to Pb leading to a blood concentration of 10 mg/dl (a concentration considered as safe for human) promotes Tau phosphorylation in forebrain, cerebellum and hippocampus (Gassowska et al., 2016).</p>
<p>However, adult exposure may also increase the risk of neurodegeneration, as suggested by the two following studies:</p>
<p>- human Tg-SwD1 APP transgenic mice treated with Pb (27 mg/kg/day by gavage) for 6 weeks beginning at 8 weeks of age showed increased accumulation of Abeta and amyloid plaques (Gu et al., 2012).</p>
<p>- former organolead workers had increased tibia Pb level associated with peristent brain damage measured by MRI (Stewart et al., 2006).</p>
<p>Some in vitro and in vivo experiments show also that neuroinflammation can lead to degeneration:</p>
<p>- the conditioned medium of Pb-treated microglial cells (10 microM for 12h) caused the death of neuroblastoma cells (Kumawak et al., 2014).</p>
<p>- immature 3D cultures treated with Pb for 10 days exhibited neuroinflammation and neuronal death was exacerbated 10 days after the end of treatment, supporting the fact that neuroinflammation leads to neurodegeneration (Zurich et al., 2002).</p>
<p>- In vivo and in vitro experiments showed that Pb cause microglial activation, which upregulate the levels of pro-inflammatory cytokines (IL-1b, TNF-a) and of iNOS and cause neuronal injury and neuronal death in hippocampus. These effects are significantly reversed by minocycline, an antibiotic blocking microglial reactivity, showing the essential role of neuroinflammation in hippocampal neurodegeneration (Liu et al., 2012)</p>
<p>- gestational exposure of mouse to Pb (0.1 mM in drinking water) led at PND 21 to increased brain mRNA expression of IL-6 and glial finbrillary acidic protein (GFAP) as marker of astrogliosis, as well as of caspase 1 and NOS 2, suggesting a link between Pb-induced neuroinflammation and deletrious effects on neurons (Kasten-Jolly et al., 2011, 2012)</p>
<p> </p>
<p>Domoic acid</p>
<p>DomA promotes the expression of inflammatory genes in the brain, such as cyclooxygenase 2 (COX2) and the development of neurodegeneration (Ryan et al., 2005). By using COX2 inhibitors that causes decrease the appearance of DomA-induced neurodegeneration, they have concluded that neuroinflammation contributes towards the development of neurodegeneration (Ryan et al., 2011).</p>
<p>Long-term treatments with NSAIDs (non-steroidal anti-inflammatory drugs) have a preventive effect on Alzheimer's disease development (Piertrzick and Behl, 2005; Wang et al., 2015), but such treatment has no effect or is even detrimental if administered once the disease is at an advanced stage (Lichtenstein et al., 2010), This may be due to the dual protective/destructive effects of neuroinflammation and to its complexity.</p>
<p>Serum Pb level negatively correlates with verbal memory score, but not with abnormal cognition in Alzheimer's disease (Park et al., 2014). Epidemiologic studies are not well-suited to accomodate the long latency period between exposures during early life and late onset of Alzheimer's disease, even if bone Pb content is an accurate measurement of historical Pb exposure in adult (Bakulski et al., 2012).</p>
<p>Besides neuroinflammation or effects associated with neuroinflammation, other mechanisms may be involved in neurodegeneration with Abeta and tau accumulation: Pb-induced epigenetic modifications of genes involved in the amyloid cascade or tau expression may contribute to the accumulation of Abeta and tau accumulation following developmental exposure to Pb (Zawia and Basha, 2005; Basha and Reddy, 2010). Also oxidative damage to DNA was shown to be involved in delayed effects observed in old rats (PD 600), if exposed early postanatally (PD 1 to 20) (Bolin et al., 2006)</p>
<p><br />
Gap of knowledge: there are no studies showing that glufosinate-induced neuroinflammation leads to neurodegeneration.</p>
<p><em>Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? </em></p>
<p>There are few studies where markers of neuroinflammation are measured simultaneously with markers of cell death and neurodegeneration. In addition, neuroinflammation is a complex KE, since the neurodegenerative consequences depend on the microglial phenotype, which has been measured only in very recent studies. An attempt to link KE<sub>up</sub> to KE<sub>down</sub> quantitatively is provided below.</p>
<p> </p>
<div>
<div>
<table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td style="width:140.2pt">
<p style="text-align:center"><strong><span style="font-size:9.0pt">Endpoints relevant for KE<sub>up</sub></span></strong></p>
<p style="text-align:center"><strong><span style="font-size:9.0pt">Neuroinflammation</span></strong></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><strong><span style="font-size:9.0pt">Endpoints relevant for KE<sub>down</sub></span></strong></p>
<p style="text-align:center"><strong><span style="font-size:9.0pt">Neurodegeneration</span></strong></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><strong><span style="font-size:9.0pt">Model and treatments</span></strong></p>
</td>
<td style="width:140.25pt">
<p style="text-align:center"><strong><span style="font-size:9.0pt">Reference</span></strong></p>
</td>
</tr>
<tr>
<td style="width:140.2pt">
<p style="text-align:justify"><strong><span style="font-size:9.0pt"> </span></strong></p>
</td>
<td style="width:140.2pt">
<p style="text-align:justify"><strong><span style="font-size:9.0pt"> </span></strong></p>
</td>
<td style="width:140.2pt">
<p style="text-align:justify"><strong><span style="font-size:9.0pt"> </span></strong></p>
</td>
<td style="width:140.25pt">
<p style="text-align:justify"><strong><span style="font-size:9.0pt"> </span></strong></p>
</td>
</tr>
<tr>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">IL-6, IL-1</span><span style="font-family:symbol; font-size:9.0pt">b</span><span style="font-size:9.0pt">, TNF-</span><span style="font-family:symbol; font-size:9.0pt">a</span><span style="font-size:9.0pt"> increased about 2x</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">in hippocampus and frontal cortex</span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Abeta 1-42 and Abeta 1-40</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">increased of 50%</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">in frontal cortex and hippocampus</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Among individual metals, Pb triggered the maxiumum induction</span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Exposure to a mixture of arsenic (0.38 ppm), cadmium (0.098 ppm) and Pb (0.22 ppm)</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">or Pb alone (2.2 ppm)</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Rat: from gestational day 05 to postnatal day 180.</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Observation in early adulthood</span></p>
</td>
<td style="width:140.25pt">
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Ashok et al., 2015</span></p>
</td>
</tr>
<tr>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Modulation of IL-6, TGF-b1 and IL-1beta</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Upregulation of GFAP (astrocyte reactivity)</span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Caspase 1 and NOS2 gene expression increased</span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Mouse treated with Pb (0.1mM) in drinking water from gestation-day 8 to PND21</span></p>
</td>
<td style="width:140.25pt">
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Kasten-Jolly et al., 2011, 2012</span></p>
</td>
</tr>
<tr>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Microglial reactivity about 3x, about 4X increase of IL-1 beta, TNF-alpha, iNOS </span></p>
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Blockade by minocycline (in vivo and in vitro)</span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">About 5x increase of neuronal death in hippocampus</span></p>
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">back to control levels in vivo and in vitro</span></p>
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Rat exposed to Pb (100ppm) from 24 to 80 days of age</span></p>
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">hippocampal neurons+ microglia co-cultures (50 </span><span style="font-family:symbol; font-size:9.0pt">m</span><span style="font-size:9.0pt">mol /L Pb for 48 h)</span></p>
</td>
<td style="width:140.25pt">
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Liu et al., 2012</span></p>
</td>
</tr>
<tr>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Microglial and astrocyte reactivities observed at the end of the 10-day treatment</span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Decrease in markers of cholinergic and GABAergic neurons that was exacerbated (30-60% increased) if harvest was performed not immediately after the 10-day treatment but after another 10-day period devoid of treatment</span></p>
</td>
<td style="width:140.2pt">
<p style="text-align:center"><span style="font-size:9.0pt">Immature 3D cultures of fetal rat brain cells</span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Pb (10<sup>-6</sup> -10<sup>-4</sup> M) applied for 10 days followed by another period of 10 days without treatment</span></p>
</td>
<td style="width:140.25pt">
<p style="text-align:center"><span style="font-size:9.0pt"> </span></p>
<p style="text-align:center"><span style="font-size:9.0pt">Zurich et al., 2002</span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
</div>
</div>
HighMixedHighDuring brain development, adulthood and agingHighHighHigh<p>The hypotheisis of developmental origin of Pb-induced neurodegeneration was tested and observed in Zebra fish by Lee and Freeman (2014).</p>
<p><br />
Ashok A, Rai NK, Tripathi S, Bandyopadhyay S., Exposure to As-, Cd-, and Pb-mixture induces Abeta, amyloidogenic APP processing and cognitive impairments via oxidative stress-dependent neuroinflammation in young rats. Toxicol Sci., 2015, 143(1): 64-80.</p>
<p>Bakulski KM, Park SK, Weisskopf MG, Tucker KL, Sparrow D, Spiro A, 3rd, et al. 2014. Lead exposure, B vitamins, and plasma homocysteine in men 55 years of age and older: the VA normative aging study. Environ Health Perspect 122(10): 1066-1074.</p>
<p>Basha MR, Murali M, Siddiqi HK, Ghosal K, Siddiqi OK, Lashuel HA, et al. 2005. Lead (Pb) exposure and its effect on APP proteolysis and Abeta aggregation. FASEB J 19(14): 2083-2084.</p>
<p>Basha R, Reddy GR. 2010. Developmental exposure to lead and late life abnormalities of nervous system. Indian journal of experimental biology 48(7): 636-641.</p>
<p>Bihaqi SW, Huang H, Wu J, Zawia NH. 2011. Infant exposure to lead (Pb) and epigenetic modifications in the aging primate brain: implications for Alzheimer's disease. J Alzheimers Dis 27(4): 819-833.</p>
<p>Bihaqi SW, Zawia NH. 2013. Enhanced taupathy and AD-like pathology in aged primate brains decades after infantile exposure to lead (Pb). Neurotoxicology 39: 95-101.</p>
<p>Bihaqi SW, Bahmani A, Subaiea GM, Zawia NH. 2014. Infantile exposure to lead and late-age cognitive decline: relevance to AD. Alzheimer's & dementia : the journal of the Alzheimer's Association 10(2): 187-195.</p>
<p>Bolin CM, Basha R, Cox D, Zawia NH, Maloney B, Lahiri DK, et al. 2006. Exposure to lead and the developmental origin of oxidative DNA damage in the aging brain. Faseb J 20(6): 788-790.</p>
<p>Brown GC, Bal-Price A., Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol., 2003, 27(3): 325-355.</p>
<p>Cagnin A, Brooks DJ, Kennedy AM, Gunn RN, Myers R, Turkheimer FE, et al., In-vivo measurement of activated microglia in dementia. Lancet, 2001, 358(9280): 461-467.</p>
<p>Gassowska M, Baranowska-Bosiacka I, Moczydlowska J, Tarnowski M, Pilutin A, Gutowska I, et al. 2016. Perinatal exposure to lead (Pb) promotes Tau phosphorylation in the rat brain in a GSK-3beta and CDK5 dependent manner: Relevance to neurological disorders. Toxicology 347-349: 17-28.</p>
<p>Gu H, Robison G, Hong L, Barrea R, Wei X, Farlow MR, et al. 2012. Increased beta-amyloid deposition in Tg-SWDI transgenic mouse brain following in vivo lead exposure. Toxicol Lett 213(2): 211-219.</p>
<p>Heneka MT, Kummer MP, Latz E., Innate immune activation in neurodegenerative disease. Nat Rev Immunol., 2014, 14(7): 463-477.</p>
<p>Hirsch EC, Hunot S., Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol., 2009, 8: 382-397</p>
<p>Janelsins MC, Mastrangelo MA, Oddo S, LaFerla FM, Federoff HJ, Bowers WJ., Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-1 expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice. J Neuroinflammation, 2005, 2: 23.</p>
<p>Kasten-Jolly J, Heo Y, Lawrence DA. 2011. Central nervous system cytokine gene expression: modulation by lead. J Biochem Mol Toxicol 25(1): 41-54.</p>
<p>Kasten-Jolly J, Pabello N, Bolivar VJ, Lawrence DA. 2012. Developmental lead effects on behavior and brain gene expression in male and female BALB/cAnNTac mice. Neurotoxicology 33(5): 1005-1020.</p>
<p>Kitazawa M, Oddo S, Yamasaki TR, Green KN, LaFerla FM., Lipopolysaccharide-induced inflammation exacerbates tau pathology by a cyclin-dependent kinase 5-mediated pathway in a transgenic model of Alzheimer's disease. J Neurosci., 2005, 25(39): 8843-8853.</p>
<p>Kraft AD, Harry GJ., Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. International Journal of Environmental research and Public Health., 2011, 8(7): 2980-3018.</p>
<p>Krstic, D., A. Madhusudan, et al., 2012. Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice. J Neuroinflammation, 2012, 9: 151.</p>
<p>Krstic D, Knuesel I. 2013. Deciphering the mechanism underlying late-onset Alzheimer disease. Nature reviews Neurology 9(1): 25-34.</p>
<p>Kumawat KL, Kaushik DK, Goswami P, Basu A. 2014. Acute exposure to lead acetate activates microglia and induces subsequent bystander neuronal death via caspase-3 activation. Neurotoxicology 41: 143-153.</p>
<p>Lee J, Freeman JL. 2014. Zebrafish as a model for investigating developmental lead (Pb) neurotoxicity as a risk factor in adult neurodegenerative disease: a mini-review. Neurotoxicology 43: 57-64.</p>
<p>Lichtenstein MP, Carriba P, Masgrau R, Pujol A, Galea E., Staging anti-inflammatory therapy in Alzheimer's disease. Frontiers in Aging Neuroscience, 2010, 2: 142.</p>
<p>Liu MC, Liu XQ, Wang W, Shen XF, Che HL, Guo YY, et al. 2012. Involvement of microglia activation in the lead induced long-term potentiation impairment. PLoS One 7(8): e43924.</p>
<p>McGeer PL, McGeer EG., Glial cell reactions in neurodegenerative diseases: Pathophysiology and therapeutic interventions. Alzheimer DisAssocDisord, 1998, 12 Suppl. 2: S1-S6.</p>
<p>McNaull BB, Todd S, McGuinness B, Passmore AP., Inflammation and Anti-Inflammatory Strategies for Alzheimer's Disease - A Mini-Review. Gerontology, 2010, 56: 3-14.</p>
<p>Neumann H., Control of Glial Immune Function by Neurons. Glia, 2001, 36: 191-199</p>
<p>Park JH, Lee DW, Park KS, Joung H. 2014. Serum trace metal levels in Alzheimer's disease and normal control groups. American journal of Alzheimer's disease and other dementias 29(1): 76-83.</p>
<p>Pietrzik, C. and C. Behl., Concepts for the treatment of Alzheimer's disease: molecular mechanisms and clinical application. Int J Exp Pathol., 2005, 86(3): 173-185.</p>
<p>Ryan JC, Morey JS, Ramsdell JS, Van Dolah FM. Acute phase gene expression in mice exposed to the marine neurotoxin domoic acid. Neuroscience 2005. 136: 1121-1132.</p>
<p>Ryan JC, Cross CA, Van Dolah FM. Effects of COX inhibitors on neurodegeneration and survival in mice exposed to the marine neurotoxin domoic acid. Neurosci Lett. 2011. 487: 83-87.</p>
<p>Sheng JG, Bora SH, Xu G, Borchelt DR, Price DL, Koliatsos VE., Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice. Neurobiol Dis., 2003, 14(1): 133-145.</p>
<p>Stewart WF, Schwartz BS, Davatzikos C, Shen D, Liu D, Wu X, et al. 2006. Past adult lead exposure is linked to neurodegeneration measured by brain MRI. Neurology 66(10): 1476-1484.</p>
<p>Taetzsch T, Block ML., Pesticides, microglial NOX2, and Parkinson's disease. J Biochem Mol Toxicol., 2013, 27(2): 137-149.</p>
<p>Tansey MG, Goldberg MS., Neuroinflammation in Parkinson's disease: Its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis., 2010, 37(3):510-8.</p>
<p>Thundyil J, Lim KL. 2015. DAMPs and neurodegeneration. Ageing research reviews 24(Pt A): 17-28.</p>
<p>Wang J, Tan L, Wang HF, Tan CC, Meng XF, Wang C, Tang SW, Yu JT (2015) Anti-inflammatory drugs and risk of Alzheimer's disease: an updated systematic review and meta-analysis. J Alzheimers Dis 44: 385-96</p>
<p>Zawia NH, Basha MR. 2005. Environmental risk factors and the developmental basis for Alzheimer's disease. Rev Neurosci 16(4): 325-337.</p>
<p>Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.</p>
<p><span style="color:#3498db"><strong>Sars-CoV-2-related references:</strong></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Debnath M et al. Changing dynamics of psychoneuroimmunology during COVID-19 pandemic. Brain Behav Immun Health. 2020 May;5:100096.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Jacomy H, et al. Human coronavirus OC43 infection induces chronic encephalitis leading to disabilities in BALB/C mice. Virology. (2006) 349:335–46</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Mesci P et al. Sofosbuvir protects human brain organoids against SARS-CoV-2. bioRxiv. 2020. Available at: doi: </span><a href="https://doi.org/10.1101/2020.05.30.125856" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.05.30.125856</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Mohammadi S. et al. Understanding the Immunologic Characteristics of Neurologic Manifestations of SARS-CoV-2 and Potential Immunological Mechanisms. Mol Neurobiol. 2020 Dec;57(12):5263-5275.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Montalvan V, Lee J, Bueso T, De Toledo J, Rivas K (2020) Neurological manifestations of COVID-19 and other coronavirus infections: a systematic review. Clin Neurol Neurosurg. 2020 Jul;194:105921.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Netland J, et al. Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2. J Virol. 2008;82:7264–7275.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Song et al. Neuroinvasive potential of SARS-CoV-2 revealed in a human brain organoid model. bioRxiv. 2020. Available at: </span><a href="https://www.biorxiv.org/content/10.1101/2020.06.25.169946v1" style="color:blue; text-decoration:underline">https://www.biorxiv.org/content/10.1101/2020.06.25.169946v1</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Wu Y, et al. Nervous system involvement after infection with COVID19 and other coronaviruses. Brain Behav Immun. 2020 Jul;87:18-22.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Zanin L, et al. SARS-CoV-2 can induce brain and spine demyelinating lesions. Acta Neurochir (Wien). 2020 Jul;162(7):1491-1494.</span></span></span></p>
2016-11-29T18:41:332021-02-23T05:47:35920190dd-6d6a-43ab-86a2-21af66b6c4c08367d882-24f4-4763-a5d9-999a7d5b4bfc<p>According to its definition, neurodegeneration includes the death of neurons. Therefore, the KER describing the link between cell death and neuroinflammation is also applicable to this KER. However in neurodegenerative diseases and in Alzheimer's disease in particular, neurodegneration is associated with accumulation of modified/aggregated proteins (insoluble amyloid; hyperphosphorylated tau), which are recognised as potential triggers of neuroinflammation:</p>
<p>Proteinopathies associated with neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD) may be sensed as damage associated molecular patterns (DAMPs) and thus activate microglia within the CNS. In animal neurodegeneration models and post-mortem brain samples from patients suffering from neurodegenerative disorders often revealed the presence of activated microglia and the accumulation of inflammatory mediators at the lesion sites, which suggests a continuous crosstalk between the brain immune system and the injured neurons during neurodegeneration. Microglial are typically activated acutely in response to an initial triggering insult, but their continued presence in large numbers around the lesion areas may actually promote neuronal death despite the absence of the initial triggering insult. Inflammatory factors being released by dying neurons and/or actively secreted from the activated microglia aid in maintaining the vicious cycle between activated microglia and damaged neurons (Thundyil and Lim 2015).</p>
<p>The fact that neuronal death can trigger neuroinflammation and that neuroinflammation can, in turn, cause neuronal degeneration, is known as a vicious circle, which is involved in the pathogeny of neurodegenerative diseases (Griffin et al., 1998; McGeer and Mc Geer, 1998; Blasko et al., 2004; Cacquevel et al., 2004; Barbeito et al., 2010; Rubio-Perez and Morillas-Ruiz, 2012; Thundyil and Lim, 2015).</p>
<p>Microglial cells are involved in the clearance of amyloid plaques (Querfurth and LaFerla, 2010), but can also be responsible for amyloid plaque formation (Streit and Sparks, 1997). As aging microglia seem to lose their ability to phagocytose (Floden and Combs, 2011), impaired clearance, as well as active deposition, can both contribute to amyloid plaque accumulation. </p>
<p>In Alzheimer's disease, Griffin and coworkers (1997) described the presence of reactive microglial cells inside the amyloid plaques and of reactive astrocytes around the plaques. Intra-cerebroventricular injections of beta-amyloid resulted in age-related increase in cholinergic loss and microglial activation (Nell et al., 2014). Increased neuronal expression of presequence protease (PreP) decreased the accumulation of beta-amyloid in synaptic mitochondria and decreases the neuroinflammatory response (Du Fang et al., 2015), showing a link between the accumulation of insoluble proteins and neuroinflammation. In addition, presence of the apolipoprotein E4 (APOE) allele, which is the strongest genetic risk factor for the development of Alzheimer's disease, increases microglial reactivity in the amyloid plaques of a mouse model of beta-amyloid deposition, suggesting a role for APOE in modulation beta-amyloid-induced neuroinflammation in Alzheimer's disease progression (Rodriguez et al., 2014).</p>
<p><em>Include consideration of temporal concordance here </em></p>
<p><strong>Binding of agonists to N-methyl-D-aspartate receptor (NMDAR) in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to reduction (or loss) of cognitive function</strong></p>
<p>DomA (0.75 mg/kg body weight) when administered intravenously in adult rats reveals neuronal degeneration followed by glial activation (Ananth et al., 2001; 2003). More specifically, 5 days after DomA administration, Nissl staining of brain sections derived from DomA-treated animals have shown extensive neuronal damage in the pyramidal neurons of CA1, CA3 subfields and hilus of the dentate gyrus in the hippocampus. In the same brain areas, neuroinflammation has also been evident characterised by increased GFAP and OX-42 immunoreactivity at 5 days after DomA administration but not earlier (24 h) (Ananth et al., 2003). Previously, the same research team has shown increased number of stained degenerated neurons in the hippocampus by Nissl staining as early as 24 h following the administration of DomA, however, the degeneration has been found to be more severe after 5 days (Ananth et al., 2001).</p>
<p><br />
</p>
<p>Gap of knowledge: there are no studies showing that glufosinate-induced neurodegeneration leads to neuroinflammation.</p>
<p><em>Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? </em></p>
<p>Quantitative evaluation of these KERs, when KEup and KEdown are measured in the same experiment in a dose and time dependent manner following exposure to DomA or GLF is not available.</p>
<p> </p>
<p>Ananth C, Thameem Dheen S, Gopalakrishnakone P, Kaur C. Domoic acid-induced neuronal damage in the rat hippocampus: changes in apoptosis related genes (bcl-2, bax, caspase-3) and microglial response. J Neurosci Res., 2001. 66: 177-190.</p>
<p>Ananth C, Gopalakrishnakone P, Kaur C. Protective role of melatonin in domoic acid-induced neuronal damage in the hippocampus of adult rats. Hippocampus, 2003. 13:375-87.</p>
<p>Barbeito AG, Mesci P, Boillee S., Motor neuron-immune interactions: the vicious circle of ALS. J Neural Transm., 2010, 117(8): 981-1000.</p>
<p>Blasko I, Stampfer-Kountchev M, Robatscher P, Veerhuis R, Eikelenboom P, Grubeck-Loebenstein B. 2004. How chronic inflammation can affect the brain and support the development of Alzheimer's disease in old age: the role of microglia and astrocytes. Aging cell 3(4): 169-176.</p>
<p>Cacquevel M, Lebeurrier N, Cheenne S, Vivien D. Cytokines in neuroinflammation and Alzheimer's disease. Curr Drug Targets., 2004, 5(6): 529-534.</p>
<p>Fang D, Wang Y, Zhang Z, Du H, Yan S, Sun Q, et al., Increased neuronal PreP activity reduces Abeta accumulation, attenuates neuroinflammation and improves mitochondrial and synaptic function in Alzheimer disease's mouse model. Human molecular genetics, 2015, 24(18): 5198-5210.</p>
<p>Griffin.W.S.T., Sheng J, Mrak R., Inflammatory Pathways. Implications in Alzheimer's disease. Humana Press Inc., 1997, 0: 169-176.</p>
<p>Floden AM, Combs CK. 2011. Microglia demonstrate age-dependent interaction with amyloid-beta fibrils. J Alzheimers Dis 25(2): 279-293.</p>
<p>Griffin, W. S., J. G. Sheng, et al., Glial-neuronal interactions in Alzheimer's disease: the potential role of a 'cytokine cycle' in disease progression. Brain Pathol., 1998, 8(1): 65-72.</p>
<p>Hayward JH, Lee SJ. A Decade of Research on TLR2 Discovering Its Pivotal Role in Glial Activation and Neuroinflammation in Neurodegenerative Diseases. 2014, Experimental neurobiology, 23(2): 138-147.</p>
<p>McGeer PL, McGeer EG., Glial cell reactions in neurodegenerative diseases: Pathophysiology and Therapeutic Interventions. Alzheimer DisAssocDisord 12 Suppl. 1998, 2: S1-S6.</p>
<p>Nell HJ, Whitehead SN, Cechetto DF., Age-Dependent Effect of beta-Amyloid Toxicity on Basal Forebrain Cholinergic Neurons and Inflammation in the Rat Brain. Brain Pathol., 2015, 25(5): 531-542.</p>
<p>Querfurth HW, LaFerla FM. 2010. Alzheimer's disease. The New England journal of medicine 362(4): 329-344.</p>
<p>Rodriguez GA, Tai LM, LaDu MJ, Rebeck GW., Human APOE4 increases microglia reactivity at Abeta plaques in a mouse model of Abeta deposition. J Neuroinflammation, 2014, 11: 111.</p>
<p>Rubio-Perez JM, Morillas-Ruiz JM.,A review: inflammatory process in Alzheimer's disease, role of cytokines. Scientific World Journal, 2012: 756357.</p>
<p>Streit WJ, Sparks DL. 1997. Activation of microglia in the brains of humans with heart disease and hypercholesterolemic rabbits. JMolMed 75: 130-138.</p>
<p>Thundyil J, Lim KL., DAMPs and Neurodegeneration. Ageing research reviews. 2015, 24(Pt A):17-28</p>
<p> </p>
<p> </p>
2016-11-29T18:41:332018-06-13T09:35:05c7a3486c-aa5a-49f3-8f1c-4cdb308a6439ce45a297-1f86-4c40-aae0-f997d0a93d88<p>NMDARs can be activated indirectly through initial activation of KA/AMPARs as it happens in the case of DomA exposure. DomA is an agonist of
presynaptic and postsynaptic KARs and sustained activation of these receptors by DomA results in massive ion flux and excessive release of glutamate
from excitatory terminals causing depolarization of the postsynaptic neuron (as descibed in MIE). Upon this depolarization the Mg2+ block is removed
from the pore of NMDARs, resulting in their activation allowing sodium, potassium, and, importantly, calcium ions to enter into a cell. The sustained
exposure to DomA causes pathological overactivation of NMDARs.
In the case of exposure to glufosinate NMDARs activation is triggered by direct, sustained binding of glufosinate to the NMDARs.
</p><p>NMDARs are unique among ligand-gated ion channels in that their activation requires binding of two co-agonists, glycine and endogenous
neurotransmitter, L-glutamate. Physiologically, however, glycine and glutamate have distinct functions. While L-glutamate is released from specific
presynaptic terminals, low concentrations of ambient glycine present at the synapse are thought to be sufficient to allow receptor activation.
There is a clear understanding that binding of glutamate or its analogue will activate NMDA receptor (accepted dogma). The prolonged activation of
NMDARs will lead to a pathological over-activation of a receptor leading to excitotoxicity (minor role of KA/AMPARs), allowing high levels of calcium
ions to enter the cell. However, KA/AMPARs play an important role for indirect NMDAR activation since (almost always) an initial activation of these
receptors triggers depolarization of postsynaptic neurons that relieves the block of the channel pore by Mg2+, resulting in NMDAR activation.
NMDA receptors are formed by a ligand binding domain (LBD) and an ion channel that are considered the core structural and functional elements of
the receptors. There is a clear understanding of how agonist binding leads to channel opening that relies on structural (e.g. crystallography or NMR)
and functional (e.g. UV and IR spectrometric measurements) experimental studies of the water-soluble LBD combined with functional studies of the
intact receptor. After the initial agonist binding, a conformational change—so-called clam shell closure—that prevents agonist dissociation occurs
followed by a conformational change in the ion channel that is tightly coupled to that in the LBD (reviewed in Traynelis et al., 2010).
Consequently it can be stated that there is a clear structural and functional mechanistic understanding in this KER between MIE (Binding of agonist to
glutamate ionotropic receptors) and KE1, NMDAR overactivation that, as explained above, can be triggered by direct binding to NMDAR or indirectly,
through initial activation of KA/AMPARs as it happens in the case of exposure to glufosinate and DomA respectively, two stressors described in this
AOP.
</p><p>Indeed, domoic acid has a very strong affinity for the ionotropic glutamate receptors, the activation of which results in excitotoxicity, initiated by an
integrative action of ionotropic receptors at both sides of the synapse blocking the channel from rapid desensitization. It has a synergistic effect with
endogenous glutamate and it acts mainly as an agonist for presynaptic and postsynaptic kainate receptors. Activation of ionotropic receptors leads to
the influx of Na+, K+ and Ca2+, particulary after activation of NMDARs. In combination with the inhibitory GABA neurotransmitter, glutamate
contributes to the control of overall neuronal excitability.
</p><p>Gufosinate (GLF) triggers alterations in glutamatergic signaling through direct binding and activation of NMDARs (Lantz et al., 2014: Matsumura et al.,
2001). GLF agonist action at the NMDAR is expected to occur through interaction with the glutamate binding site and requires binding of the glycine
co-agonist as well as release of the magnesium block from the channel pore. Additionally, the possible inhibition by GLF of the high affinity glutamate
re-uptake transporter, especially GLT-I was studied to determine whether GLF could increase the levels of endogenous glutamate at the synaptic cleft,
resulting in over activation of NMDARs. Such mechanism was excluded by Lantz (Lantz et al., 2014) but suggested by other studies (Watanabe and
Sano, 1998).
</p><p><em>
Include consideration of temporal concordance here
</em>
</p><p>There is well established understanding of NMDAR activation by endogenous agonist glutamate that happens in the absence of the Mg2+ block under
conditions of depolarized post-synaptic membrane(accepted dogma) (Blanke et al., 2009a and b; Enoki R, et al.,2004).
</p><p>Single channel behavior of NMDARs from hippocampal CA1 neurons was studied using very low glutamate concentrations to improve temporal
resolution of individual glutamate binding events. Openings resulting from individual receptor activations showed surprising complexity: they consist of
a long cluster of bursts of openings. Furthermore, the NMDARs appeared to have different gating modes, occasionally entering periods of very high
open probability (Gibb et al., 1991).
Single channel analysis also provided insight in how NMDARs function at the synapse. In response to a brief pulse of glutamate, mimicking synaptic
release, NMDARs activate slowly over hundreds of milliseconds and continue activating long after all glutamate has been removed from the synaptic
cleft, thereby briefly “memorizing” the occurrence of a synaptic input. Single channel analysis of NR1 and NR2A receptors indicates that after a brief
pulse of glutamate, receptors enter a high affinity closed state from which either channel opening or agonist unbinding occurs with approximately
equal probability (Popescu et al., 2004). A single synaptic event is therefore expected to only partially activate NMDARs. Consequently, a closely
spaced second pulse of agonist is able to further increase the open probability, endowing the NMDAR with an ability to decode synaptic input
frequency.
</p><p><b>Domoic acid</b> is an agonist for presynaptic and postsynaptic kainate receptors, however indirectly also activates NMDA receptors. Kainate receptors
are localized both at presynaptic and postsynaptic sites. At presynaptic sites, they directly affect transmitter release from both excitatory and inhibitory
neuron terminals. At postsynaptic sites, kainate receptors lead to cell depolarization, which would bring the neuron closer to its spike firing threshold.
By having this dual localization, kainate receptors help in the control of neuronal excitability. However, sustained activation of postsynaptic kainate
receptors by domoic acid results in massive ion flux and excessive release of glutamate from excitatory terminals. The released glutamate in turn
activates NMDA receptors, which have lost their physiologic Mg2++ block because of domoic acid–induced depolarization. The final event is an
increase of NMDA-mediated Ca2++ flux and subsequent activation of intracellular pro-oxidative cascades and ion imbalances, eventually leading to
excitotoxicity-mediated neuronal death (Babot et al., 2005;Giordano et al., 2006).
</p><p>Kainate receptors are widely expressed in the hippocampus. Glutamatergic granule cells in the hippocampus express these receptors, suggesting that
cell death found after domoic acid intoxication may be produced by hyperstimulation of NMDA receptor after glutamate is released in excess. In
agreement with this hypothesis, the seriously damaged CA3 area of the hippocampus receives projections from hippocampal granule cells. Qiu and
Curras-Collazo (Qiu et al., 2006a) elegantly demonstrated that domoic acid first targets kainate receptors in the hippocampus by blocking its effects in
vivo with a kainate receptor antagonist. The sequential involvement of distinct glutamate receptors was confirmed and further elucidated in rat mixed
cortical cell and hippocampal slice cultures (Jakobsen et al., 2002; Qiu et al., 2006b).
</p><p>Using primary cultures of rodent cerebellar granule cells, an in vitro model mainly constituted by glutamatergic neurons that express both NMDA and
kainate receptors it was proved that domoic acid increased glutamate release, intracellular calcium, and cell death, which were prevented by kainate
and NMDA receptor antagonists (Berman and Murray, 1997; Vale-Gonzalez et al., 2006) confirming that DomA toxicity is mediated by both KA and
NMDARs.
</p><p><b>Glufosinate</b> (GLF) and its primary metabolite N-acetylglufosinate (NAcGLF) interaction with NMDARs was studied in the primary culture of rat cortical
neurons by performing [3H]CGP 39653 binding experiments. The results showed that their binding affinity to NMDAR (IC50, GLF 668 uM and NAcGLF
approximately 100 uM) corresponded to the concentration that produce the highest increase of mean firing rate. Furthermore, they produced biphasic
MFR profile, specific to NMDAR agonists. The obtained results suggest that GLF and NAcGLF can produce both effects,excitatory and inhibitory on
network activity through direct activation of NMDARs (Lantz et al., 2014).
</p><p>Direct activation of NMDARs by GLF is also suggested by in vivo studies where three NMDA receptor antagonists, dizocilpine, LY235959, and Compound
40, and AMPA/KA antagonist, NBQX, were co-administrated with glufosinate ammonium (80 mg/kg, intraperitoneally) in mice. Statistical analyses
showed that the NMDA receptor antagonists markedly inhibited the GLF-induced convulsions, while the AMPA/KA receptor antagonist had no effect.
These results suggest that the convulsion caused by glufosinate ammonium were mediated through activation of NMDA receptors (Matsumura et al.,
2001).
</p><p>The increase in MFR induced by GLF in neuronal networks was significantly blocked by MK-801 but not entirely suggesting that GLF can increase
activity in the MEA system through non-synaptic NMDARs, since these are not blocked by MK-801.
It is not entirely clear whether GLF can work through an inhibition of the glutamate reuptake transporter, GLT-I, increasing the concentration of
endogenous glutamate at the synaptic cleft and subsequently resulting in over activation of NMDARs (Lantz et al., 2014: Watanabe and Sano, 1998).
Further studies are necessary to determine whether this alternative mechanism of GLF-induced NMDAR overactivation takes place.
Additionally GLF also modulates glutamine synthetase (GS) activity. Since, astrocytic GS in the brain participates in the metabolic regulation of glutamate (endogenous agonist of NMDAR) it is not clear if this pathway contributes to NMDAR activation too.
</p><p><em>
Is it known how much change in the first event is needed to impact the second?
Are there known modulators of the response-response relationships?
Are there models or extrapolation approaches that help describe those relationships?
</em>
</p><p>To predict how potent an agonist can be, it is usually based on the half maximal effective concentration (EC50) that induces the currents through
NMDA receptors of brain slices and cells (or in recombinantly expressed proteins of these receptors). Traynelis et al. 2010 summarised the IC50 values
for agonists of the different NMDA receptor subunits.
The activation effect (efficacy) of agonist on NMDA receptor have been found to be dependent on:
-the type of subunits that form the NMDA receptor
-the chemical structure of the agonist
-the binding site of a receptor that the agonist prefers
-how tightly an agonist binds to the receptor (affinity)
Glufosinate and its primary metabolite N-acetylglufosinate NAcGLF bind to the NMDAR with the following affinity: the IC50 value for GLF was 668 mM
and for NAcGLF was about 100 mM.
</p><p>Various studies suggest the existence of functional NMDA-like receptors in invertebrates (Xia et al., 2005). Fly and rodent NMDARs exhibit several
important differences (Murphy and Glanzman, 1997). The expression and function of NMDA receptors in rodent and primates is well characterized in
the existing literature.
</p><p><br />
Babot Z, Cristofol R, Sun˜ol C.,Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is
dependent on GABAA receptors and niflumic acidsensitive chloride channels. Eur J Neurosci., 2005, 21: 103–112.
</p><p>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; 2009a. Chapter 13. Frontiers in Neuroscience.
</p><p>Blanke ML., and Antonius M.J. VanDongen, Activation Mechanisms of the NMDA Receptor in Biology of the NMDA Receptor,2009b, Chapter 13, Van Dongen AM, editor. Boca Raton (FL): CRC Press.
</p><p>Berman FW, Murray TF., 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. J Neurochem., 1997, 69: 693–703.
</p><p>Enoki R, et al., NMDAR-mediated depolarizing after-potentials in the basal dendrites of CA1 pyramidal neurons. Neurosci Res., 2004, 48: 325-337.
</p><p>Gibb AJ, Colquhoun D. Glutamate activation of a single NMDAR-channel produces a cluster of channel openings. Proc. R. Soc. Lond. (Biol.) 1991, 243:
39-47.
</p><p>Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG., Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic
model of glutathione deficiency. Mol Pharmacol., 2006, 70: 2116–2126.
</p><p>Jakobsen B, Tasker A, Zimmer J., Domoic acid neurotoxicity in hippocampal slice cultures. Amino Acids, 2002, 23: 37–44.
</p><p>Lantz Stephen R , Cina M. Mack , Kathleen Wallace, Ellen F. Key , Timothy J. Shafer , John E. Casida, Glufosinate binds to N-methyl-D-aspartate
receptors and increases neuronal network activity in vitro. NeuroToxicology, 2014, 45: 38–47.
</p><p>Matsumura N1, Takeuchi C, Hishikawa K, Fujii T, Nakaki T., Glufosinate ammonium induces convulsion through N-methyl-D-aspartate receptors in
mice. Neurosci Lett., 2001, 304(1-2): 123-5.
</p><p>Murphy GG, Glanzman DL., Mediation of classical conditioning in Aplysia californica by long-term potentiation of sensorimotor synapses. Science, 1997,
278: 467-78.
</p><p>Popescu G, et al. Reaction mechanism determines NMDAR response to repetitive stimulation. Nature. 2004, 430: 790-799.
</p><p>Qiu S, Curras-Collazo MC., Histopathological and molecular changes produced by hippocampal microinjection of domoic acid. Neurotoxicol Teratol.,
2006a, 28: 354–362.
</p><p>Qiu S, Pak CW, Curras-Collazo MC., Sequential involvement of distinct glutamate receptors in domoic acid-induced neurotoxicity in rat mixed cortical
cultures: Effect of multiple dose/duration paradigms, chronological age, and repeated exposure. Toxicol Sci., 2006b, 89: 243–256.
</p><p>Traynelis SF, 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(3):405-96.
</p><p>Vale-Gonzalez C, Alfonso A, Sun˜ol C, Vieytes MR, Botana LM., Role of the plasma membrane calcium adenosine triphosphatase on domoate-induced
intracellular acidification in primary cultures of cerebellar granule cells. J Neurosci Res., 2006, 84: 326–337.
</p><p>Watanabe T1, Sano T., Neurological effects of glufosinate poisoning with a brief review. Hum Exp Toxicol. 1998, 17: 35-9.
</p><p>Xia S, et al., NMDARs mediate olfactory learning and memory in Drosophila. Curr Biol., 2005, 15:603-618.
</p>2016-11-29T18:41:352016-11-29T20:44:06d58bcf47-999b-4505-b1c5-696e1ad94129dde9cb60-607b-444f-8e58-8da1bafc6e71<p>One of the mitochondrial functions is to buffer intracellular Ca2+ levels facilitating the maintenance of Ca2+ homeostasis in the cell. In the case of Ca2+ overload, mitochondria are not able to buffer the excess of Ca2+ that leads to mitochondrial dysfunction measured by the increased generation of reactive oxygen species (ROS), triggering mitochondrial permeability transition pore opening (Choi et al.,2013) and reduced ATP production (reviewed in Gleichmann and Mattson, 2011).
</p><p>There is functional and structural mechanistic understanding supporting the relationship between KE "Ca2+ influx, increased" and KE "Mitochondrial dysfunction".
</p><p>The increase in cytoplasmic Ca2+ can cause the activation of plasma membrane and endoplasmic reticulum (ER) Ca2+-ATPases that results in higher ATP demand. At the same time elevated Ca2+ can cause reduced levels of ATP by the direct uptake of the cation into the matrix that utilizes the proton circuit and directly competes with mitochondrial ATP synthesis (reviewed in Nicholls, 2009).
</p><p>Ca2+ overload besides of being detrimental to mitochondrial energy production can also induce mitochondrial ROS generation. A number of possible mechanisms have been suggested by which Ca2+ overload can increase ROS production including: 1) stimulated increase of metabolic rate by Ca2+, 2) stimulated nitric oxide production by Ca2+, 3) Ca2+ induced cytochrome c dissociation, 4) Ca2+ induced cardiolipin peroxidation, 5) Ca2+ induced mitochondrial permeability transition pore (MPTP)opening with release of cytochrome c (leading to apoptosome formation and caspase-3 activation)and apoptosis inducing factor (AIF), decreased level of reduced glutathione (GSH), the antioxidative enzymes, and 6) Ca2+-calmodulin dependent protein kinase activation (reviewed in Peng and Jou, 2010; Gleichmann and Mattson, 2011). It is worth mentioning that mitochondrial ROS increase is capable of modulating Ca2+ dynamics causing further increase of Ca2+ levels.
</p><p>The cytoplasmic and mitochondrial Ca2+ levels, the oxidative stress and the energy production are very closely inter-related. For example, decreased (or lack) of ATP production can affect the function of plasma membrane Ca2+ pump activity causing Ca2+ overload, oxidative stress and further restriction in ATP generating capacity (reviewed in Nicholls, 2009). Prolonged oxidative stimuli cause further mitochondrial dysfunction, including the decrease of mitochondrial transmembrane potential (ΔΨm), further overload of mitochondrial calcium, and opening of mitochondrial permeability transition pore (MPTP) (Choi et al., 2013).
</p><p>Mitochondria within dendrites are elongated and perform extensive directional and lateral movement at physiological conditions. Under an excitotoxic exposure to glutamate, mitochondrial movement has been found to be inhibited and mitochondria change morphology becoming rounded and swollen. Although blocking mitochondrial ATP production is sufficient to inhibit mitochondrial movement (Rintoul et al., 2003), research has shown that the collapse of mitochondrial structure requires extracellular Ca2+ influx via NMDA receptors (Rintoul et al., 2003; Pivovarova et al., 2004; Shalbuyeva et al., 2006), suggesting that structural, mechanistic understanding is also available supporting this KER.
</p><p>In neurons, the high mitochondrial content in axons and dendrites closely correlates with the high energy demand in these structures that is needed to pump the ions that underlie the generation of action potentials mediated by the electrochemical gradients (Attwell and Laughlin, 2001).
</p><p><em>
Include consideration of temporal concordance here
</em>
</p>
<ul>
<li>DomA has been shown to cause a significant time- and concentration-dependent increase of ROS production in mouse cerebellar granule neurons (CGNs) and the maximal effect (2.5 fold increase) has been recorded 1 h after exposure (Giordano et al., 2006). The time course involved the measurement of oxidant-sensitive fluorescent dye DCFH2-DA from 15 to 120 min and the concentrations assessed are 1 and 10 µM DomA (Giordano et al., 2006). ROS production is higher in Gclm (-/-) neurons lacking glutathione (21.97 pmol DCF/mg of protein) than in Gclm (+/+) neurons (10.23 pmol DCF/mg of protein) after treatment with DomA (Giordano et al., 2006). In the same study, treatment of mouse CGNs with 1 and 10 µM DomA elevate intracellular Ca2+ by approximately 5 or 8 fold higher compared to controls, respectively (Giordano et al., 2006), showing that the cytosolic Ca2+ increase (upstream KE) is higher than the down-stream KE (ROS production due to mitochondrial dysfunction).
</li>
</ul>
<ul>
<li>The same research group have measured intracellular Ca2+ concentrations at different time points after DomA treatment of cerebellar granule neuons (CGNs) from mice lacking the modifier subunit of glutamate-cysteine ligase (Gclm). Glutamate-cysteine ligase (Glc) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis. CGNs from Gclm (-/-) mice have very low levels of GSH and are 10-fold more sensitive to DomA-induced toxicity than CGNs from Gclm (+/+) mice (Giordano et al., 2007). The low DomA dose (0.1μM) causes a small and delayed increase in intracellular Ca2+ concentration with a full recovery by 20 min, whereas, the higher DomA concentration (10 μM) causes a rapid and robust increase in intracellular Ca2+, which lasts even after 25 min, revealing that upstream KE (cytosolic Ca2+) happens much earlier than the down-stream KE (ROS production). Interestingly, in the same study the mitochondrial Ca2+ concentration has been measured and showed that 0.1μM DomA causes an increase by about 3-fold, with a delay of about 15 min, but no changes in mitochondrial Ca2+ concentration have been observed at 10 μM of DomA (Giordano et al., 2007).
</li>
</ul>
<ul>
<li>Mice injected intraperitoneally (i.p.) at a dose of 2 mg/kg of DomA once a day for 3 weeks show markedly lowered (1.5-2 fold) respiratory control ratio, mitochondrial ATP production rate, electron transport chain activity and cellular ATP concentration (Lu et al., 2012; Wu et al., 2013). In Lu et al. 2013 the same treatment in mice causes a 3 or 1.8-fold decrease in electron transport chain activity and mitochondrial ATP content, respectively. Western blot analysis demonstrate that the level of complex I-V proteins (mt-Nd6, Sdha, Uqcrc1, mt-Co1, and Atp5a1) in the hippocampus of DomA-treated mice is significantly decreased compared to controls (Lu et al., 2012). In the same study, DomA treatment significantly elevate the expression of NOX subunits (p47phox and gp91phox), of ROS (3.2 fold increase) and protein carbonyl levels, as well as the production of superoxide anion radicals (Lu et al., 2012). Under the same experimental conditions an increase of NOX activity (2 fold) has been reported in the hippocampus of DomA-treated mice (Lu et al., 2013). Furthermore, DomA exposure induces ER stress by increasing the levels of phosphorylated pancreatic endoplasmic reticulum-resident kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), glucose-regulated protein 78, C/EBP homologous protein (CHOP), X-box binding protein 1 (XBP1) and the phosphorylated inositol-requiring enzyme 1 (IRE1) (Lu et al., 2012).
</li>
</ul>
<ul>
<li>DomA (0.75 mg/kg body weight) administered intravenously in adult rats reveals no remarkable changes at the mRNA level of iNOS expression but demonstrates significant induction in the expression of iNOS protein level in the neurons and astrocytes of the hippocampus (Ananth et al., 2003).
</li>
</ul>
<p><br />
</p>
<table border="1" style="border-collapse:collapse;font-size:75%">
<tr>
<td align="center" style="background:#f0f0f0;"><b>Stressor</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Experimental Model</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Tested concentrations</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Exposure route</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Exposure duration</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Increased intracelllular Ca 2+ levels (KE up) (measurements, quantitative if available)</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Mitochondrial dysfunction (KE down) (measurements, quantitative if available)</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>References</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Temporal Relationship</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Dose-response relationship</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Incidence</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Comments</b>
</td></tr>
<tr>
<td> DomA</td>
<td>Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice </td>
<td>0.01 to 10 µM</td>
<td></td>
<td>Time course (15 to 120 min)</td>
<td>5 and 8 fold increase of [Ca2+]i compared to controls.</td>
<td>Increase in ROS production (2.5 fold) after 1 h of exposure.</td>
<td>Giordano et al., 2006</td>
<td></td>
<td>Same dose</td>
<td>Incidence of upstream KE (Increased intracelllular Ca 2+ levels) is higher than the incidence of downstream KE (mitochondrial dysfunction)</td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>CGNs from Gclm (+/+) and Gclm (−/−) mice </td>
<td>0.01 to 10 µM </td>
<td></td>
<td>Time course (0 to 25 min)</td>
<td>0.1μM domoic acid caused a small and delayed increase (4 fold) in [Ca2+]i, with a full recovery by 20 min. In contrast, the higher concentration of domoic acid (10μM) caused a rapid and robust increase (8 fold) in [Ca2+]i, which was still elevated after 25 min. 0.1μM DomA increases [Ca2+]M by about 3 fold, with a delay of about 15 min. In contrast, no changes in [Ca2+]M were observed following 10μM of DomA.</td>
<td>DomA (0.1μM) caused a 3 fold increase in DHR fluorescence, which accumulates in mitochondria and fluoresces when oxidized by ROS or reactive nitrogen species. This occurred between 1 and 2 h and was higher in CGNs from Gclm (−/−) mice.</td>
<td>Giordano et al., 2007</td>
<td>Yes</td>
<td>Same dose</td>
<td></td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>Adult mice </td>
<td>2 mg/kg</td>
<td>intraperitoneally (i.p.)</td>
<td>Once a day for 3 weeks </td>
<td></td>
<td>Decreased respiratory control ratio (1.5-2 fold), mitochondrial ATP production rate, electron transport chain activity, cellular ATP concentration, electron transport chain activity (3 fold) and mitochondrial ATP content (1.8 fold).</td>
<td>Lu et al., 2012; Lu et al. 2013; Wu et al., 2013</td>
<td></td>
<td></td>
<td></td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>Adult rats</td>
<td>0.75 mg/kg </td>
<td></td>
<td></td>
<td></td>
<td>Induction in the expression of iNOS protein level.</td>
<td>Ananth et al., 2003</td>
<td></td>
<td></td>
<td></td>
<td>
</td></tr>
<tr>
<td>
</td></tr></table>
<p><br />
</p><p>Gap of knowledge: there are no studies available showing that Glufosinate (GLF) increases intra-cellular calcium levels causing mitochondrial dysfunction. Such a mechanism of toxicity can be assumed taking into consideration that GLF neurotoxicity is induced by direct activation of NMDARs.
</p><p><em>
Is it known how much change in the first event is needed to impact the second?
Are there known modulators of the response-response relationships?
Are there models or extrapolation approaches that help describe those relationships?
</em>
</p><p><br />
It was established that the dendritic calcium levels could underlie the differential vulnerability of C57BL/6 (resistant to kainite excitotoxicity) and C57BL/10 strains (vulnerable) mice to triggered neuronal degeneration induced by increased Ca2+ levels (Shuttleworth and Connor, 2001). A striking difference was found in dendrite calcium responses in hippocampus after kainate exposure of C57BL/6 (resistant to kainite excitotoxicity) and C57BL/10 strains (vulnerable). Ca2+ signals in distal dendrites were large in C57BL/10 neurons, and, if a threshold concentration of 1.5 uM was reached, a region of sustained high Ca2+ was established in the distal dendritic tree. This region then served as an initiation site for a degenerative cascade, producing high Ca2+ levels that slowly spread to involve the entire neuron and led to neuronal cell death. Dendritic Ca2+ signals in C57BL/6 neurons were much smaller and did not trigger these propagating secondary responses. Neurons from both strains had similar membrane properties and responded to kainate with intense action potential firing.
Degenerative Ca2+ responses were seen in both strains if soma calcium level was above 1.5 uM serving <a href="/wiki/index.php?title=As_a_threshold&action=edit&redlink=1" class="new" title="As a threshold (page does not exist)">as a threshold</a> that if exceeded triggered excitotoxic neuronal cell death (Shuttleworth and Connor, 2001).
</p><p>DomA toxicosis in California sea lions (CSLs, Zalophus californianus) is accompanied by increased expression of markers of oxidative stress such as malondialdehyde (MDA) and 3-nitrotyrosine (NT) in neurons (Madl et al., 2014).
</p><p>In Atlantic salmon (Salmo salar), the cognition function has been investigated after exposure to sub-lethal doses of DomA (6 mg DA/kg bw). In addition, 14C-2-deoxyglucose has been injected i.m. to measure brain metabolic activity by autoradiography. The three brain regions investigated telencephalon, optic tectum and cerebellum have demonstrated a clear increase of metabolic activity in DomA exposed brains (Bakke and Horsberg, 2007).
</p><p><br />
Ananth C, Gopalakrishnakone P, Kaur C., Protective role of melatonin in domoic acid-induced neuronal damage in the hippocampus of adult rats. Hippocampus, 2003, 13: 375-87.
</p><p>Attwell D, Laughlin SB, An energy budget for signaling in the grey matter of the brain. J. Cereb. Blood Flow Metab., 2001, 21: 1133–1145.
</p><p>Bakke MJ, Horsberg TE., Effects of algal-produced neurotoxins on metabolic activity in telencephalon, optic tectum and cerebellum of Atlantic salmon (Salmo salar). Aquat Toxicol., 2007, 85: 96-103.
</p><p>Choi IY, Lim JH, Kim C, Song HY, Ju C, Kim WK., 4-hydroxy-2(E)-Nonenal facilitates NMDA-Induced Neurotoxicity via Triggering Mitochondrial Permeability Transition Pore Opening and Mitochondrial Calcium Overload. Exp Neurobiol., 2013, 22 :200-207.
</p><p>Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG. Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol., 2006, 70 :2116-26.
</p><p>Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG. Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.
</p><p>Gleichmann M, Mattson MP., Neuronal calcium homeostasis and dysregulation. Antioxid Redox Signal., 2011, 14 :1261-1273.
</p><p>Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF. Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52: 646-59.
</p><p>Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ., Troxerutin counteracts domoic acid-induced memory deficits in mice by inhibiting CCAAT/enhancer binding protein β-mediated inflammatory response and oxidative stress. J Immunol., 2013, 190: 3466-3479.
</p><p>Madl JE, Duncan CG, Stanhill JE, Tai PY, Spraker TR, Gulland FM., Oxidative stress and redistribution of glutamine synthetase in California sea lions (Zalophus californianus) with domoic acid toxicosis. J Comp Pathol., 2014, 150: 306-315.
</p><p>Nicholls DG., Mitochondrial calcium function and dysfunction in the central nervous system. Biochim Biophys Acta., 2009, 1787: 1416-1424.
</p><p>Peng TI, Jou MJ. Oxidative stress caused by mitochondrial calcium overload. Ann N Y Acad Sci., 2010, 201: 183-188.
</p><p>Pivovarova NB, Nguyen HV, Winters CA, Brantner CA, Smith CL, Andrews SB., Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons. J Neurosci., 2004, 24: 5611-5622.
</p><p>Rintoul GL, Filiano AJ, Brocard JB, Kress GJ, Reynolds IJ., Glutamate decreases mitochondrial size and movement in primary forebrain neurons. J Neurosci., 2003, 23: 7881-7888.
</p><p>Shalbuyeva N, Brustovetsky T, Bolshakov A, Brustovetsky N. Calcium-dependent spontaneously reversible remodeling of brain mitochondria. J Biol Chem., 2006, 281: 37547-37558.
</p><p>Shuttleworth CW, Connor JA. Strain-dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons. J Neurosci., 2001, 15:21(12):4225-36.
</p><p>Wu DM, Lu J, Zhang YQ, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ., Ursolic acid improves domoic acid-induced cognitive deficits in mice. Toxicol Appl Pharmacol., 2013, 271: 127-36.
</p>2016-11-29T18:41:332016-11-29T20:08:05dde9cb60-607b-444f-8e58-8da1bafc6e714f8c9fd6-ed55-4019-8287-e45ac29ddde8<p>ROS generation is known to activate different pathways leading to apoptosis, whereas depletion of energy production induces necrotic cell death.
</p><p>There is functional mechanistic understanding supporting this relationship between KE3 and KE4.
</p><p>ROS are known to stimulate a number of events and pathways that lead to apoptosis, triggered by ROS-induced ER stress signalling pathway (Lu et al., 2014), caspase-dependent and -independent apoptosis (Zhou et al., 2015), mitogen-activated protein kinase (MAPK) signal transduction pathways (reviewed in Cuadrado and Nebreda, 2010, Harper and LoGrasso, 2001).
</p><p>Depletion of cellular ATP is known to cause switching from apoptotic cell death triggered by a variety of stimuli to necrotic cell death (Leist et al., 1997) suggesting that the level of intracellular ATP determines whether the cell dies by apoptosis or necrosis (Nicotera et al., 1998). There is strong proof that apoptosis requires energy, as it is a highly regulated process involving a number of ATP-dependent steps such as caspase activation, enzymatic hydrolysis of macromolecules, chromatin condensation, bleb formation and apoptotic body formation (Richter et al., 1996).
</p><p><em>
Include consideration of temporal concordance here
</em>
</p><p>In the case of DomA, in vitro studies have shown that oxidative stress and oxidative stress-induced activation of the stress-activated protein kinase/c-jun-N-terminal kinase (SAPK/JNK) pathway is implicated in DomA-mediated apoptosis (Giordano et al., 2007; 2008; 2009; Lu et al., 2010). In vivo findings also show that ROS-mediated cognitive deficits are associated with apoptosis induced by activation of the JNK pathway (Lu et al., 2010; 2011).
</p>
<ul>
<li>Mice injected intraperitoneally (i.p.) with DomA at a dose of 2 mg/kg once a day for 4 weeks have shown increase (6 fold) of the TUNEL positive cells in the hippocampus . In the same study they have found that indicators of mitochondria function are markedly decreased (1.5-2 fold) and ROS levels are elevated (3.2 fold) (Lu et al., 2012). DomA treatment also significantly decreases the levels of bcl-2, procaspase-3 and procaspase-12 and increases the activation of caspase-3 and caspase-12 in the mouse hippocampus (Lu et al., 2012). The same research group using similar dose but longer exposure (4 weeks), has shown increase of ROS (3 fold) and NOX (2 fold) and elevated (8 fold) mean value of TUNEL-positive cells in the hippocampal CA1 sections as well as increase in the activation of caspase-8 and caspase-3 (Wu et al., 2012). These two in vivo studies (Lu et al., 2012; Wu et al., 2012) suggest that both KEs are affected in response to the same dose of DomA and exposure paradigm and that the incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction).
</li>
</ul>
<ul>
<li>The cell viability has been measured by the MTT reduction assay in mouse cerebellar granule neurons (CGNs) and showed that the IC50 values for DomA are 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons (Giordano et al., 2006). This reduction in cell viability has been demonstrated to be concentration dependent after studying a range of concentrations of DomA (0.01 and 10 µM). Giordano et al. 2007 have shown that 100 nM DomA induce apoptotic cell death in mouse CGNs. In a follow-up study, the same research group has performed a dose response evaluation and showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs derived from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione are more sensitive as 10 nM DomA induces a significant increase in apoptotic cell number (Giordano et al., 2009). The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes has been caused by 100 nM DomA. Interestingly, 1 and 10 µM DomA still cause significant apoptosis in both cell types but to a lesser extent compared to 100 nM DomA. ROS have been measured only at the dose of 100 nM DomA, 30 min after treatment and showed 2.5 fold increase compared to controls in CGNs from Gclm (+/+) mice (Giordano et al., 2009). Caspase 3 activity has also been measured after 12 h with prior 1 h exposure to 100 nM DomA and found to be increased (2.2 fold). In the same study, DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h exposure. Again these in vitro studies (Giordano et al., 2007; 2009) suggest that both KEs are affected by the same dose of DomA and that the incidence of KE down (cell death) is higher than the incidence of KE up (mitochondrial dysfunction). Furthermore, KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that takes place 12-24 h later.
</li>
</ul>
<ul>
<li>Mixed cortical cultures have been treated with 3, 5, 10, or 50 μM DomA for a variety of exposure durations (10 min, 30 min, 1 h, or 2 h), after which DomA is washed out and the culture medium is replaced with conditioned medium from unexposed sister cultures (Qiu et al., 2006). In all cases neuronal death has been measured 24 h following the beginning of exposure. The results show that DomA-induced neuronal death is determined by both concentration and duration of exposure. After a 10-min exposure, 50 μM DomA produces marked neuronal death of 47.4 %, whereas by 1 h of treatment, the same concentration produces near maximal neuronal death but longer exposures do not increase neuronal death further (Qiu et al., 2006). Regarding time dependence, this study shows that low concentrations of DomA produces more neuronal death if this is measured 22 h after the washout than if measured immediately after DomA treatment, while higher concentrations of DomA (20–100 μM) produces equivalent degrees of neuronal death when measured at these two time points (Qiu et al., 2006). Based on these findings, three EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration of exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) (Qiu et al., 2006).
</li>
</ul>
<ul>
<li>The mean concentration of DomA in rat brain samples obtained at 30 min after intraperitoneal (i.p.) administration of 1 mg/kg DA is 7.2 ng/g (Tsunekawa et al., 2013). These animals have been examined and revealed after histopathological analysis neuronal shrinkage and cell death, including an increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and after 5 days (19.0 %) compared to the controls (1.7 %) (Tsunekawa et al., 2013). In the same study, indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone (Tsunekawa et al., 2013).
</li>
</ul>
<ul>
<li>Brain slices from 8-day-old pups have been treated after 2 weeks with 10 μM DomA and assessed with propidium iodine (PI) stain to determine cellular damage (Erin and Billingsley, 2004). A time course has been carried out and viable cultures have been visualized 12, 24, 48 and 92 h after DomA treatment. Changes in PI uptake has been detected after 24 h post-treatment and at 4h the average fold-increase of PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively (Erin and Billingsley, 2004). In the same study, incubation of brain slices with DomA induces degradation of α-spectrin to the 120-kDa product after 18 h of treatment but no change has been noted after 12 h incubation, whereas caspase 3 activity results have not been conclusive (Erin and Billingsley, 2004).
</li>
</ul>
<ul>
<li>Using observations of neuronal viability and morphology, exposure of cultured murine cortical neurones to DomA for 24 h have shown to induce concentration-dependent neuronal cell death and the EC50 determined to be 75 µM (Larm et al., 1997).
</li>
</ul>
<p><br />
</p>
<table border="1" style="border-collapse:collapse;font-size:75%">
<tr>
<td align="center" style="background:#f0f0f0;"><b>Stressor</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Experimental Model</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Tested concentrations</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Exposure route</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Exposure duration</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Mitochondrial dysfunction (KE up) (measurements, quantitative if available)</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Cell death (KE down) (measurements, quantitative if available)</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>References</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Temporal Relationship</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Dose-response relationship</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Incidence</b>
</td>
<td align="center" style="background:#f0f0f0;"><b>Comments</b>
</td></tr>
<tr>
<td> DomA</td>
<td>16-month-old male ICR mice</td>
<td>2 mg/kg</td>
<td>Intraperitoneally (i.p.) </td>
<td>Once a day for 4 weeks </td>
<td>Indicators of mitochondrial function were markedly decreased (1.5-2 fold) and ROS levels were elevated (3.2 fold).</td>
<td>The mean of TUNEL positive cells in the hippocampus was increased (6 fold). The levels of bcl-2, procaspase-3 and procaspase-12 were significantly decreased and the activation of caspase-3 and caspase-12 in the mouse hippocampus were increased.</td>
<td>Lu et al., 2012</td>
<td></td>
<td>Same dose</td>
<td>Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)</td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>16-month-old male ICR mice</td>
<td>2 mg/kg</td>
<td>i.p. </td>
<td>Once a day for 4 weeks </td>
<td>ROS levels were increased (3 fold) and NOX (2 fold).</td>
<td>The mean value of TUNEL-positive cells in the hippocampal CA1 sections was elevated (8 fold) and the activation of caspase-8 and caspase-3 was increased.</td>
<td>Wu et al., 2012</td>
<td></td>
<td>Same dose</td>
<td>Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)</td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice </td>
<td>0.01 to 10 µM</td>
<td></td>
<td>Time course (15 to 120 min)</td>
<td>DomA caused a significant time- and concentration-dependent increase in ROS production. The higher ROS production (2.5 fold increase) was recorded after 1 h of exposure.</td>
<td>IC50 values for DomA were 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons based on MTT assay after 24 h of exposure.</td>
<td>Giordano et al., 2006</td>
<td>KE up (mitochondrial dysfunction) happens earlier than KE down (cell death) </td>
<td>Same doses</td>
<td></td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>CGNs from Gclm (+/+) and Gclm (−/−) mice </td>
<td>0.01 to 10 µM </td>
<td></td>
<td>Time course (0 to 180 min)</td>
<td>DomA (0.1μM) caused a 3 fold increase in DHR fluorescence, which accumulates in mitochondria and fluoresces when oxidized by ROS or reactive nitrogen species. This occurred between 1 and 2 h and was higher in CGNs from Gclm (−/−) mice.</td>
<td>0.1μM DomA was maximally effective in inducing apoptosis, while a concentration causing high toxicity (10μM) induced very limited apoptosis, 24 h after exposure.</td>
<td>Giordano et al., 2007</td>
<td>KE up (mitochondrial dysfunction) happens earlier (1-2 h) than KE down (cell death) that occurs after 24 h </td>
<td>Same doses</td>
<td></td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>CGNs from Gclm (+/+) and Gclm (−/−) mice </td>
<td>0.01 to 10 µM </td>
<td></td>
<td>For ROS: 30min, Apoptosis: 12-24 h.</td>
<td>ROS levels were measured only at the dose of 100 nM DomA 30 min after treatment in CGNs from Gclm (+/+) mice and showed 2.5 fold increase compared to controls .</td>
<td>A dose response study that showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione were more sensitive as 10 nM DomA induced a significant increase in apoptotic cells number .The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes was caused by 100 nM DomA. 1 and 10 µM DA caused significant apoptosis in both cell types but to less extend compared to 100 nM DomA. Caspase 3 activity after 12 h with prior 1 h exposure to 100 nM DomA found to be increased (2.2 fold). DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h from exposure. </td>
<td>Giordano et al., 2009</td>
<td>KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that take place 12-24 h later</td>
<td>Same dose</td>
<td>Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)</td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>Mixed cortical cultures obtained from pregnant Holtzman rats on embryonic day (ED) 16–18</td>
<td>3, 5, 10, or 50 μM</td>
<td></td>
<td>10 min, 30 min, 1 h or 2 h, after which DomA was washed out and the culture medium replaced with conditioned medium from unexposed sister cultures .</td>
<td></td>
<td>EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) .</td>
<td>Qiu et al., 2006</td>
<td></td>
<td></td>
<td></td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>Rat</td>
<td>1 mg/kg DA</td>
<td> i.p.</td>
<td></td>
<td>Indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone .</td>
<td>Neuronal shrinkage and cell drop out as well as increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and 5 days (19.0 %) has been found compared with that of controls (1.7 %) .</td>
<td>Tsunekawa et al., 2013</td>
<td></td>
<td></td>
<td></td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>Rat rain slices from 8-day-old pups </td>
<td>10 μM </td>
<td></td>
<td>Time course (12, 24, 48 and 92 h) after DomA treatment.</td>
<td></td>
<td>PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively . Degradation of α-spectrin to the 120-kDa product after 18 h of DomA treatment was noted but no change was noted after 12 h incubation, whereas caspase 3 activity results were not conclusive. </td>
<td>Erin and Billingsley, 2004</td>
<td></td>
<td></td>
<td></td>
<td>
</td></tr>
<tr>
<td> DomA</td>
<td>Cultured murine cortical neurones</td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>DomA induces concentration-dependent neuronal cell death and the EC50 determined to be 75 µM .</td>
<td>Larm et al., 1997</td>
<td></td>
<td></td>
<td></td>
<td>
</td></tr>
<tr>
<td>
</td></tr></table>
<p><br />
Gap of knowledge: there are no studies showing that GLF induces neuronal cell death through mitochondrial dysfunction.
</p><p>Rats have been administered with DA at the dose of 1.0 mg/kg for 15 days. The histochemical analysis of hippocampus from these animals has revealed no presence of apoptotic bodies and no Fluoro-Jade B positive cells (Schwarz et al., 2014).
</p><p><em>
Is it known how much change in the first event is needed to impact the second?
Are there known modulators of the response-response relationships?
Are there models or extrapolation approaches that help describe those relationships?
</em>
</p><p>The experiments describing semi-quantitative effects for this KER is described in the table above.
</p>Not SpecifiedUnspecificNot SpecifiedAll life stages<p>Neuronal necrosis has been noted in sea lions accidentally exposed to DomA (Silvagni et al., 2005) that correlated well with the histopathological findings previously reported in experimental studies (Tryphonas et al., 1990).
</p><p><br />
Cuadrado A, Nebreda AR., Mechanisms and functions of p38 MAPK signalling. Biochem J., 2010, 429(3): 403–417.
</p><p>Erin N, Billingsley ML., Domoic acid enhances Bcl-2-calcineurin-inositol-1,4,5-trisphosphate receptor interactions and delayed neuronal death in rat brain slices. Brain Res., 2004, 1014: 45-52.
</p><p>Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG., Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol., 2006, 70: 2116-2126.
</p><p>Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG., Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.
</p><p>Giordano G, Klintworth HM, Kavanagh TJ, Costa LG., Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases. Neurochem Int., 2008, 52: 1100-1105.
</p><p>Giordano G, Li L, White CC, Farin FM, Wilkerson HW, Kavanagh TJ, Costa LG., Muscarinic receptors prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells. J Neurochem., 2009, 109: 525-538.
</p><p>Harper SJ, LoGrasso P., Signalling for survival and death in neurones: the role of stress-activated kinases. JNK and p38. Cell Signal., 2001, 13(5): 299–310.
</p><p>Larm JA, Beart PM, Cheung NS., Neurotoxin domoic acid produces cytotoxicity via kainate- and AMPA-sensitive receptors in cultured cortical neurones. Neurochem Int., 1997, 31: 677-682.
</p><p>Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P., Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med., 1997, 185: 1481−1486.
</p><p>Lu J, Wu DM, Zheng YL, Hu B, Zhang ZF., Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis. Brain Pathol., 2010, 20: 598-612.
</p><p>Lu J, Wu DM, Zheng ZH, Zheng YL, Hu B, Zhang ZF., Troxerutin protects against high cholesterol-induced cognitive deficits in mice. Brain., 2011, 134: 783-797.
</p><p>Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF., Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52(3): 646-59.
</p><p>Lu TH, Su CC, Tang FC, Chen CH, Yen CC, Fang KM, Lee KL, Hung DZ, Chen YW., Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway. Chem Biol Interact., 2014, 225: 1-12.
</p><p>Nicotera P, Leist M, Ferrando-May E., Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicol Lett., 1998, 102-103: 139-142.
</p><p>Qiu S, Pak CW, Currás-Collazo MC., Sequential involvement of distinct glutamate receptors in domoic acid-induced neurotoxicity in rat mixed cortical cultures: effect of multiple dose/duration paradigms, chronological age, and repeated exposure. Toxicol Sci., 2006, 89: 243-256.
</p><p>Richter C, Schweizer M, Cossarizza A, Franceschi C. Control of apoptosis by the cellular ATP level. FEBS Lett., 1996, 378: 107-110.
</p><p>Schwarz M, Jandová K, Struk I, Marešová D, Pokorný J, Riljak V. Low dose domoic acid influences spontaneous behavior in adult rats. Physiol Res., 2014, 63: 369-76.
</p><p>Silvagni PA, Lowenstine LJ, Spraker T, Lipscomb TP, Gulland FMD., Pathology of Domoic Acid Toxicity in California Sea Lions (Zalophus californianus). Vet Path., 2005, 42: 184-191.
</p><p>Tryphonas L, Truelove J, Iverson F, Todd EC, Nera EA. Neuropathology of experimental domoic acid poisoning in non-human primates and rats. Can Dis Wkly Rep. 1990 Sep;16 Suppl 1E:75-81.
</p><p>Tsunekawa K, Kondo F, Okada T, Feng GG, Huang L, Ishikawa N, Okada S., Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation. BMC Neurosci., 2013, 14: 4-16.
</p><p>Wu DM, Lu J, Zheng YL, Zhang YQ, Hu B, Cheng W, Zhang ZF, Li MQ., Small interfering RNA-mediated knockdown of protein kinase C zeta attenuates domoic acid-induced cognitive deficits in mice. Toxicol Sci., 2012, 128: 209-222.
</p><p>Zhou Q, Liu C, Liu W, Zhang H, Zhang R, Liu J, Zhang J, Xu C, Liu L, Huang S, Chen L., Rotenone induction of hydrogen peroxide inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, leading to neuronal apoptosis. Toxicol Sci., 2015, 143: 81-96.
</p>2016-11-29T18:41:332016-11-29T20:08:104f8c9fd6-ed55-4019-8287-e45ac29ddde88367d882-24f4-4763-a5d9-999a7d5b4bfc<p>The pioneering work of Kreutzberg and coworkers (1995, 1996) has shown that neuronal injury leads to neuroinflammation, with microglia and astrocyte reactivities. Several chemokines and chemokines receptors (fraktalkine, CD200) control the neuron-microglia interactions, and a loss of this control can trigger microglial reactivity (Blank and Prinz, 2013; Chapman et al., 2000; Streit et al., 2001). Upon injury causing neuronal death (mainly necrotic), signals termed Damage-Associated Molecular Patterns (DAMPs) are released by damaged neurons and promote microglial reactivity (Marin-Teva et al., 2011; Katsumoto et al., 2014). Toll-like receptors (TLRs) are pattern-recognition receptors that recognize specific pathogen- and danger-associated molecular signatures (PAMPs and DAMPs) and subsequently initiate inflammatory and immune responses. Microglial cells express TLRs, mainly TLR-2, which can detect neuronal cell death (for review, see Hayward and Lee, 2014). TLR-2 functions as a master sentry receptor to detect neuronal death and tissue damage in many different neurological conditions including nerve trans-section injury, traumatic brain injury and hippocampal excitotoxicity (Hayward and Lee, 2014). Astrocytes, the other cellular mediator of neuroinflammation (Ranshoff and Brown, 2012) are also able to sense tissue injury via TLR-3 (Farina et al., 2007; Rossi, 2015).</p>
<p>It is widely accepted that cell/neuronal injury and death lead to neuroinflammation (microglial and astrocyte reactivities) in adult brain. In the developing brain, neuroinflammation was observed after neurodegeneration induced by excitotoxic lesions (Acarin et al., 1997; Dommergues et al., 2003) or after ethanol exposure (Tiwari et al., 2012; Ahmad et al., 2016). It is important to note that physiological activation of microglial cells is observed during normal brain development for removal of apoptotic debris (Ashwell 1990, 1991). But exposure to toxicant (ethanol), excitotoxic insults (kainic acid) or traumatic brain injury during development can also induce apoptosis in hippocampus and cerebral cortex, as measured either by TUNEL, BID or caspase 3 upregulation associated to an inflammatory response, as evidenced by increased level of pro- inflammatory cytokines IL-1b, TNF-a, of NO, of p65 NF-kB or of the marker of astrogliosis, glial fibrillary acidic protein (GFAP), suggesting that, during brain development, neuroinflammation can also be triggerred by apoptosis induced by several types of insult (Tiwari and Chopra, 2012; Baratz et al., 2015; Mesuret et al., 2014).</p>
<p><em>Include consideration of temporal concordance here </em></p>
<p>Pb</p>
<p>In 3D cultures prepared from fetal rat brain cells exposed to Pb (10<sup>-6</sup> - 10<sup>-4</sup> M for 10 days), Pb-induced neuronal death was evidenced by a decrease of cholinergic and GABAergic markers associated to a decrease in protein content, and was accompanied by microglial and astrocyte reactivities (Zurich et al., 2002). These effects were more pronounced in immature than in differentiated cultures (Zurich et al., 2002). In adult rats, exposure to 100 ppm of Pb for 8 weeks caused neuronal death, evidenced by an increase in apoptosis (TUNEL) that was associated with microglial reactivity and an increase in IL-1b, TNF-a and i-NOS expression (Liu et al., 2012). Acute exposure to Pb (25 mg/kg, ip, for 3 days) increased GFAP and glutamate synthetase expression with impaiment of glutamate uptake and probable neuronal injury (Struzynska, 2000; Struzynska et al., 2001).</p>
<p>It is interesting to note that glial cells and in particular astrocytes are able to accumulate lead, suggesting that thes cells may be also a primary target of lead neurotoxic effects (Zurich et al., 1998; Lindhal et al., 1999). </p>
<p>Domoic acid</p>
<ul>
<li>Astrogliosis is one of the histopathological findings revealed by the assessment of brains derived from patients diagnosed with Amnesic Shellfish Poisoning (ASP) (reviewed in Pulido, 2008). In a reference study, where the brain of a patient after acute <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">domoic acid (kainic acid-type neurotoxin that causes amnesic shellfish poisoning) </span></span>intoxication has been examined in great detail gliosis has been detected in the overlying cortex, dorsal and ventral septal nuclei, the secondary olfactory areas and the nucleus accumbens (Cendes et al., 1995). Reactive astrogliosis has also been confirmed in the sixth cortical layer and subjacent white matter in the orbital and lateral basal areas, the first and second temporal gyri, the fusiform gyrus, the parietal parasagittal cortex, and the insula (Cendes et al., 1995).</li>
</ul>
<ul>
<li>Adult rats have been assessed seven days after the administration of DomA (2.25 mg/kg i.p.) and revealed astrocytosis identified by glial fibrillary acidic protein (GFAP)-immunostaining and activation of microglia by GSI-B4 histochemistry (Appel et al., 1997). More investigators have suggested that DomA can activate microglia (Ananth et al., 2001; Chandrasekaran et al., 2004).</li>
</ul>
<ul>
<li>DomA treatment (2 mg/kg once a day for 3 weeks) in mice significantly stimulates the expression of inflammatory mediators, including IL-1β (1.7 fold increase), TNF-α (2 fold increase), GFAP (1.4 fold increase), Cox-2 (3 fold increase), and iNOS (1.6 fold increase) compared to controls (Lu et al, 2013).</li>
</ul>
<ul>
<li>Adult female and male mice have been injected i.p. with 4mg/kg (LD50) of DomA and Real-time PCR has been performed in the brain derived at 30, 60 and 240 min post-injection. The inflammatory response element cyclooxygenase 2 (COX-2) has been found to be 8 fold increased at the 30 and 60 min time points and then showed a descent back toward basal expression levels by 240 min (Ryan et al., 2005).</li>
</ul>
<ul>
<li>Adult male rats treated with 2 mg/kg DomA i.p. have been sacrificed after 3 or 7 d and shown that GFAP and lectin staining could identify regions of reactive gliosis within areas of neurodegeneration however observed at higher magnifications compared to the ones used for neurodegeneration (Appel et al., 1997; Scallet et al., 2005).</li>
</ul>
<ul>
<li>At 5 days and 3 months following DomA administration of male Wistar rats, a large number of OX-42 positive microglial cells exhibiting intense immunoreactivity in CA1 and CA3 regions of the hippocampus have been detected. With an antibody against GFAP, immunoreactive astrocytes have been found to be sparsely distributed in the hippocampus derived from DomA treated rats after 3 months' time interval (Ananth et al., 2003). At 5 days after the administration of DomA, GFAP positive astrocytes have been found increased in the hippocampus (Ananth et al., 2003).</li>
</ul>
<p> </p>
<p><strong>Mercury</strong></p>
<p>Young mice receiving a fish diet (MeHgCl) for 3 months exhibited in cortex a decrease of the chemokine Ccl<sub>2</sub> and neuronal death, as measured by a decrease in cell density, as well as microglial reactivity (increase in Iba1-labelled cells) (<strong>Godefroy et al., 2012</strong>)</p>
<p>Perinatal exposure to MeHgCl (GD7-PD21, 0.5 mg/kg bw/day in drinking water) lead to a delayed decrease (PD 36) of cholinergic muscarinic receptors in cerebellum accompanied by astrogliosis (<strong>Roda et al., 2008</strong>).</p>
<p>Immature rat brain cell cultures maintained in 3D conditions were exposed to either MeHgCl or HgCl<sub>2</sub> (10<sup>-9</sup> – 10<sup>-6</sup> M, for 10 days). This treatment caused microglial and astrocyte activation without neuronal death, but a reversible decrease of the expression of the neuronal marker MAP2 (<strong>Monnet-Tschudi et al., 1996 ; Eskes et al., 2002</strong>).</p>
<p>Adult marmoset exposed acutely to 5 mg Hg/kg/day p.o. exhibited apoptosis in occipital cortex, as well as glial reactivity (GFAP and Iba1 increased). Mercury content in occipital cortex was 31 mg/g (<strong>Yamamoto et al., 2012</strong>).</p>
<p>Monkeys exposed to MeHgCl (50 mg/bw for 6,12,18 months) showed microglial and astrocyte activation without any change in neuronal number. Both astrocyte and microglia accumulated elevated levels of inorganic mercury, suggesting a direct effect of mercury on glial cells (<strong>Charleston et al., 1996</strong>).</p>
<p>Human LUHMES cells as model of dopaminergic neurons and the human astrocyte cell line CFF-STTG1 were exposed to MeHgCl (0.25 -5 mM), thiomersal (0.25 – 5 mM) or HgCl2 (5-35 mM), what affected their cell viability. Neurons were much more sensitive than astrocytes (<strong>Lohren et al., 2015</strong>).</p>
<p>A direct activation of rat primary microglial cells and astrocytes was observed after exposure to MeHgCl (10<sup>-10</sup>-10<sup>-6</sup> M, for 5 days). (<strong>Eskes et al., 2002</strong>).</p>
<p>Astrocyte + microglia in co-cultures exposed to mercury (1-5 mM for 30 min to 6 days) showed lower levels of GSH in microglia than in astrocytes (<strong>Ni et al., 2011 ; 2012</strong>).</p>
<p>Human primary astrocyte cell line exposed to MeHgCl (1.125 mM) for 24h and 72h did not exhibit an increase of GFAP, but of NfkB after the 72h (<strong>Malfa et al., 2014</strong>).</p>
<p> </p>
<p><em>Sex dependency</em></p>
<p>In prairie voles 10 weeks exposure to 600 ppm HgCl<sub>2</sub> in drinking water lead to an increase of TNF-a in hippocampus of male, but not in female (<strong>Curtis et al., 2011</strong>).</p>
<p> </p>
<p><strong>Acrylamide</strong> (acrylamide is a common food contaminant generated by heat processing)</p>
<p>Adult mice received 10, 20, 30 mg/kg bw for 4 weeks. The dose of 20 mg/kg bw caused neurological symptoms (ex. cognitive impairment) associated to an increased oxidative stress, a decrease of GSH and glial reactivity (GFAP and Iba1 increased) in cortex, hippocampus and striatum. An increase in TNF-a, IL-1b and i-NOS expression in all 3 brain regions was also observed. (<strong>Santhanasabepathy et al., 2015</strong>)</p>
<p>Isolated and/or co-cultures of microglial cells or astrocytes treated with acrylamide 0-1mM for 24-96h exhibited an increased release of TNF-a, IL-1b, IL-6 and G-CSF, suggesting a direct effect of acrylamide on glial cells (<strong>Zhao et al., 2017a,b,c</strong>).</p>
<p>Neonatal rat astrocytes treated with acrylamide (0.1-1mM) for 7, 11, 15, or 20 days increased their proliferation rate as measured by PCNA staining. Astrocyte proliferation is also a sign of reactivity. (<strong>Aschner et al., 2005)</strong>.</p>
<p> </p>
<p>Pb</p>
<p>It is interesting to note that glial cells and in particular astrocytes are able to accumulate lead, suggesting that thes cells may be also a primary target of lead neurotoxic effects (Zurich et al., 1998; Lindhal et al., 1999). </p>
<p>Sobin and coworkers (2013) described a Pb-induced decrease in dentate gyrus volume associated with microglial reactivity at low dose of Pb (30 ppm), but not at high doses (330 ppm), plausibly due to the death of microglial cells at the high dose of Pb.</p>
<p>Pb decreased IL-6 secretion by isolated astrocytes (Qian et al., 2007). Such a decrease was also observed in isolated astrocytes treated with methylmercury, and was reverted in microglia astrocyte co-cultures, suggesting that cell-cell interactions can modify the response to a toxicant and that cultures of a single cell type may not be representative of the organ toxicity (Eskes et al., 2002). </p>
<p><br />
Domoic acid</p>
<p>Adult male and female Sprague Dawley rats have received a single intraperitoneal (i.p.) injection of DomA (0, 1.0, 1.8 mg/kg) and have been sacrificed 3 h after the treatment. Histopathological analysis of these animals has shown no alterations for GFAP immunostaining in the dorsal hippocampus and olfactory bulb, indicating absence of reactive gliosis (Baron et al., 2013).</p>
<p>The exposed zebrafish from the 36-week treatment with DomA showed no neuroinflammation in brain (Hiolski et al., 2014). At the same time, microarray analysis revealed no significant changes in <em>gfap</em> gene expression, a marker of neuroinflammation and astrocyte activation (Hiolski et al., 2014).</p>
<p><strong>Mercury</strong></p>
<p>Mouse developmental exposure to 50 mM of HgCl<sub>2</sub> in maternal drinking water from GD8 to PD21 did not induce any change in GM-CSF, IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70. IL-13, IL-17, MCP1, MIP2 and TNF-a measured by Luminex in brain slices of PD21 and PD70. No sex differences, but brain increase of IgG and increased sociability in females (Zhang et al., 2012).</p>
<p>3D rat brain cell cultures treated for 10 days with HgCl2 or MeHgCl (10-10 - 10-6 M) exhibited increased apotosis measured by TUNEL, but exclusively in immature cultures. The proportion of cells undergoing apoptotis was highest for astrocytes than for neurons. But the apoptotic nuclei were not associated with reactive microglial cells as evidenced by double staining (Monnet-Tschudi, 1998).</p>
<p><strong>Acrylamide</strong></p>
<p>A 2 weeks exposure to acrylamide in drinking water (44mg/kg/day) induced behavioral effects, such a decreased in locomotor activity, but with no effect at gene level on neuronal and inflammatory markers analyzed in somatosensory and motor cortex (Bowyer et al., 2009).</p>
<p><em>Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? </em></p>
<p>Quantitative evalutation of this KER does not exist (gap of knowledge).</p>
Not SpecifiedMaleNot SpecifiedFemaleHighAll life stagesHighHighHighHigh<p>California sea lions that have been exposed to the marine biotoxin DomA developed an acute or chronic toxicosis marked by seizures, whereas histopathological analysis revealed neuroinflammation characterised by gliosis (Kirkley et al., 2014).</p>
<p>Neuroinflammation has been described in mammals (rat, mouse, monkey, human).</p>
<p>Acarin L, González B, Castellano B, Castro AJ. 1997. Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain. ExpNeurol 147: 410-417.</p>
<p>Ahmad A, Shah SA, Badshah H, Kim MJ, Ali T, Yoon GH, et al. 2016. Neuroprotection by Vitamin C Against Ethanol-Induced Neuroinflammation Associated Neurodegeneration in the Developing Rat Brain. CNS Neurol Disord Drug Targets 15(3): 360-370.</p>
<p>Ananth C, Thameem DS, Gopalakrishnakone P, Kaur C. Domoic acid-induced neuronal damage in the rat hippocampus: changes in apoptosis related genes (bcl-2, bax, caspase-3) and microglial response. J Neurosci Res., 2001, 66: 177-190.</p>
<p>Ananth C, Gopalakrishnakone P, Kaur C. Induction of inducible nitric oxide synthase expression in activated microglia following domoic acid (DA)-induced neurotoxicity in the rat hippocampus. Neurosci Lett., 2003, 338: 49-52.</p>
<p>Appel NM, Rapoport SI, O’Callaghan JP, Bell JM, Freed LM. Sequelae of parenteral domoic acid administration in rats: comparison of effects on different metabolic markers in brain. Brain Res., 1997, 754: 55-64.</p>
<p>Aschner, M., Wu, Q., Friedman, M.A., 2005. Effects of acrylamide on primary neonatal rat astrocyte functions. Ann N Y Acad Sci. 1053<strong>,</strong> 444-54.</p>
<p>Ashwell K. 1990. Microglia and cell death in the developing mouse cerebellum. DevBrain Res 55: 219-230.</p>
<p>Ashwell K. 1991. The distribution of microglia and cell death in the fetal rat forebrain. DevBrain Res 58: 1-12.</p>
<p>Baratz R, Tweedie D, Wang JY, Rubovitch V, Luo W, Hoffer BJ, et al. 2015. Transiently lowering tumor necrosis factor-alpha synthesis ameliorates neuronal cell loss and cognitive impairments induced by minimal traumatic brain injury in mice. J Neuroinflammation 12: 45.</p>
<p>Baron AW, Rushton SP, Rens N, Morris CM, Blain PG, Judge SJ. Sex differences in effects of low level domoic acid exposure. Neurotoxicology, 2013, 34: 1-8.</p>
<p>Blank T, Prinz M. Microglia as modulators of cognition and neuropsychiatric disorders. Glia, 2013, 61: 62-70.</p>
<p>Bowyer, J.F., et al., 2009. The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water. Toxicol Appl Pharmacol. 240<strong>,</strong> 401-11.</p>
<p>Cendes F, Andermann F, Carpenter S, Zatorre RJ, Cashman NR. Temporal lobe epilepsy caused by domoic acid intoxication: evidence for glutamate receptor-mediated excitotoxicity in humans. Ann Neurol., 1995, 37: 123-126.</p>
<p>Chandrasekaran A, Ponnambalam G, Kaur C. Domoic acid-induced neurotoxicity in the hippocampus of adult rats. Neurotox Res., 2004, 6:1 05-117.</p>
<p>Chapman GA, Moores K, Harrison D, Campbell CA, Stewart BR, Strijbos PJLM. Fractalkine Cleavage from Neuronal Membrans Represents an Acute Event in Inflammatory Response to Excitotoxic Brain Damage. J Neurosc., 2000, 20 RC87: 1-5.</p>
<p>Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM: Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. <em>NeuroToxicology </em>1996, <strong>17:</strong>127-138.</p>
<p>Curtis, TJ., et al., 2011. Chronic inorganic mercury exposure induces sex-specific changes in central TNFalpha expression: importance in autism? Neurosci Lett. 504<strong>,</strong> 40-4.</p>
<p>Dommergues MA, Plaisant F, Verney C, Gressens P. 2003. Early microglial activation following neonatal excitotoxic brain damage in mice: a potential target for neuroprotection. Neuroscience 121(3): 619-628.</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Farina C, Aloisi F, Meinl E. Astrocytes are active players in cerebral innate immunity. Trends Immunol, 2007, 28(3): 138-145.</p>
<p>Godefroy, D., et al., 2012. The chemokine CCL2 protects against methylmercury neurotoxicity. Toxicol Sci. 125<strong>,</strong> 209-18.</p>
<p>Hayward JH, Lee SJ. A Decade of Research on TLR2 Discovering Its Pivotal Role in Glial Activation and Neuroinflammation in Neurodegenerative Diseases. Experimental Neurobiology, 2014, 23(2): 138-147.</p>
<p>Hiolski EM, Kendrick PS, Frame ER, Myers MS, Bammler TK, Beyer RP, Farin FM, Wilkerson HW, Smith DR, Marcinek DJ, Lefebvre KA., Chronic low-level domoic acid exposure alters gene transcription and impairs mitochondrial function in the CNS. Aquat Toxicol., 2014, 155: 151-159.</p>
<p>Katsumoto A, Lu H, Miranda AS, Ransohoff RM. Ontogeny and functions of central nervous system macrophages. J Immunol., 2014, 193(6): 2615-2621.</p>
<p>Kirkley KS, Madl JE, Duncan C, Gulland FM, Tjalkens RB. Domoic acid-induced seizures in California sea lions (Zalophus californianus) are associated with neuroinflammatory brain injury. Aquat Toxicol., 2014, 156C: 259-268.</p>
<p>Kreutzberg GW. Microglia, the first line of defence in brain pathologies. Arzneimttelforsch, 1995, 45: 357-360.</p>
<p>Kreutzberg GW. Microglia : a sensor for pathological events in the CNS. Trends Neurosci., 1996, 19: 312-318.</p>
<p>Lindhal LS, Bird L, Legare ME, Mikeska G, Bratton GR, Tiffany-Castiglioni E. 1999. Differential ability of astroglia and neuronal cells to accumulate lead: Dependence on cell type and on degree of differentiation. ToxSci 50: 236-243.</p>
<p>Liu MC, Liu XQ, Wang W, Shen XF, Che HL, Guo YY, et al., Involvement of microglia activation in the lead induced long-term potentiation impairment. PLoS One, 2012, 7(8): e43924.</p>
<p>Lohren, H., et al., 2015. Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes. J Trace Elem Med Biol. 32<strong>,</strong> 200-8.</p>
<p>Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ. Troxerutin counteracts domoic acid-induced memory deficits in mice by inhibiting CCAAT/enhancer binding protein β-mediated inflammatory response and oxidative stress. J Immunol., 2013, 190: 3466-3479.</p>
<p>Malfa, G.A., et al., 2014. "Reactive" response evaluation of primary human astrocytes after methylmercury exposure. J Neurosci Res. 92<strong>,</strong> 95-103.</p>
<p>Marin-Teva JL, Cuadros MA, Martin-Oliva D, Navascues J., Microglia and neuronal cell death. Neuron glia biology, 2011, 7(1): 25-40.</p>
<p>Mesuret G, Engel T, Hessel EV, Sanz-Rodriguez A, Jimenez-Pacheco A, Miras-Portugal MT, et al. 2014. P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage. CNS neuroscience & therapeutics 20(6): 556-564.</p>
<p>Monnet-Tschudi F, Zurich MG, Honegger P (1996) Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon. Brain Res 741:52-59</p>
<p>Monnet-Tschudi F (1998) Induction of apoptosis by Mercury Compounds depends on maturation and is not associated with microglial activation. JNeurosciRes 53:361-367</p>
<p>Ni, M., et al., 2011. Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity. Glia. 59<strong>,</strong> 810-20.</p>
<p>Ni, M., et al., 2012. Glia and methylmercury neurotoxicity. J Toxicol Environ Health A. 75<strong>,</strong> 1091-101.</p>
<p>Pulido OM. Domoic acid toxicologic pathology: a review. Mar Drugs, 2008, 6: 180-219.</p>
<p>Qian Y, Zheng Y, Weber D, Tiffany-Castiglioni E. 2007. A 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes. American journal of physiology Cell physiology 293(3): C897-905.</p>
<p>Ransohoff RM, Brown MA. Innate immunity in the central nervous system. J Clin Invest., 2012, 122(4): 1164-1171.</p>
<p>Roda, E., et al., 2008. Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat. J Chem Neuroanat. 35<strong>,</strong> 285-94.</p>
<p>Rossi D. Astrocyte physiopathology: At the crossroads of intercellular networking, inflammation and cell death. Prog Neurobiol., 2015, 130: 86-120.</p>
<p>Ryan JC, Morey JS, Ramsdell JS, Van Dolah FM. Acute phase gene expression in mice exposed to the marine neurotoxin domoic acid. Neuroscience, 2005, 136: 1121-1132.</p>
<p>Santhanasabapathy, R., et al., 2015. Farnesol quells oxidative stress, reactive gliosis and inflammation during acrylamide-induced neurotoxicity: Behavioral and biochemical evidence. Neuroscience. 308<strong>,</strong> 212-27.</p>
<p>Scallet AC, Schmued LC, Johannessen JN. Neurohistochemical biomarkers of the marine neurotoxicant, domoic acid. Neurotoxicol Teratol., 2005, 27: 745-752.</p>
<p>Sobin C, Montoya MG, Parisi N, Schaub T, Cervantes M, Armijos RX. 2013. Microglial disruption in young mice with early chronic lead exposure. Toxicol Lett 220(1): 44-52.</p>
<p>Streit WJ, Conde J, Harrison JK. Chemokines and Alzheimer's disease. Neurobiol Aging., 2001, 22: 909-913.</p>
<p>Struzynska L. 2000. The protective role of astroglia in the early period of experimental lead toxicity in the rat. Acta Neurobiol Exp (Wars) 60(2): 167-173.</p>
<p>Struzynska L, Bubko I, Walski M, Rafalowska U. 2001. Astroglial reaction during the early phase of acute lead toxicity in the adult rat brain. Toxicology 165: 121-131.</p>
<p>Tiwari V, Chopra K. 2012. Attenuation of oxidative stress, neuroinflammation, and apoptosis by curcumin prevents cognitive deficits in rats postnatally exposed to ethanol. Psychopharmacology (Berl) 224(4): 519-535.</p>
<p>Wang, Y.T., et al., 2017. Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of alpha-synuclein aggregation and programmed cell death. Sci Rep. 7<strong>,</strong> 45741.</p>
<p>Yamamoto, M., et al., 2012. Increased expression of aquaporin-4 with methylmercury exposure in the brain of the common marmoset. J Toxicol Sci. 37<strong>,</strong> 749-63.</p>
<p>Zhang, Y., Bolivar, V.J., Lawrence, D.A., 2012. Developmental exposure to mercury chloride does not impair social behavior of C57BL/6 x BTBR F(1) mice. J Immunotoxicol. 9<strong>,</strong> 401-10.</p>
<p>Zhao, M., et al., 2017a. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model. Toxicol In Vitro. 39<strong>,</strong> 119-125.</p>
<p>Zhao, M., et al., 2017b. Acrylamide-induced neurotoxicity in primary astrocytes and microglia: Roles of the Nrf2-ARE and NF-kappaB pathways. Food Chem Toxicol. 106<strong>,</strong> 25-35.</p>
<p>Zhao, W.Z., et al., 2017c. Neuroprotective Effects of Baicalein on Acrolein-induced Neurotoxicity in the Nigrostriatal Dopaminergic System of Rat Brain. Mol Neurobiol.</p>
<p>Zurich MG, Monnet-Tschudi F, Berode M, Honegger P. 1998. Lead acetate toxicity in vitro: Dependence on the cell composition of the cultures. Toxicol In Vitro 12(2): 191-196.</p>
<p>Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.</p>
2016-11-29T18:41:332022-07-15T08:26:25d70d314e-a83f-4253-bdc5-d65b49b8821f61e734b0-62b8-4d8b-b3e4-17e28f2e9621<p>It is well established in the existing literature that NMDA receptor–dependent synaptic potentiation (LTP) and depression (LTD) are two forms of activity directly linked to long-term changes in synaptic efficacy and plasticity, the fundamental processes underlying learning and memory. The best characterized form of LTP occurs in the CA3-CA1 region of the hippocampus, in which LTP is initiated by transient activation of NMDARs that leads to a persistent increase in synaptic transmission through AMPA receptors (Benke et al., 1998) that can be achieved either through increasing the number of AMPA receptors at the post-synaptic surface or by increasing the single channel conductance of the receptors expressed. It has been shown that LTP in the CA1 region of the hippocampus could be accounted for by these two mechanisms (Benke et al 1998).
The degree of activity of NMDARs is determined in part by extracellular Mg(2+) and by the co-agonists for this receptor, glycine and D-serine. During strong stimulation, a relief of the voltage-dependent block of NMDARs by Mg(2+) provides a positive feedback for NMDAR Ca(2+) influx into postsynaptic CA1 spines. The induction of LTP at CA3-CA1 synapses requires further signal amplification of NMDAR activity. Src family kinases (SFKs) play a "core" role in the induction of LTP by enhancing the function and expression of NMDARs. At CA3-CA1 synapses, NMDARs are largely composed of NR1 (NMDA receptor subunit 1)-NR2A or NR1-NR2B containing subunits. Recent, but controversial, evidence has correlated NR1-NR2A receptors with the induction of LTP and NR1-NR2B receptors with LTD. However, LTP can be induced by activation of either subtype of NMDAR and the ratio of NR2A:NR2B receptors has been proposed as an alternative determinant of the direction of synaptic plasticity. Many transmitters and signal pathways can modify NMDAR function and expression and, for a given stimulus strength, they can potentially lead to a change in the balance between LTP and LTD (MacDonald et al., 2006).
</p><p>Mammalian learning and memory is one of the outcomes of the functional expression of neurons connected into neural networks. Neuronal damage or cell death induced by chemical compounds disrupts integration and transmission of information through neural networks thereby setting the stage for subsequent impairment of learning and memory. Exposure to chemicals that will increase the risk of functional neuronal network damage lead to learning and memory impairment.
</p><p>Long-term potentiation (LTP) is a long-lasting increase in synaptic efficacy after high-frequency stimulation of afferent fibers, and its discovery
potentiated the idea that individual synapses possess the properties expected for learning and memory (reviewed in Lynch et al., 2014). Moreover, LTP
is intimately related to the theta rhythm, an oscillation long-associated with learning. Learning-induced enhancement in neuronal excitability, a
measurement of neural network function, has also been shown in hippocampal neurons following classical conditioning in several experimental approaches (reviewed in Saar and Barkai, 2003).
On the other hand, memory requires the increase in magnitude of EPSCs to be developed quickly and to be persistent for a at least a few weeks without
disturbing already potentiated contacts. Once again, a substantial body of evidence have demonstrated that tight connection between LTP and diverse
instances of memory exist (reviewed in Lynch et al., 2014).
</p><p>The recent studies suggest that NMDA receptor-dependent long-term depression of both LTD and LTP is usually accompanied by morphological changes in spines. LTD is characterized by long lasting dendritic spine shrinkage and reduced F-actin polymerization, in addition to reduced numbers of synaptic AMPA receptors. Moreover, the actin binding protein cofilin has been implicated in mediating such synaptic structural plasticity (Chen et al., 2007). If sustained, such LTD-changes in hippocampus or cortex, triggered by NMDARs overactivation could lead to synaptic dysfunction, contributing to learning and memory damage (Calabrese et al., 2014).
</p><p><em>
Include consideration of temporal concordance here
</em>
</p><p>A series of important findings support that the biochemical changes that happen after induction of LTP also occur during memory acquisition, showing
temporality between the two KEs (reviewed in Lynch et al., 2014).
Furthermore, a review of Morris water maze (MWM)data as a tool to investigate spatial learning and memory in laboratory rats also pointed to the
disconnection between neuronal networks rather than brain damage of certain regions is responsible for the impairment of MWM performance(D'Hooge and De Deyn, 2001). Functional integrated neural networks that involve the coordination action of different brain regions are consequently important for spatial learning and MWM performance.
Morris et al. 1986 found that blocking the NMDA receptor with AP5 inhibits spatial learning in rats. More importantly, in the same study they measured
brain electrical activity and recorded that this agent also inhibits LTP, however, they did not prove that spatial learning and LTP inhibition are
causally related (Morris et al., 1986). Since then a number of NMDA receptor antagonists have been studied for their ability to induce impairment
of learning and memory. It is worth mentioning that similar findings have been found in human subjects, where by combining behavioural and electrophysiological data from patients with temporal lobe epilepsy exposed to ketamine, the involvement of NMDA receptors in human memory processes was demonstrated (Grunwald et al., 1999).
</p><p><b>Domoic Acid (DomA):</b>
Mice exposed to DomA (2.0 mg/kg) showed impairment of the acquisition of the place task in the Morris water maze (Petrie et al., 1992). These animals also failed to select the appropriate problem-solving strategies in their attempt to search for the underwater platform (longer escape latencies than controls) (Petrie et al., 1992). This impairment of acquisition and retention of this spatial navigation task by DomA, was associated with neuronal damage not only in the hippocampus, but also in limbic brain regions (Petrie et al., 1992).
Similar results were obtained in a different study that also utilized the Morris water maze but in rats (Kuhlmann and Guilarte, 1997). These animals
received DomA (1.5 or 3.0 mg/kg i.p.) and exhibited significant learning deficits while animals treated with a lower dose (0.75 mg/kg) showed no deficits.
By incorporating in the test a visual cue trial (a black flag was positioned over the goal platform to eliminate the learning and memory components), it was possible to rule out sensory and motor deficits in the treated animals unable to solve the task (Kuhlmann and Guilarte, 1997).
</p><p>Rats were examined using a neurobehavioral test battery (passive avoidance, auditory startle and conditioned avoidance) after DomA treatment with 0, 0.22, 0.65, or 1.32 mg/kg i.p. that caused hardly any measurable brain injury (Sobotka et al., 1996). Approximately 25% of the animals that received the higher dose (1.32 mg/kg) DomA died or were euthanized. Surviving animals were assessed three days after exposure and showed changes on the auditory startle test (Sobotka et al., 1996). This effect was limited to exaggerated startle responses as measured by mean-response amplitude changes and did not include changes in habituation, suggesting the presence of behavioural hyper-reactivity rather than memory impairment. No change on the other two measures of avoidance learning was detected for all DomA treated groups compared to controls (Sobotka et al., 1996).
</p><p>In an experimental approach aiming to evaluate single versus repeated doses of DomA on learning and memory, adult mice were treated with single or four i.p. injections of 1.0 or 2.0 mg/kg over a seven day period. All animals were tested on a spatial delayed matching-to-sample test in Morris water maze (Clayton et al., 1999). Animals given a single dose of 2.0 mg/kg DomA performed more poorly than controls on “nonalternation” test days; sessions in which the correct response was the same as the preceding day (Clayton et al., 1999). This finding implies that DomA-treated animals had difficulty recalling information after a 24 h delay period and were exhibiting behavioural signs similar to human antegrade amnesia. Animals exposed to multiple doses displayed initially greater general symptomatology but after recovery, did not show greater cognitive impairment than subjects treated
with a single injection (Clayton et al., 1999).
</p><p>In the only study where DomA i.v. administration was tested in relation to impairment of learning and memory, rats were dosed with 0.04 μg DomA/kg (and
diazepam, ip). Radial arm maze tests revealed severe learning impairment, suggesting deficits in working memory. In subjects that were able to ultimately solve the maze, performance deficits were documented on re-learning the same task. The memory-based deficits observed in these animals are similar to human antegrade amnesia that has been reported after DomA exposure (Nakajima and Potvin, 1992).
In humans, the hallmark of DomA-induced neurotoxicity is the rather dramatic disruption of memory processing. Clinical evaluation of 14 adult
human subjects poisoned by DomA (after the outbreak in Canada) revealed that the majority of them (12/14) had severe antegrade memory deficits with
relative preservation of higher cortical functions, since the patients were unable to remember events that occurred after DomA intoxication and had difficulty
recalling new information (reviewed in Pulido, 2008; Grant et al., 2010).
</p><p>In a separate clinical report of one case, a patient that received treatment at
the hospital for DomA poisoning showed memory impairment which was resolved after three weeks (reviewed in Pulido, 2008; Grant et al., 2010). Other
symptoms of the syndrome include nausea, vomiting, abdominal cramps, diarrhoea, headache, unstable blood pressure, cardiac arrhythmias and
neurological dysfunction, including coma and seizures (reviewed in Pulido, 2008; Grant et al., 2010).
</p><p>Treatment of female Sprague-Dawley rats with 0, 1, 2, 4, or 7.5 mg domoic acid/kg of body weight for 24 h caused neurobehavioural changes at doses above 4 mg/kg of body weight characterised by unequivocal behavioural and neurological signs leading to partial seizures and status epilepticus(Tryphonas et al., 1990).Similar effects were observed in other in vivio studies ( Fuquay et al., 2012; Muha and Ramsdell, 2011).
</p><p>The exposed zebrafish from the 36-week treatment with DomA showed no visible signs of neurobehavioral excitotoxicity (i.e.circle- or spiral-swimming) when observed over a 30–45 min period following each injection (Hiolski et al., 2014).
The latencies measured during training sessions did not differ among treated and not treated animals but the step-through latency in the 24 h retention trial was significantly lowered in DomA-exposed mice (2 mg/kg once a day for 3 weeks) (Lu et al., 2012). DomA-treated mice had longer escape latencies compared to controls in Morris water maze. The same group reported that DomA-treated mice (2 mg/kg once a day for 3 weeks)compared to controls showed a decrease (4.5 fold) in the step-through latency in the 24 h retention trial, a decrease (4 fold) in the number of crossings over the exact former location of the platform and a reduction (3 fold) in the time spent in the target quadrant (where the platform was located during hidden platform training) during the MWM task probe test (Wu et al., 2013, Wu et al, 2012). In this study FoxO1 knockdown reversed the cognitive deficits induced by DA in mice (Wu et al., 2013).
</p><p>Adult male and female Sprague Dawley rats received a single intraperitoneal injection of DomA (0, 1.0, 1.8 mg/kg). These low levels of DomA showed that
males may be more susceptible to severe neurotoxicity, whereas females are affected more quickly as it increased locomotor and grooming activity after monitoring behaviour for 3h (Baron et al., 2013).
</p><p><b>Glufosinate (GLF):</b>
</p><p>1. <b>GLF impairs neuronal network function</b>.
The networks of cortical rat neurons were exposed to glufosinate (GLF) and its primary metabolite N-acetylglufosinate (NAcGLF) and electrical activity was measured using microelectrode array (MEA). The MEA recordings showed the concentration-responses for GLF and NAcGLF on network mean firing rates (MFR) that were biphasic, increasing at lower concentrations, decreasing below control levels at higher concentrations (similarly to NMDA). Increases in MFR occurred between 100–300 uM for NAcGLF (190% control, maximum) and 10–1000 uM for GLF (340% control, maximum) indicating that GLF is affecting neuronal network function (Lantz et al., 2014). Toxicokinetic data from reports of intentional human poisonings indicate that GLF concentrations in the CNS could reach levels high enough to lead to effects mediated via NMDARs (Watanabe and Sano, 1998).
</p><p>Direct activation of NMDARs by GLF is also suggested by in vivo studies where three NMDA receptor antagonists, dizocilpine, LY235959, and Compound 40, and AMPA/KA antagonist, NBQX, were co-administrated with glufosinate ammonium (80 mg/kg, intraperitoneally) in mice. Statistical analyses showed that the NMDA receptor antagonists markedly inhibited the GLF-induced convulsions, while the AMPA/KA receptor antagonist had no effect. These results suggest that the convulsion caused by glufosinate ammonium were mediated through NMDA receptors (Matsumura et al., 2001).
</p><p>2. <b>GLF exposure triggers convulsions and memory impairment.</b>
GLF expsoure produces moderate to severe convulsions and memory loss (Koyama et al., 1994; Watanabe and Sano, 1998; Ohtake et al., 2001; Park et al., 2006, 2013; Mao et al., 2011a and b, 2012), as well as causes structural changes to several brain regions, including the cortex and hippocampus (Calas et al., 2008; Meme et al., 2009; Park et al., 2006 ), two brain structures rich in NMDARs that play an important role in learning and memory processes.
</p><p>• A 64 year old patient who ingested GLF suffered mental disturbances and later developed generalized convulsions, impaired respiration and circulatory failure. During recovery he exhibited loss of short-term memory (retrograde and anterograde amnesia) (Watanabe and Sano, 1998).
</p><p>• Similarly, a 34-yr-old man who ingested glufosinate ammonium developed mental deterioration (Park et al., 2013).
</p><p>• Retrograde amnesia has been reported following acute GLF toxicity in humans (Park et al., 2006).
</p><p>• Chronic GLA (glufosinate-ammonium) treatments with 5 and 10mg/kg induce mild memory impairments and a modification
of hippocampal texture in mice. It is suggested that these modifications may be causally linked one to another.
Hippocampal MRI texture and spatial memory alterations might be the consequences of hippocampal glutamate homeostasis modification (Calas et al., 2008).
</p><p>• Spatial memory impairment was observed in mice after chronic exposure to as low as 2.5 mg/kg of GLF (Calas et al.,2008; Meme et al., 2009).
</p><p>One of the most difficult issues for neuroscientists is to link neuronal network function to cognition, including learning and memory. It is still unclear
exactly what modifications in neuronal circuits need to happen in order to alter motor behaviour as it is recorded in a learning and memory test
(Mayford et al., 2012), meaning that there is no clear understanding about how these two KEs are connected.
</p><p>It is unclear whether GLF affects only glutamatergic systems since other potential mechanisms underlying GLF neurotoxicity have not been widely investigated.
Based on the existing data it is understood that exposure to GLF or NAcGLF could disrupt the neuronal network function through disruption of glutamatergic neurotransmission but further work is required to clarify molecular mechanisms that cause impairment of memory.
</p><p><em>
Is it known how much change in the first event is needed to impact the second?
Are there known modulators of the response-response relationships?
Are there models or extrapolation approaches that help describe those relationships?
</em>
</p><p>Quantitative evaluation of this KER does not exist.
</p>HighHighHigh<p>Administration of DomA (9.0 mg DomA kg(-1) bw, i.p.) to Sparus aurata (seabream) caused neurological disturbances such as swimming in a circle, in a
spiral, or upside down, that were reversed 24 hours after exposure (Nogueira et al., 2010).
In rainbow trout (Oncorhynchus mykiss), DomA (0.75 mg/kg bw) administration caused increased aggressive behaviour 30 min after exposure compared
to controls (Bakke et al., 2010).
</p><p><br />
Bakke MJ, Hustoft HK, Horsberg TE., Subclinical effects of saxitoxin and domoic acid on aggressive behaviour and monoaminergic turnover in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol., 2010, 99: 1-9.
</p><p>Baron AW, Rushton SP, Rens N, Morris CM, Blain PG, Judge SJ., Sex differences in effects of low level domoic acid exposure. Neurotoxicology, 2013, 34: 1-8.
</p><p>Benke TA1, Lüthi A, Isaac JT, Collingridge GL., Modulation of AMPA receptor unitary conductance by synaptic activity. Nature, 1998, 25: 793-7.
</p><p>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.
</p><p>Calas AG, Richard O, Même S, Beloeil JC, Doan BT, Gefflaut T, Même W, Crusio WE, Pichon J, Montécot C. Chronic exposure to glufosinate-ammonium induces spatial memory impairments, hippocampal MRI modifications and glutamine synthetase activation in mice. Neurotoxicology. 2008, 29(4): 740-7
</p><p>Chen LY, Rex CS, Casale MS, Gall CM, Lynch G., Changes in synaptic morphology accompany actin signaling during LTP. J Neurosci., 2007, 27: 5363–5372.
</p><p>Clayton EC, Peng YG, Means LW, Ramsdell JS. Working memory deficits induced by single but not repeated exposures to domoic acid. Toxicon. 1999, 37: 1025-1039.
</p><p>D'Hooge R, De Deyn PP., Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev., 2001, 36: 60-90.
</p><p>Fuquay JM, Muha N, Pennington PL, Ramsdell JS., Domoic acid induced status epilepticus promotes aggressive behavior in rats. Physiol Behav., 2012, 105: 315-320.
</p><p>Grant KS, Burbacher TM, Faustman EM, Gratttan L., Domoic acid: neurobehavioral consequences of exposure to a prevalent marine biotoxin. Neurotoxicol Teratol., 2010, 32: 132-141.
</p><p>Grunwald T, Beck H, Lehnertz K, Blümcke I, Pezer N, Kurthen M, Fernández G, Van Roost D, Heinze HJ, Kutas M, Elger CE.. Evidence relating human verbal memory to hippocampal N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A., 1999, 96: 12085-12089.
</p><p>Hiolski EM, Kendrick PS, Frame ER, Myers MS, Bammler TK, Beyer RP, Farin FM, Wilkerson HW, Smith DR, Marcinek DJ, Lefebvre KA., Chronic low-level domoic acid exposure alters gene transcription and impairs mitochondrial function in the CNS. Aquat Toxicol., 2014, 155: 151-9.
</p><p>Koyama K, Andou Y, Saruki K, Matsuo H., Delayed and severe toxicities of a herbicide containing glufosinate and a surfactant. Vet Hum Toxicol., 1994, 36: 17–8.
</p><p>Kuhlmann AC, Guilarte TR., The peripheral benzodiazepine receptor is a sensitive indicator of domoic acid neurotoxicity. Brain Res., 1997, 751: 281-288.
</p><p>Lantz SR, Mack CM, Wallace K, Key EF, Shafer TJ, Casida JE., Glufosinate binds to N-methyl-D-aspartate receptors and increases neuronal network activity in vitro. Neurotoxicology, 2014, 45:38-47.
</p><p>Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF., Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52: 646-59.
</p><p>Lynch G, Cox CD, Gall CM., Pharmacological enhancement of memory or cognition in normal subjects. Front Syst Neurosci., 2014, 8: 90-103.
</p><p>MacDonald JF1, Jackson MF., Beazely MAHippocampal long-term synaptic plasticity and signal amplification of NMDA receptors. Crit Rev Neurobiol., 2006, 18: 71-84.
</p><p>Mao Y-C, Hung D-Z, Wu M-L, Tsai W-J, Wang L-M, Ger J, et al. Acute human glufosinatecontaining herbicide poisoning. Clin Toxicol., 2012, 5: 1–7.
</p><p>Mao Y-C, Wang J-D, Hung D-Z, Deng J-F, Yang C-C., Hyperammonemia following glufosinate-containing herbicide poisoning: a potential marker of severe neurotoxicity. Clin Toxicol (Phila), 2011a, 49: 48–52.
</p><p>Mao Y-C, Yang C-C. Response to ‘‘Hyperammonemia following glufosinate-containing herbicide poisoning: A potential marker of severe neurotoxicity’’ by Yan-Chido Mao et al., Clin Toxicol (Phila) 2011b; 49: 48–52. Clin Toxicol 2011;49(July (6)): 513.
</p><p>Mayford M, Siegelbaum SA, Kandel ER., Synapses and memory storage. Cold Spring Harb Perspect Biol., 2012:4(6). pii: a005751.
</p><p>Meme S, Calas A-G, Monte´ cot C, Richard O, Gautier H, Gefflaut T, et al., MRI characteri- zation of structural mouse brain changes in response to chronic exposure to the glufosinate ammonium herbicide. Toxicol Sci 2009, 111: 321–30.
</p><p>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(1-2): 123-5.
</p><p>Morris RG, Anderson E, Lynch GS, Baudry M.,Selective impairment of learning and blockade of long-term potentiation by an N-methyl-Daspartate receptor antagonist, AP5. Nature, 1986, 319: 774-776.
</p><p>Muha N, Ramsdell JS., Domoic acid induced seizures progress to a chronic state of epilepsy in rats. Toxicon., 2011, 57: 168-171.
</p><p>Nakajima S, Potvin JL., Neural and behavioural effects of domoic acid, an amnesic shellfish toxin, in the rat. Can J Psychol., 1992, 46: 569-581.
</p><p>Nogueira I, Lobo-da-Cunha A, Afonso A, Rivera S, Azevedo J, Monteiro R, Cervantes R, Gago-Martinez A, Vasconcelos V., Toxic effects of domoic acid in the seabream Sparus aurata. Mar Drugs, 2010, 8: 2721-232.
</p><p>Ohtake T, Yasuda H, Takahashi H, Goto T, Suzuki K, Yonemura K, et al., Decreased plasma and cerebrospinal fluid glutamine concentrations in a patient with bialaphos poisoning. Hum Exp Toxicol., 2001, 20: 429–34.
</p><p>Park JS1, Kwak SJ, Gil HW, Kim SY, Hong SY., Glufosinate herbicide intoxication causing unconsciousness, convulsion, and 6th cranial nerve palsy. J Korean Med Sci., 2013, 28:1687-9.
</p><p>Park HY, Lee PH, Shin DH, Kim GW., Anterograde amnesia with hippocampal lesions following glufosinate intoxication. Neurology, 2006, 67:914–5.
</p><p>Petrie BF, Pinsky C, Standish NM, Bose R, Glavin GB., Parenteral domoic acid impairs spatial learning in mice. Pharmacol Biochem Beh., 1992, 41: 211-214.
</p><p>Pulido OM., Domoic acid toxicologic pathway: a review. Mar Drugs, 2008, 6:180-219.
</p><p>Saar D, Barkai E. (2003) Long-term modifications in intrinsic neuronal properties and rule learning in rats. Eur J Neurosci. 17: 2727-2734.
</p><p>Sobotka TJ, Brown R, Quander DY, Jackson R, Smith M, Long SA, Barton CN, Rountree RL, Hall S, Eilers P, Johannessen JN, Scallet AC., Domoic acid: neurobehavioral and neurohistological effects of low-dose exposure in adult rats. Neurotoxicol Teratol., 1996, 18: 659-670.
</p><p>Tryphonas L, Truelove J, Nera E, Iverson F., Acute neurotoxicity of domoic acid in the rat. Toxicol Pathol., 1990, 18: 1-9.
</p><p>Watanabe T, Sano T., Neurological effects of glufosinate poisoning with a brief review. Hum Exp Toxicol., 1998, 17(1): 35-9.
</p><p>Wu DM, Lu J, Zheng YL, Zhang YQ, Hu B, Cheng W, Zhang ZF, Li MQ., Small interfering RNA-mediated knockdown of protein kinase C zeta attenuates domoic acid-induced cognitive deficits in mice. Toxicol Sci., 2012, 128: 209-222.
</p><p>Wu DM, Lu J, Zhang YQ, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ., Ursolic acid improves domoic acid-induced cognitive deficits in mice. Toxicol Appl Pharmacol., 2013, 271: 27-36.
</p><p>Retrieved from <a rel="nofollow" target="_blank" class="external free" href="https://aopkb.org/aopwiki/index.php/?oldid=23557">https://aopkb.org/aopwiki/index.php/?oldid=23557</a>
</p>2016-11-29T18:41:342016-11-29T20:23:40920190dd-6d6a-43ab-86a2-21af66b6c4c0d70d314e-a83f-4253-bdc5-d65b49b8821f<p>Neurodegeneration (retraction of dendrites or axons) or neuronal cell death decreases the number of synaptic connections affecting the neuronal network function (Seeley et al., 2009). Based on neuropathology (Braak and Braak, 1991), neuroimaging (Buckner et al., 2005 and Greicius et al., 2004), and evidence from transgenic animal models (Palop et al., 2007a), it is suggested that neurodegeneration leads to neural network dysfunction (Buckner et al., 2005 and Palop et al., 2006). In human spongiform encephalopathies, which cause rapidly progressive dementia, direct evidence supports disease propagation along affected trans-synaptic connections (Scott et al., 1992). For all other neurodegenerative diseases, there are limited human experimental data supporting the “network degeneration hypothesis.”
It is demonstrated as a class-wide phenomenon, with major mechanistic significance, predicting that the spatial patterning of disease relates to some structural, metabolic, or physiological aspect of neural network biology dysfunction. Confirming the network degeneration hypothesis has clinical impact, stimulating development of new network-based diagnostic and disease-monitoring assays.
</p><p>Based on neuropathological findings and neuroimaging from patients suffering from neurodegeneration as well as from evidence derived by transgenic animal models of neurodegeneration, it has been suggested that neurodegeneration is related to neural network dysfunction (Palop et al., 2007b; Seeley et al., 2009). Neurodegeneration leads to impairment of retrograde axonal transport that prohibits the growth factor supply to long-range projection neurons, causing synapse loss, and post-synaptic dendrite retraction that leads to decreases of the neuronal network (Seeley et al., 2009).
</p><p><em>
Include consideration of temporal concordance here
</em>
</p><p>The effective concentration of DomA causing a decrease to 50% of control mean firing rate (MFR) values (EC50) in rat primary cultures (13-30 DIV) is 0.28 μM (Mack et al., 2014). Decrease of MFR has also been reported before by Hogberg et al. 2011, where mature cultures (28-35 DIV) have been exposed acutely to a wide range of concentrations of DomA. The concentration of 0.5 μM DomA significantly reduces MFR (77 %), the MBR (78 %)and the number of spikes per burst (71 %). Higher concentrations of DomA (1 and 2 μM) also significantly decrease the MFR, whereas concentrations up to 0.1 μM of DomA do not cause any effect on MFR (Hogberg et al., 2011). In primary rat cortical neurons (12-22 DIV), DomA (50 μM) has been
reported to reduce MFR by more than 90% (McConnell et al., 2012).
</p><p>Ten-minute exposure of rat hippocampal CA1 region slices to 400 nM DA causes depression of fEPSP (Qiu et al., 2009). After 1 h washout, fEPSP
gradually has been gradually recovered. DomA-potentiated slices have shown also less tetanus-induced LTP compared with control slices when tested with
either original stimulus or reset stimulus (Qiu et al., 2009). In addition, prolonged application of 400 nM DA reversibly depresses CA1 fEPSP and
impairs the subsequent development of tetanus LTP (Qiu et al., 2009).
</p><p>Gap of knowledge: there are no experiments to support such a KE relationship after exposure to GLF.
</p><p>Administration of high dose DomA (4.4 mg/kg) to adult male Sprague-Dawley rats causes elevation of electrocorticogram (ECoG) beginning 30 min post
injection, whereas at a lower dose (2.2 mg/kg) ECoG becomes elevated after 110 min (Binienda et al., 2011).
</p><p><em>
Is it known how much change in the first event is needed to impact the second?
Are there known modulators of the response-response relationships?
Are there models or extrapolation approaches that help describe those relationships?
</em>
</p><p>Not enough information exists to understand this linkage quantitativly.
</p><p>It has been shown at the neuromascular junction of D. melanogaster that quisqualate-type glutamate receptors are blocked by DomA (1 mM) (Lee et al.,
2009). However, in crayfish (Procambarus clarkia) the same concentration of DomA has no effect in spike activity (Bierbower and Cooper, 2013).
</p><p><br />
Bierbower SM, Cooper RL. The mechanistic action of carbon dioxide on a neural circuit and NMJ communication. J Exp Zool A Ecol Genet Physiol., 2013, 319: 340-54.
</p><p>Binienda ZK, Beaudoin MA, Thorn BT, Ali SF. Analysis of electrical brain waves in neurotoxicology: γ-hydroxybutyrate. Curr Neuropharmacol., 2011, 9: 236-9.
</p><p>Braak H., E. Braak, Neuropathological staging of Alzheimer-related changes, Acta Neuropathol., 1991, 82: 239–259.
</p><p>Buckner R.L., A.Z. Snyder, B.J. Shannon, G. LaRossa, R. Sachs, A.F. Fotenos, Y.I. Sheline, W.E. Klunk, C.A. Mathis, J.C. Morris, M.A.Molecular, structural, and functional characterization of Alzheimer's disease: evidence for a relationship between default activity, amyloid, and memory. J. Neurosci., 2005, 25:7709–7717.
</p><p>Greicius M.D., G. Srivastava, A.L. Reiss, V. Menon, Default-mode network activity distinguishes Alzheimer's disease from healthy aging: evidence from functional MRI. Proc. Natl. Acad. Sci. USA, 2004, 101: 4637–4642.
</p><p>Hogberg HT, Sobanski T, Novellino A, Whelan M, Weiss DG, Bal-Price AK. 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, 2011, 32: 158-168.
</p><p>Lee JY, Bhatt D, Bhatt D, Chung WY, Cooper RL. Furthering pharmacological and physiological assessment of the glutamatergic receptors at the Drosophila neuromuscular junction. Comp Biochem Physiol C Toxicol Pharmacol., 2009, 150(4): 546-57.
</p><p>Mack CM, Lin BJ, Turner JD, Johnstone AF, Burgoon LD, Shafer TJ. Burst and principal components analyses of MEA data for 16 chemicals describe at least three effects classes. Neurotoxicology, 2014, 40: 75-85.
</p><p>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.
</p><p>Palop J.J., J. Chin, L. Mucke, A network dysfunction perspective on neurodegenerative diseases. Nature, 2006, 443: 768–773.
</p><p>Palop JJ, Chin J, Roberson ED, Wang J, Thwin MT, Bien-Ly N, Yoo J, Ho KO, Yu GQ, Kreitzer A, et al. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron, 2007a, 55: 697-711.
</p><p>Palop J.J, J. Chin, E.D. Roberson, J. Wang, M.T. Thwin, N. Bien-Ly, J. Yoo, K.O. Ho, G.Q. Yu, A. Kreitzer, et al., Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron, 2007b, 55: 697–711.
</p><p>Qiu S, Jebelli AK, Ashe JH, Currás-Collazo MC. Domoic acid induces a long-lasting enhancement of CA1 field responses and impairs tetanus-induced long-term potentiation in rat hippocampal slices. Toxicol Sci., 2009, 111: 140-150.
</p><p>Scott R.S., D. Davies, H. Fraser. Scrapie in the central nervous system: neuroanatomical spread of infection and Sinc control of pathogenesis. J. Gen. Virol., 1992, 73: 1637–1644.
</p><p>Seeley WW, Crawford RK, Zhou J, Miller BL, Greicius MD. Neurodegenerative diseases target large-scale human brain networks. Neuron, 2009, 62: 42-52.
</p>2016-11-29T18:41:342016-11-29T20:24:52Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.ionotropic glutamatergic receptors and cognition<p>Magdalini Sachana, Sharon Munn, Anna Bal-Price</p>
<p>European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy</p>
<p>Corresponding author: anna.price@ec.europa.eu</p>
Open for citation & commentWPHA/WNT EndorsedIncluded in OECD Work Plan1.23<p>Under physiological conditions activation of glutamate ionotropic receptors such as N-methyl-D-aspartate (NMDARs), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPARs) and kainate (KARs) is responsible for basal excitatory synaptic transmission and main forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) that are fundamental for learning and memory processes (Schrattenholz and Soskic, 2006). However, sustained (direct or indirect) over-activation of these receptors can induce excitotoxic neuronal cell death. Indeed, mainly increased Ca2+ influx through NMDARs promotes many pathways of toxicity due to generation of free radical species, reduced ATP production, endoplasmic reticulum (ER) stress and protein aggregation. Neuronal injury induced by over-activation of these receptors and the excessive Ca2+ influx is considered an early key event of excitotoxicity. Additionally, the excessive activation of NMDARs has been found to play a significant role in a variety of neurological disorders ranging from acute hypoxic-ischemic brain injury (Barenger et al., 2001) to chronic neurodegenerative diseases (Mehta et al., 2013). The proposed AOP is relevant to adult neurotoxicity testing. A molecular initiating event (MIE) has been defined as a direct binding of agonists to NMDARs or indirect, through prior activation of AMPARs and/or KARs resulting in sustained NMDARs over-activation causing excitotoxic neuronal cell death, mainly in hippocampus and cortex, two brain structures fundamental for learning and memory processes. The AOP is based on the empirical support describing (1) domoic acid (DomA) induced excitotoxicity triggered by indirect (through KARs/AMPARs) NMDARs over-activation leading to impaired learning and memory and (2) glufosinate (GLF) induced excitotoxicity that through direct binding to NMDARs causes convulsions and memory loss (Lanz et al., 2014). GLF is the methylphosphine analog of L-glutamate, used as a component of bactericidal and fungicidal herbicidal. DomA, a natural toxin that accumulates in mussels and shellfish is also an analogue of L-glutamate and among the most prominent features described after human exposure to DomA is memory impairment (Lefebvre and Robertson, 2010). DomA and GLF are described as the examples of the stressors due to large amounts of published data (especially in the case of DomA), however this AOP is relevant to any agonist that directly or indirectly cause NMDARs over-activation. Some of the known agonists selective for the NMDARs are derived from the naturally occurring amino acids such as ibotenic acid, homocysteine and l-aspartate and polyamines like spermidine.</p>
<p> </p>
<p>Evidence for Chemical Initiation of this Molecular Initiating Event
</p><p>L-Glutamate and glycine (or D-serine) are endogenous agonists that bind to the LBD of specific NMDA receptor subunits. Here listed some known agonists for NMDA receptor, some of them are specific to the NR1 subunit and some others to the NR2 subunit (reviewed in Traynelis et al., 2010):
</p><p><b>Specific to NR1</b>
</p><p>Glycine,
l-Serine,
d-Serine,
l-Alanine,
d-Alanine,
d-Cycloserine,
HA 966, (+)-(1-hydroxy-3-aminopyrrolidine-2-one,)
β-Cl-d-Alanine,
β-F-dl-Alanine,
tri-F-dl-Alanine,
ACPC, 1-aminocyclopropane-1-carboxylic acid,
ACBC, 1-aminocyclobutane-1-carboxylic acid,
GLYX-13.
</p><p><b>Specific to NR2</b>
</p><p>l-Glutamate,
d-Glutamate,
l-Aspartate,
d-Aspartate,
N-Methyl-l-aspartate,
N-Methyl-d-aspartate,
SYM208,1
l-Homocysteinsulfinate,
d-Homocysteinsulfinate,
l-Homocysteate,
d-Homocysteate,
l-Cysteinesulfinate,
l-Cysteate,
d-Cysteate,
Homoquinolinate,
Ibotenate,
(R,S)-(Tetrazol-5-yl)glycine,
L-CCG-IV, (2S,3R,4S)-2-(carboxycyclopropyl)glycine,
trans-ACBD, trans-1-aminocyclobutane-1,3-dicarboxylate,
cis-ADA, cis-azetidine-2,4-dicarboxylic acid,
trans-ADC, azetidine-2,4-dicarboxylic acid,
cis-ACPD, (1R,3R)-aminocyclopentane-cis-dicarboxylate,
cis-2,3-Piperidinedicarboxylic acid,
(R)-NHP4G, 2-(N-hydroxylpyrazol-4-yl)glycine,
(R,S)-Ethyl-NHP5G, 2-(N-hydroxypyrazol-5-yl)glycine,
(R)-Propyl-NHP5G, 2-(N-hydroxypyrazol-5-yl)glycine.
</p><p><b>Domoic acid (DomA)</b> is structurally similar to kainic acid (KA) and both of them are analogues of the excitatory neurotransmitter L-glutamate. DomA induces excitotoxicity by an integrative action on ionotropic glutamate receptors at pre- and post-synaptic sides. DomA directly activates KA/AMPARs receptors followed by indirect activation of the NMDARs. Indeed, indirect activation of NMDARs by DomA is linked to the fact that KA and AMPA receptors activated by DomA induce increased levels of intracellular Ca2+ and Na+ which, in turn, causes endogenous glutamate release that subsequently potentiates activation of NMDARs (Berman and Murray, 1997; Berman et al., 2002; Watanabe et al., 2011). DomA has been demonstrated through both in vitro and in vivo approaches to indirectly activate the NMDARs (reviewed in Pulido et al., 2008).
</p><p><b>Glufosinate (GLF)</b>((RS)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid, phosphinothricin) is a phosphorus containing amino acid herbicide that is naturally occurring as a component of the bacteria-derived bactericidal and fungicidal tripeptides bialaphos and phosalacine (Lanz et al., 2014). There are studies suggesting that convulsive and amnesic effects of GLF are mediated through direct binding and activation of NMDAR (Lantz et al., 2014; Matsumura et al., 2001). GLF agonist action at the NMDAR is expected to occur through direct interaction with the glutamate binding site and requires binding of the glycine co-agonist as well as release of the magnesium block from the channel pore.
</p><p>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) <span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">as well as OECD TG 443 (OECD, 2018)</span></span> both require testing of learning and memory (USEPA, 1998; OECD, 2007) advising to use the following tests passive avoidance, delayed-matching-to-position for the adult rat and for the infant rat, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and acquisition and retention of schedule-controlled behaviour. These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009).</p>
<p>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).</p>
adjacentNot SpecifiedModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedHighadjacentModerateHighadjacentLowModerateadjacentLowLowadjacentLowModerateadjacentNot SpecifiedLow<p>1) <strong>Essentiality of KE "NMDARs, Overactivation" for the KE "Cell death"</strong> is <em><strong>MODERATE</strong></em>. NMDARs play a central role in excitotoxic neuronal injury. Over-activation of these receptors causes disruption of Ca2+ homeostasis that through mitochondrial dysfunction triggers signals leading to apoptotic or necrotic death. However, the ways that cells respond to mitochondrial injury vary and often are considered unclear and controversial (Pivovarova and Andrews, 2010). However, NMDAR antagonists failed to reverse these Ca2+ induced cell deaths, leading to suggestions that NMDAR-independent pathways that involve α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), acid-sensing channels and transient receptor potential channels might be also responsible for excitotoxic neuronal injury (Pivovarova and Andrews, 2010). Several agonists have higher affinity than NMDA itself but are not relevant for behavioural studies as NMDA activation leads to epilepsy and cell death, a common approach to induce neurotoxic lesions.</p>
<p>2) <strong>Essentiality of KE "Calcium influx, Increased" for the KE "Cell death"</strong> is <em><strong>MODERATE</strong></em>. Ca2+ plays important role in excitotoxicity but the mechanisms involved in excitotoxic cell death are still debated (Berliocchi et al., 2005). Depending on the extent and the duration of the Ca2+ influx, neurons survive, die through apoptotic mechanisms in case of sustained slow Ca2+ influx, or undergo necrosis when rapid high Ca2+ influx occurs. Over-expression of the endogenous calpain inhibitor, calpastatin, or the calpain-resistant isoform the Na+/Ca2+ exchanger 2 (NCX2) prevents Ca2+ overload and protects neurons from excitotoxicity (Bano et al., 2005).</p>
<p>3) <strong>Essentiality of KE "Mitochondria dysfunction" for the AO "Impairment of learning and memory"</strong> is <em><strong>STRONG</strong></em>. ROS is known to have a negative effect on synaptic plasticity and learning and memory (reviewed in Lynch, 2004). H2O2 inhibits LTP both in vitro and in vivo, which is associated with increased ROS. A negative correlation has been found between ROS concentration in hippocampus and ability of rats to sustain LTP. Administration of antioxidants, vitamins E and C, reverses the inhibitory effects of stress on LTP and prevents the increase of ROS in hippocampus. In transgenic mice that overexpress superoxide dismutase (SOD), the enzyme which catalyzes the conversion of superoxide to H2O2, the LTP in CA1 is inhibited. Intracerebroventricular injection of H2O2, at a concentration which increases ROS levels in hippocampus, impairs LTP that is prevented after pretreatment with the antioxidant phenylarsine oxide. Knocking down Forkhead box protein O1 (FoxO1) in mice, which is an important regulator of mitochondrial function, reverses mitochondrial abnormalities and cognitive impairment induced by DA in mice (Wu et al., 2013).</p>
<p>4) <strong>Essentiality of KE "Mitochondria dysfunction" for the KE "Cell death"</strong> is <em><strong>MODERATE</strong></em>. There is a considerable number of mitochondrial associated processes that lead to necrotic or apoptotic cell death such as uncoupling of oxidative phosphorylation, activation of the mitochondrial permeability transition pore (MPTP), release of pro-apoptotic proteins, activation of poly(ADP-ribose) polymerase-1 and proteases such as calpain, increased levels of and delayed Ca2+ de-regulation (Pivovarova and Andrews, 2010). Although the understanding of these mechanisms is clearly established, the cascade of events and the significance of them are less clear (Pivovarova and Andrews, 2010). A significant body of evidence, both clinical and experimental, supports a role for the mitochondrial permeability transition pore in excitotoxicity (reviewed in Pivovarova and Andrews, 2010). However, the effects of cyclosporin A, the classical MPTP inhibitor, on neuronal mitochondria are inconsistent raising doubts about its role in neural cell death. However, ADP/ATP translocator deficiency, which is not essential for MPTP but does regulate pore opening, protects neurons against excitotoxicity. Furthermore, MPTP opening renders neurons vulnerable to excitotoxicity.</p>
HighMaleHighFemaleHighAdultsHighHighHigh<p>The aim of the present AOP is to construct a linear pathway that captures the KEs and KERs that occur after binding of agonist to NMDA receptor in hippocampal and cortical neurons of adults. The majority of the KEs of the AOP are characterised by MODERATE essentiality for the AO (loss or reduction of cognitive function )or other KEs that follow. The biological plausibility in the majority of KERs is rated STRONG as there is extensive mechanistic understanding. However, the empirical support for the majority of presented KERs cannot be rated high as in most occasions the KEup and KEdown of a KER has not been investigated simultaneously, under the same experimental protocol or not in the suggested brain regions (cortex and hippocampus).</p>
<p><strong>Life Stage Applicability:</strong> This AOP is applicable for adults. However, studies exploring the neurotoxic effects of DomA on the developing nervous system demonstrate that DomA can cause neurobehavioral, biochemical and morphological effects similar to the ones observed in adult animals (reviewed in Costa et al., 2010). The DomA doses required to cause these effects in developing organisms are one to two orders of magnitude lower than those needed for loss or reduction of cognitive function in adults. This difference has been attributed to toxicokinetic and/or toxicodynamic particularities that exist between adults and children.</p>
<p><strong>Taxonomic Applicability:</strong> The data used to support the KERs in this AOP derives from experimental studies conducted in primates, rats and mice or cell cultures of similar origin as well as from human epidemiological studies or clinical cases of DomA poisoning. The majority of the KEs in this AOP seem to be highly conserved across species. It remains to be proved if these KERs of the present AOP are also applicable for other species rather than human, primates, rats or mice. Increasing evidence from sea lions exposed to DomA further supports some of the KERs of the present AOP.</p>
<p><strong>Sex Applicability:</strong> The majority of the studies addressing the KEs and KERs of this AOP have been carried out mainly in male laboratory animals. Few studies are available in females and some of them compare the effects between females and males. It appears that this AOP is applicable for both females and males.</p>
<p>1) <strong>Essentiality of KE "NMDARs, Overactivation" for the KE "Cell death"</strong> is <em><strong>MODERATE</strong></em>. NMDARs play a central role in excitotoxic neuronal injury. Over-activation of these receptors causes disruption of Ca2+ homeostasis that through mitochondrial dysfunction triggers signals leading to apoptotic or necrotic death. However, the ways that cells respond to mitochondrial injury vary and often are considered unclear and controversial (Pivovarova and Andrews, 2010). However, NMDAR antagonists failed to reverse these Ca2+ induced cell deaths, leading to suggestions that NMDAR-independent pathways that involve α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), acid-sensing channels and transient receptor potential channels might be also responsible for excitotoxic neuronal injury (Pivovarova and Andrews, 2010). Several agonists have higher affinity than NMDA itself but are not relevant for behavioural studies as NMDA activation leads to epilepsy and cell death, a common approach to induce neurotoxic lesions.</p>
<p>2) <strong>Essentiality of KE "Calcium influx, Increased" for the KE "Cell death"</strong> is <em><strong>MODERATE</strong></em>. Ca2+ plays important role in excitotoxicity but the mechanisms involved in excitotoxic cell death are still debated (Berliocchi et al., 2005). Depending on the extent and the duration of the Ca2+ influx, neurons survive, die through apoptotic mechanisms in case of sustained slow Ca2+ influx, or undergo necrosis when rapid high Ca2+ influx occurs. Over-expression of the endogenous calpain inhibitor, calpastatin, or the calpain-resistant isoform the Na+/Ca2+ exchanger 2 (NCX2) prevents Ca2+ overload and protects neurons from excitotoxicity (Bano et al., 2005).</p>
<p>3) <strong>Essentiality of KE "Mitochondria dysfunction" for the AO "Impairment of learning and memory"</strong> is <em><strong>STRONG</strong></em>. ROS is known to have a negative effect on synaptic plasticity and learning and memory (reviewed in Lynch, 2004). H2O2 inhibits LTP both in vitro and in vivo, which is associated with increased ROS. A negative correlation has been found between ROS concentration in hippocampus and ability of rats to sustain LTP. Administration of antioxidants, vitamins E and C, reverses the inhibitory effects of stress on LTP and prevents the increase of ROS in hippocampus. In transgenic mice that overexpress superoxide dismutase (SOD), the enzyme which catalyzes the conversion of superoxide to H2O2, the LTP in CA1 is inhibited. Intracerebroventricular injection of H2O2, at a concentration which increases ROS levels in hippocampus, impairs LTP that is prevented after pretreatment with the antioxidant phenylarsine oxide. Knocking down Forkhead box protein O1 (FoxO1) in mice, which is an important regulator of mitochondrial function, reverses mitochondrial abnormalities and cognitive impairment induced by DA in mice (Wu et al., 2013).</p>
<p>4) <strong>Essentiality of KE "Mitochondria dysfunction" for the KE "Cell death"</strong> is <em><strong>MODERATE</strong></em>. There is a considerable number of mitochondrial associated processes that lead to necrotic or apoptotic cell death such as uncoupling of oxidative phosphorylation, activation of the mitochondrial permeability transition pore (MPTP), release of pro-apoptotic proteins, activation of poly(ADP-ribose) polymerase-1 and proteases such as calpain, increased levels of and delayed Ca2+ de-regulation (Pivovarova and Andrews, 2010). Although the understanding of these mechanisms is clearly established, the cascade of events and the significance of them are less clear (Pivovarova and Andrews, 2010). A significant body of evidence, both clinical and experimental, supports a role for the mitochondrial permeability transition pore in excitotoxicity (reviewed in Pivovarova and Andrews, 2010). However, the effects of cyclosporin A, the classical MPTP inhibitor, on neuronal mitochondria are inconsistent raising doubts about its role in neural cell death. However, ADP/ATP translocator deficiency, which is not essential for MPTP but does regulate pore opening, protects neurons against excitotoxicity. Furthermore, MPTP opening renders neurons vulnerable to excitotoxicity.</p>
<p>The table provides a summary of the biological plausibility and the empirical support for each KER described in this AOP based on "Annex 1: Guidance for assessing relative level of confidence in the overall AOP based on rank ordered elements" found in the User's Handbook.</p>
<p>More information about the evidence that support these KERs and the relevant literature can be found in each KER description.</p>
<p>The main base for the overall scoring is that the empirical support coming from the experiments with one stressor (domoic acid, DomA). However this AOP is not specific for DomA, it is applicable to any chemicals that act as NMDARs agonists.</p>
<table>
<tbody>
<tr>
<td><strong>KERs WoE</strong></td>
<td><strong>Biological plausibility</strong></td>
<td><strong>Does KEup occurs at lower doses than KEdown?</strong></td>
<td><strong>Does KEup occurs at earlier time points than KE down?</strong></td>
<td><strong>Is there higher incidence of KEup than of KEdown?</strong></td>
<td><strong>Inconsistencies/Uncertainties</strong></td>
</tr>
<tr>
<td><a href="/wiki/index.php/Relationship:360" title="Relationship:360">Binding of agonist to NMDARs directly leads to NMDARs overactivation</a></td>
<td>Extensive understanding</td>
<td>N/A</td>
<td>Yes</td>
<td>N/A</td>
<td>Limited conficting data</td>
</tr>
<tr>
<td><a href="/wiki/index.php/Relationship:361" title="Relationship:361">NMDARs overactivation directly leads to increased calcium influx</a></td>
<td>Extensive understanding</td>
<td>Same dose</td>
<td>Yes</td>
<td>Not investigated</td>
<td>Limited conficting data</td>
</tr>
<tr>
<td>Increased calcium influx indirectly leads to mitochondrial dysfunction</td>
<td>Extensive understanding</td>
<td>Same dose</td>
<td>Yes</td>
<td>Yes</td>
<td>No conflicting data</td>
</tr>
<tr>
<td>Mitochondrial dysfunction directly leads to cell death</td>
<td>Extensive understanding</td>
<td>Same dose</td>
<td>Yes</td>
<td>Yes</td>
<td>Limited conficting data</td>
</tr>
<tr>
<td>Cell death leads to Neurodegeneration</td>
<td>Extensive understanding</td>
<td>Same dose</td>
<td>Yes</td>
<td>Yes</td>
<td>Limited conficting data</td>
</tr>
<tr>
<td>Cell death leads to Neuroinflammation</td>
<td>Extensive understanding</td>
<td>Not investigated</td>
<td>Not investigated</td>
<td>Not investigated</td>
<td>N/A</td>
</tr>
<tr>
<td>Neurodegeneration directly leads to Decreased neuronal network function</td>
<td>Extensive understanding</td>
<td>Not investigated</td>
<td>Not investigated</td>
<td>Not investigated</td>
<td>N/A</td>
</tr>
<tr>
<td>Decreased neuronal network function indirectly leads to loss or reduction of cognitive function</td>
<td>Scientific understanding is not completely established</td>
<td>Not investigated</td>
<td>Not investigated</td>
<td>Not investigated</td>
<td>N/A</td>
</tr>
<tr>
<td> </td>
</tr>
</tbody>
</table>
<p>Exposure to xenobiotics can potentially affect the nervous system resulting in neurobehavioral alterations and/or neurological clinical symptoms. To assess the neurotoxic properties of compounds, current testing largely relies on neurobehavioural tests in laboratory animals, histopathological analysis, neurochemical and occasionally electrophysiological observations. Throughout the years, a significant number of methods have been developed to assess neurobehaviour in laboratory animals and a comprehensive summary of them can be found in OECD Series on testing and assessment, number 20, Guidance Document for Neurotoxicity Testing (2004). This document is considered an essential supplement to a substantial number of already existing OECD Test Guidelines that are applied to gain information on the neurotoxicity properties of chemical compounds. Namely, these are: general Test Guidelines such as single dose toxicity (e.g. OECD 402, 403, 420, 423 and 425), repeated dose toxicity (e.g. OECD 407 and 408), chronic exposure (e.g. OECD 452) as well as Test Guidelines specifically developed for the study of neurotoxicity in adult laboratory animals, such as OECD Test Guideline for Neurotoxicity (424).</p>
<p>Learning and memory is an important endpoint and a wide variety of tests to assess chemical effects on cognitive functions is available and used for the study of neurotoxicity. Some of these tests that allow the appreciation of cognitive function in laboratory animals are: habituation, ethologically based anxiety tests (elevated plus maze test, black and white box test, social interaction test), conditioned taste aversion (CTA), active avoidance, passive avoidance, spatial mazes (Morris water maze, Biel water maze, T-maze), conditional discrimination (simple discrimination, matching to sample), delayed discrimination (delayed matching-to-sample, delayed alternation) and eye-blink conditioning.</p>
<p>The present AOP can potentially provide the basis for development of a mechanistically informed IATA for neurotoxicity. The construction of IATA for predicting neurotoxic effects in adults is expected to make use of more than one AOP within an interconnected network in order to take into consideration all critical biological processes that may contribute to impairment of learning and memory in adults. Through this network, identification of KEs and KERs common across multiple AOPs can emerge that should be considered during IATA construction and that may inform also in vitro assay development. The development of alternative assays would allow screening of chemicals for potential NMDAR activators and reducing the use of in vivo studies.</p>
<p>Results from assays based on the KEs of this AOP can serve to interpret and accept results that derive from non-standard test methods. Omics data from toxicogenomic, transcriptomic, proteomic, and metabolomic studies can be interpreted in a structured way using this AOP that is relevant to adult neurotoxicity. Currently learning and memory testing is not required by the OECD TG 424. This AOP could serve as a base for chemical evaluation with potential to cause impairment of learning and memory. The assay development would refer to the identified in this AOP KEs that could form a testing strategy for identifying chemicals with potential to cause cognitive deficit. Finally, this AOP could provide the opportunity to group chemicals using not only chemical properties but also mechanistic information that can later inform data gap filling by read-across and predict neurotoxic properties of a target substance.</p>
<p><br />
Bano D, Young K.W, Guerin C.J, Lefeuvre R, Rothwell N.J, Naldini L, Rizzuto R, Carafoli E, Nicotera P. Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity. Cell, 2005, 120: 275-285.</p>
<p>Berliocchi L, Bano D, Nicotera P. Ca2+ signals and death programmes in neurons. Philos Trans R Soc Lond B Biol Sci., 2005, 360: 2255-2258.</p>
<p>Costa LG, Giordano G, Faustman EM. Domoic acid as a developmental neurotoxin. Neurotoxicology, 2010, 31(5):409-23.</p>
<p>Health Effects Test Guidelines OPPTS 870.6300 Developmental Neurotoxicity Study, US EPA, Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-96, 239, 1996, 1-14.</p>
<p>Lynch MA. Long-term potentiation and memory. Physiol Rev. 2004, 84(1):87-136.</p>
<p>OECD (2004) Series on testing and assessment number 20, Guidance document for neurotoxicity testing.</p>
<p>OECD (2007). Test Guideline 426. OECD Guideline for Testing of Chemicals. Developmental Neurotoxicity Study. <a class="external free" href="http://www.oecd.org/document/55/0,3343,en_2649_34377_2349687_1_1_" rel="nofollow" target="_blank">http://www.oecd.org/document/55/0,3343,en_2649_34377_2349687_1_1_</a> 1_1,00.html</p>
<p>OECD (2008) Nr 43 GUIDANCE DOCUMENT ON MAMMALIAN REPRODUCTIVE TOXICITY TESTING AND ASSESSMENT. ENV/JM/MONO(2008)16</p>
<p>Pivovarova NB, Andrews SB. Calcium-dependent mitochondrial function and dysfunction in neurons. FEBS J., 2010, 277: 3622-3636.</p>
<p>Wu DM, Lu J, Zhang YQ, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ. Ursolic acid improves domoic acid-induced cognitive deficits in mice. Toxicol Appl Pharmacol., 2013, 271:127-36.</p>
2016-11-29T18:41:162023-04-29T16:02:56