<p>Katherine von Stackelberg <span style="font-size:12px"><em>(Harvard Center for Risk Analysis, Boston, MA, USA.) (Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)</em></span></p>
<p>Elizabeth Guzy <em><span style="font-size:12px">(Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)</span></em></p>
<p>Tian Chu <span style="font-size:12px"><em>(Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.)</em></span></p>
<p>Birgit Claus Henn <span style="font-size:12px"><em>(Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA.) (Department of Environmental Health, Boston University School of Public Health, Boston, MA, USA.)</em></span></p>
<p><u>Of the content populated in the AOP-Wiki:</u></p>
<p>Travis Karschnik <span style="font-size:12px"><em>(General Dynamics Information Technology, Duluth, MN, USA.)</em></span></p>
<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p>Metal mixture activation of ERK1/2 and JNK1/2 in astrocytes leads to increased C<sup>a2+</sup> release (Asit Rai and others 2010). Alterations to calcium, an essential nutrient which is required in multiple cellular and physiological functions, such as cell adhesion, signal transduction, and neurotransmission can be expected to have downstream effects in those functions (Antonio et al., 2002). Changes in neurotransmission can then lead to changes in learning and cognitition (Neal and Guilarte 2010).</p>
<p>MEK-ERK1/2 is important in understanding uptake of metals into the brain and its relationship to deficits in learning and cognition from exposure to metals commonly detected at Superfund sites including lead, cadmium, manganese, and arsenic. Current risk assessment guidance dictates a largely chemical-by-chemical evaluation of exposures and risks, which fails to adequately address potential interactions with other chemicals, nonchemical stressors, and genetic factors. Cumulative risk assessment methods and approaches are evolving to meet regulatory needs, (MacDonell et al., 2013; Backhaus and Faust 2012; IPCS Workshop 2009) but significant challenges remain. As our understanding of complex exposures and interactions continues to grow, synthesis and integration across disciplines and studies focused on different aspects of the environmental fate–exposure–toxicology–health outcome continuum are required to assess the likelihood of adverse effects and to support cumulative risk assessment. Environmental exposures are virtually always to complex mixtures (Katherine von Stackelberg et al., 2015).</p>
</div>
<div id="background">
<h3>Background</h3>
<p>An examination of neurodevelopmental disorders and subclinical effects using multi-domain global neurodevelopment assessments is warranted as they can have profound population level implications. In the context of neurotoxicity, neurodevelopmental pathways in the developing human brain are not fully understood (Schubert et al., 2015; Bal-Price et al., 2015) although there are a number of commonly observed phenomena which may take part in those pathways e.g. changes in intracellular calcium, ROS generation, apoptosis, and neurotransmitter disruption. This AOP highlights a specific set of response-response relationships using a subset of those commonly observed phenonema related to metals and metal mixture exposures leading to deficits in learning and cognition.</p>
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p>This AOP was developed as part of an Environmental Protection Agency effort to increase the impact of AOPs published in the peer-reviewed literature, but heretofore unrepresented in the AOP-Wiki, by facilitating their entry and update. The originating work for this AOP was <strong>Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework, Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.</strong> This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.</p>
<p>An examination of neurodevelopmental disorders and subclinical effects using multi-domain global neurodevelopment assessments is warranted as they can have profound population level implications. In the context of neurotoxicity, neurodevelopmental pathways in the developing human brain are not fully understood (Schubert et al., 2015; Bal-Price et al., 2015) although there are a number of commonly observed phenomena which may take part in those pathways e.g. changes in intracellular calcium, ROS generation, apoptosis, and neurotransmitter disruption. This AOP highlights a specific set of response-response relationships using a subset of those commonly observed phenonema related to metals and metal mixture exposures leading to deficits in learning and cognition. </p>
<p>The focus of the originating work was to conduct a review of the literature on relationships between prenatal and early life exposure to mixtures of lead (Pb), arsenic (As), cadmium (Cd), and manganese (Mn) with neurodevelopmental outcomes and then use an AOP framework to integrate lines of evidence from multiple disciplines based on evolving guidance developed by the Organization for Economic Cooperation and Development (OECD). Importantly, the review considered whether exposures to mixtures of metals was associated with neurodevelopment effects that were greater or less than effects from exposure to each individual metal.</p>
</div>
</div>
<div id="development_strategy">
<h3>Strategy</h3>
<p>The originating authors conducted a literature search to develop a database of publications categorized by discipline or field of study: toxicology, epidemiology, exposure, and gene-environment interaction. The literature search relied on standard search engines such as PubMed, Web of Science, Google Scholar, Environmental Index, Scopus, Toxline, and Toxnet and the search strategy included terms related to metal mixtures, individual metals (e.g., arsenic, lead, manganese, and cadmium), neurodevelopmental health outcomes, and associated Medical Subject Headings (MeSH) terms. The originating authors reviewed references from individual citations to identify additional studies not captured through the literature search itself. They then included all relevant publications through September 2013. Only studies focused primarily on developmental or neurotoxic endpoints were included; those focused on carcinogenesis or other systemic effects were not included unless there was a particular relevance to a neurotoxic or developmental outcome.</p>
<p>The scope of the aforementioned EPA project was limited to re-representing the AOP(s) as presented in the originating publication. The literature used to support this AOP and its constituent pages began with the originating publication and followed to the primary, secondary, and tertiary works cited therein. </p>
<p>KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred. Once a baseline level of information was populated for the AOP the authors of the originating publication were contacted for collaboration.</p>
<p>Efforts were made not to editorialize or otherwise add any content to the AOP or its constituent pages that weren’t provided in the primary, secondary, or tertiary literature. In some cases, however, descriptive content was added to pages e.g., assays on a KE page, even if they weren’t specifically provided in the literature stemming from the originating publication.</p>
<p><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><strong><span style="font-size:8.0pt">1. Support for Biological Plausibility of KERs</span></strong></span></span></p>
<p style="margin-left:14px; margin-right:20px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Is there a mechanistic relationship between KE</span><span style="font-size:5.0pt">up </span><span style="font-size:8.0pt">and KE</span><span style="font-size:5.0pt">down </span><span style="font-size:8.0pt">consistent with established biological knowledge?</span></span></span></p>
<p style="margin-left:14px; margin-right:6px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Extensive</span> <span style="font-size:8.0pt">understanding of the KER based on extensive previous documentation and broad acceptance.</span></span></span></p>
<p style="margin-left:14px; margin-right:10px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete</span></span></span></p>
<p style="margin-left:14px; margin-right:18px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Empirical support for association between KEs , but the structural or functional relationship between them is not understood.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Empirical evidence indicates a complex relationship between MEK, ERK1/2 activation and inhibition and Ca2+ response including Ca2+ feeding back into a ERK1/2 activation. This relationship appears to vary across species and cell type.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Intracellular calcium regulation is broadly known as being an important aspect of a number of processes in a variety of cells and is particularly critical in nerve cell terminals where it mediates transmitter release.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The role of various neurotransmitters and receptors in cognitive function and memory formation are well studied.</span></span></span></p>
<p>Life stages applicable to this AOP encompass the full life cycle. Many of the key events are measured in pregnant females with the adverse outcome (impairment, learning and memory) measured at all life stages.</p>
<p><strong>Taxonomic Applicability</strong></p>
<p>Most evidence for this AOP is derived from rodents and humans where rodents were selected with their ability to model human responses. </p>
<p style="margin-left:7px; margin-right:6px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></span></p>
<p style="margin-left:7px; margin-right:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs</span></span></span></p>
<p style="margin-left:7px; margin-right:9px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE</span></span></span></p>
<p style="margin-left:7px; margin-right:8px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">No or contradictory experimental evidence of the essentiality of any of the KEs.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">MEK, ERK1/2 activation is fundamental in delivering signals which regulate the cell cycle, proliferation, differentiation, adhesion, and more. Disruptions in this activation have wide reaching effects however, there is evidence that downstream KEs can also activate this KE. </span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Calcium, as a primary intracellular messenger in neurons and regulator of cell responses to stress has been shown to directly affect neurotransmitter release with manipulation.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Neurotransmitter receptor blocking experiments have shown to directly impair learning and memory tasks in rodents.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">There is direct evidence contained KER 2955.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><strong><span style="font-size:8.0pt">3. Empirical Support for KERs</span></strong></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup > than that for KEdown? Inconsistencies?</span></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">if there is dependent change in both events following exposure to a wide range of specific stressors (extensive evidence for temporal, dose- response and incidence concordance) and no or few data gaps or conflicting data</span></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">if there is demonstrated</span> <span style="font-size:8.0pt">dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with the expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories,</span> <span style="font-size:8.0pt">etc.</span></span></span></p>
<p style="margin-left:14px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">if there are limited or no studies reporting dependent</span> <span style="font-size:8.0pt">change in both events following exposure to a specific stressor (i.e., endpoints never measured in the same study or not at all), and/or lacking evidence of temporal or dose- response concordance, </span><span style="font-size:8.0pt">or identification</span> <span style="font-size:8.0pt">of significant inconsistencies</span> <span style="font-size:8.0pt">in empirical support across taxa and species that don’t align with the expected pattern for the hypothesized</span> <span style="font-size:8.0pt">AOP</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure.</span></span></span></p>
<p style="margin-left:7px"><span style="font-size:11pt"><span style="font-family:"Cambria",serif"><span style="font-size:8.0pt">The evidence collection strategy for this AOP focused mainly on metal and metal mixture exposures, of which, there were many that showed dependent change in both these events following exposure. </span></span></span></p>
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<p>Developmental neurotoxicity (DNT) is an adverse outcome of concern to multiple regulatory agencies. In vitro screening assays for MEK-ERK1/2 activation would not be recommended as a direct alternative or replacement to established DNT assays like OECD Test No. 426 (OECD 2007). However, detection of MEK-ERK1/2 activation in neuronal cell types may be used to prioritize chemicals with potential to elicit neurotoxicity and flag them for testing in ortogonal assays for evaluating DNT, including proposed alternative test methods (Bal-Price et al. 2018; Crofton et al 2022). </p>
</div>
<div id="references">
<h2>References</h2>
<p>Antonio, M. Teresa, Noelia López, and M. Luisa Leret. "Pb and Cd poisoning during development alters cerebellar and striatal function in rats." Toxicology 176.1-2 (2002): 59-66.</p>
<p>Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266</p>
<p>Backhaus T, Faust M. Predictive environmental risk assessment of chemical mixtures: A conceptual framework. Environmental Science & Technology, 2012; 46(5):2564–2573.</p>
<p>Bal-Price A, Crofton KM, Sachana M, Shafer TJ, Behl M, Forsby A, Hargreaves A, Landesmann B, Lein PJ, Louisse J, Monnet-Tschudi F, Paini A, Rolaki A, Schrattenholz A, Sunol C, van Thriel C, Whelan M, Fritsche E. Putative adverse outcome pathways relevant to neurotoxicity. Critical Reviews in Toxicology, 2015; 45(1):83–91.</p>
<p>Bal-Price A, Hogberg HT, Crofton KM, Daneshian M, FitzGerald RE, Fritsche E, Heinonen T, Hougaard Bennekou S, Klima S, Piersma AH, Sachana M, Shafer TJ, Terron A, Monnet-Tschudi F, Viviani B, Waldmann T, Westerink RHS, Wilks MF, Witters H, Zurich MG, Leist M. Recommendation on test readiness criteria for new approach methods in toxicology: Exemplified for developmental neurotoxicity. ALTEX. 2018;35(3):306-352. doi: 10.14573/altex.1712081. Erratum in: ALTEX. 2019;36(3):506. </p>
<p>Crofton KM, Bassan A, Behl M, Chushak YG, Fritsche E, Gearhart JM, Marty MS, Mumtaz M, Pavan M, Ruiz P, Sachana M, Selvam R, Shafer TJ, Stavitskaya L, Szabo DT, Szabo ST, Tice RR, Wilson D, Woolley D, Myatt GJ. Current status and future directions for a neurotoxicity hazard assessment framework that integrates <em>in silico</em> approaches. Comput Toxicol. 2022 May;22:100223. doi: 10.1016/j.comtox.2022.100223. </p>
<p>International Programme on Chemical Safety (IPCS),World Health Organization (WHO). Assessment of combined exposures to multiple chemicals. Report of a WHO/IPCS International Workshop, 2009.</p>
<p>Izquierdo, Ivan. Role of NMDA receptors in memory. Trends in Pharmacological Sciences 12.4 (1991): 128-129</p>
<p>Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. "Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework," Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.</p>
<p>Lupușoru CE, Popa EG, Sandu RB, Buca BR, Mititelu-Tarțău L, Lupușoru RV, The influence of Bidens tripartita extracts on psychomotor abilities and cognitive functions in rats. Farmacia, 2017; 65(2): 284-288.</p>
<p>MacDonell MM, Haroun LA, Teuschler LK, Rice GE, Hertzberg RC, Butler JP, Chang Y-S, Clark SL, John AP, Perry CS, Garcia SS, Jacob JH, Scofield MA. 2013. Cumulative risk assessment toolbox:Methods and approaches for the practitioner. Journal of Toxicology, 2013; Article ID 310904, doi:10.1155/2013/310904.</p>
<p>Navarette M, Perea G, Maglio L, Pastor J, de Sola RG, Araque A. Astrocyte calcium signal and gliotransmission in human brain tissue. Cerebral Cortex, 2013; 23:1240–1246.</p>
<p>Neal, A.P., Guilarte, T.R. Molecular Neurobiology of Lead (Pb2+): Effects on Synaptic Function. Mol Neurobiol 42, 151–160 (2010). https://doi.org/10.1007/s12035-010-8146-0</p>
<p>OECD (2007), <em>Test No. 426: Developmental Neurotoxicity Study</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264067394-en">https://doi.org/10.1787/9789264067394-en</a>.</p>
<p>Schubert D, Martens GJM, Kolk SM. Molecular underpinnings of prefrontal cortex development in rodents provide insights into the etiology of neurodevelopmental disorders. Molecular Psychiatry, 2013; 2014:1–15.</p>
</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/2146">Event: 2146: Activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase 1/2</a></h4>
<p>ERK1 and ERK2 are proteins of 43 and 41 kDa that are nearly 85% identical overall, with much greater identity in the core regions involved in binding substrates (Boulton et al., 1990; 1991). The two phosphoacceptor sites, tyrosine and threonine, which are phosphorylated to activate the kinases, are separated by a glutamate residue in both ERK1 and ERK2 to give the motif TEY in the activation loop (Payne et al., 1991). Both are ubiquitously expressed, although their relative abundance in tissues is variable. For example, in many immune cells ERK2 is the predominant species, while in several cells of neuroendocrine origin they may be equally expressed (Gray Pearson and others 2001). They are stimulated to some extent by a vast number of ligands and cellular perturbations, with some cell type specificity (Lewis et al., 1998). In fibroblasts (the cell type in which the generalizations about their behavior and functions have been developed) they are activated by serum, growth factors, cytokines, certain stresses, ligands for G protein-coupled receptors (GPCRs), and transforming agents, to name a few (Gray Pearson and others 2001). They are highly expressed in postmitotic neurons and other highly differentiated cells (Boulton et al., 1991). In these cells they are often involved in adaptive responses such as long-term potentiation (English and Sweatt 1996; Atkins et al., 1998; Rossi-Arnaud et al., 1997).</p>
<h4>How it is Measured or Detected</h4>
<p>Western blotting and immunoblotting.</p>
<h4>References</h4>
<p>Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM, Sweatt JD 1998 The MAPK cascade is required for mammalian associative learning. Nat Neurosci 1 :602 –609</p>
<p>Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD 1991 ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65 :663 –675</p>
<p>Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw C, Hsu J, Cobb MH 1990 An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249 :64 –67</p>
<p>English JD , Sweatt JD 1996 Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 271 :24329 –24332</p>
<p>Gray Pearson and others, Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions, Endocrine Reviews, Volume 22, Issue 2, 1 April 2001, Pages 153–183, https://doi.org/10.1210/edrv.22.2.0428</p>
<p>Lewis TS, Shapiro PS, Ahn NG 1998 Signal transduction through MAP kinase cascades. Adv Cancer Res 74 :49 –139</p>
<p>Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shananowitz J, Hunt DF, Weber MJ, Sturgill TW 1991 Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10 :885 –892</p>
<p>Rossi-Arnaud C, Grant SG, Chapman PF, Lipp HP, Sturani E, Klein R 1997 A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature 390 :281 –286</p>
<p>Calcium is arguably the most versatile and important intracellular messenger in neurons<span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span>(Berridge et al., 2000). Interestingly, although calcium may often promote neuronal death, it can also activate pathways that promote survival. For example, calcium can promote survival through a pathway involving activation of protein kinase B (PKB/Akt) by calcium/calmodulin-dependent protein kinase (Yano et al., 1998). Calcium is a prominent regulator of cellular responses to stress, activating transcription through the cyclic-AMP response element-binding protein (CREB), which can promote neuron survival in experimental models of developmental cell death (Hu et al., 1999). Calcium can also activate a rapid neuroprotective signalling pathway in which the calcium-activated actin-severing protein gelsolin induces actin depolymerization, resulting in suppression of calcium influx through membrane NMDA (N-methyl-d-aspartate) receptors and voltage-dependent calcium channels (Furukawa et al., 1997). This may occur through intermediary actin-binding proteins that interact with NMDA receptor and calcium channel proteins. Finally, signals such as calcium and secreted amyloid precursor protein-α (sAPP-α), which increase cyclic GMP production, can induce activation of potassium channels and the transcription factor NF-κB, and thereby increase resistance of neurons to excitotoxic apoptosis (Furukawa et al., 1996).</p>
<h4>How it is Measured or Detected</h4>
<p>An increase in [Ca<sup>2+]</sup>i was measured using Fluo3 AM as an indicator dye after the addition of metals (single or in mixture) to the culture wells following an optimized protocol (Arey et al., 2022). The fluorescent signals were read by fluorescence imaging plate reader Synergy HT (BioTek, Winooski, VT) (Rai and others 2010).</p>
<p>Briefly, Ca2+ levels in human astrocytes were monitored by fluorescence microscopy using the Ca2+ indicator fluo-4. Slices were incubated with fluo-4-AM (2–5 µL of 2 mM dye were dropped over the tissue, attaining a final concentration of 2–10 µM and 0.01% of pluronic) and Sulforhodamine 101 (100 µM) for 30–60 min at room temperature (Navarrete and others 2013). In these conditions, most of the Fluo-4-loaded cells were astrocytes as indicated by their SR101 staining (Nimmerjahn et al., 2004; Dombeck et al., 2007; Kafitz et al., 2008; Takata and Hirase 2008), and confirmed in some cases by their electrophysiological properties. Astrocytes were imaged with an Olympus FV300 laser-scanning confocal microscope or a CCD camera (Retiga EX) attached to the Olympus BX50WI microscope (Navarrete and others 2013).</p>
<p>Diversity of endogenous Ca2+ activity in a mature hippocampal astrocyte in situ: Ca2+ signals in cell body and processes are different. (A) Cumulative Ca2+ activity recorded in an astrocyte over a 165 s period revealed by the calcium indicator Fluo4-AM. The visible boundaries of the astrocyte are shown in white. Note the different intensities of spatially-<br />
confined local activity in the astrocyte cell body (s), primary process (p1) stemming from the soma and secondary processes (p2) branching from a primary process. Intensity of the<br />
normalized cumulative activity is expressed in arbitrary units (a.u.) and shown in pseudocolour, from dark (lowest) to white (highest). (B) Frequency map of the Ca2+ activity in the astrocyte during the 165 s period as in A. Activity is measured in individual pixels, expressed in mHz and color-coded from black (never active) to dark red (frequently active). Most of the activity is within the white boundaries and the most frequently active pixels are in defined small regions (arrowheads) of the primary and secondary processes (30 mHz), whereas pixels of the soma are less active (~10 mHz) (Volterra et al., 2014). </p>
<p>Free intracellular calcium ions were measured using the fluorescent calcium indicator FLUO-3/AM (Molecular probes, Eugene, OR, USA). Cells (4 × 10<sup>4</sup> cells/cm<sup>2</sup>) were seeded in 24-well plates for 24 h to reach 60%–70%, and then treated for 24 h with As(III) (0.5 and 1 mg/l), or coexposed to As(III) (1 mg/l) and F (2.5, 5, and 10 mg/l). After treatment, supernatant was collected and combined with trypsinized cells. Pelleted samples were resuspended in 500 μl of FLUO-3/AM (4 μmol/l) and incubated at 37 °C for 30 min. After centrifugation, cells were washed with HBSS (Hank's Buffered Salt Solution, Sigma), made up to 400 μl with HBSS and analyzed by flow cytometry. The signal from FLUO-3/AM bound to Ca<sup>2+</sup> was recorded using the Fl-1 channel (Rocha et al., 2011).</p>
<p>Fluo-4/AM was used as an intracellular free Ca<sup>2+</sup> fluorescent probe to analyze [Ca<sup>2+</sup>]<sub>i</sub> in Cd-exposed cerebral cortical neurons. In short, the harvested cells were incubated with Fluo-4/AM (5 µmol/L final concentration) for 30 min at 37°C in the dark, washed with PBS, and analyzed on a BD-FACS Aria flow cytometry. Intracellular [Ca<sup>2+</sup>]<sub>i</sub> levels were represented by fluorescent intensity. Fluorescent intensity was recorded by excitation at 494 nm and emission at 516 nm. The data were analyzed by Cell Quest program (Becton Dickinson), and the mean fluorescence intensity was obtained by histogram statistics (Yuan et al., 2013).</p>
<h4>References</h4>
<p>Arey BJ Seethala R Ma Z Fura A Morin J Swartz J Vyas V Yang W Dickson JK JrFeyen JH A novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo Endocrinology 2005 146 2015 2022</p>
<p>Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266</p>
<p>Berridge, M. J., Lipp, P. & Bootman, M. D. The versatility and universality of calcium signaling. Nature Rev. Mol. Cell Biol. 1, 11– 21 (2000).</p>
<p>Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW. Imaging large-scale neural activity with cellular resolution in awake, mobile mice, Neuron, 2007, vol. 56 (pg. 43-57)</p>
<p>Furukawa, K. et al. The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J. Neurosci. 17, 8178– 8186 (1997).</p>
<p>Furukawa, K., Barger, S. W., Blalock, E. M. & Mattson, M. P. Activation of K+ channels and suppression of neuronal activity by secreted β-amyloid-precursor protein. Nature 379, 74–78 (1996).</p>
<p>Hu, S. C., Chrivia, J. & Ghosh, A. Regulation of CBP-mediated transcription by neuronal calcium signaling. Neuron 22, 799– 808 (1999).</p>
<p>Kafitz KW, Meier SD, Stephan J, Rose CR. Developmental profile and properties of sulforhodamine 101-labeled glial cells in acute brain slices of rat hippocampus, J Neurosci Methods, 2008, vol. 169 (pg. 84-92)</p>
<p>Marta Navarrete and others, Astrocyte Calcium Signal and Gliotransmission in Human Brain Tissue, Cerebral Cortex, Volume 23, Issue 5, May 2013, Pages 1240–1246, https://doi.org/10.1093/cercor/bhs122</p>
<p>Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo, Nat Methods, 2004, vol. 1 (pg. 31-37)</p>
<p>R.A. Rocha, J.V. Gimeno-Alcañiz, R. Martín-Ibañez, J.M. Canals, D. Vélez, V. Devesa, Arsenic and fluoride induce neural progenitor cell apoptosis, Toxicology Letters, Volume 203, Issue 3, 2011, Pages 237-244, ISSN 0378-4274, https://doi.org/10.1016/j.toxlet.2011.03.023.</p>
<p>Takata N, Hirase H. Cortical layer 1 and layer 2/3 astrocytes exhibit distinct calcium dynamics in vivo., PLoS ONE, 2008, vol. 3 pg. e2525</p>
<p>Volterra, Andrea, Nicolas Liaudet, and Iaroslav Savtchouk. "Astrocyte Ca2+ signalling: an unexpected complexity." Nature Reviews Neuroscience 15.5 (2014): 327-335.</p>
<p>Yano, S., Tokumitsu, H. & Soderling, T. R. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 396, 584–587 (1998).</p>
<p>Yuan Y, Jiang C-y, Xu H, Sun Y, Hu F-f, Bian J-c, et al. (2013) Cadmium-Induced Apoptosis in Primary Rat Cerebral Cortical Neurons Culture Is Mediated by a Calcium Signaling Pathway. PLoS ONE 8(5): e64330. https://doi.org/10.1371/journal.pone.0064330</p>
<td><a href="/aops/499">Aop:499 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via disrupted neurotransmitter release</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/471">Aop:471 - Various neuronal effects induced by elavl3, sox10, and mbp</a></td>
<td><a href="/aops/471">Aop:471 - Neuron defect induced early behavioral change</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/564">Aop:564 - DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<p>Any of various neurotransmitters or indicators of neurotransmission. </p>
</div>
<h4>How it is Measured or Detected</h4>
<div>
<p>Weighed brain tissues were homogenized in a Potter-Elvehjem type A homogenizer with a teflon pestle using cold acidified n-butanol. The biogenic amines were extracted and estimated according to the procedure of Sadavongvivad (1970). The recovery experiments were done simultaneously. Recoveries for different standards were 92 + 3% for dopamine (DA), 80+ 5% for norepinephrine (NE) and 90 + 5% for 5-hydroxytryptamine (5-HT). Fluorescence was measured in a Aminco SPF-500 spectrofluorometer (Chandra et al., 1981).</p>
<p>BDNF quantitative real-time PCR. Hippocampal neuronal cultures were exposed to normal bath solution or 1.0 or 2.0μM Pb2+ for 5 days, and subsequently RNA was harvested according to manufacturer’s instructions (RNeasy; Qiagen), quantified by reading the absorbance at 260 nm, and converted to complementary DNA (cDNA) using 1 μg RNA according to manufacturer’s instructions (High Capacity Reverse Transcription Kit 4368814; Applied Biosystems). Quantitative real-time PCR (q-rtPCR) was performed in triplicate using TaqMan Gene Expression Assays (Applied Biosystems) with 50 ng cDNA using the following probes: Actin (Rat, Rn00667869_m1; Applied Biosystems) and BDNF exon I, exon II, exon IV, and exon IX (Applied Biosystems). Data were analyzed as previously described (Livak and Schmittgen, 2001), and results were expressed as fold change in gene expression relative to<sup> control <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">(Stansfield and others 2012)</span></span>.</sup></p>
<p>BDNF release via ELISA. Sandwich ELISAs were performed on DIV12 cell culture media using the BDNF Emax ImmunoAssay System kit (Promega, Madison, WI) according to the manufacturer’s instructions. BDNF content was interpolated from standard curve runs for each plate (linear range of 7.8–500 pg/ml). BDNF protein content was divided by total protein for each sample to determine the number of picograms of peptide per microgram of total protein (Stansfield and others 2012).</p>
<p>In vivo microdialysis is a well-established method for monitoring the extracellular levels of neurotransmitters in the CNS. This technique has been used extensively in neuroscience for almost 30 years (Westerink 1995; Ungerstedt 1991; Robinson 1991; Benveniste 1989; Benveniste and Huttemeier 1990; Di Chiara 1990). Microdialysis allows online estimates of neurotransmitters in living animals and is a suitable method for monitoring the extracellular levels of neurotransmitters during local administration of pharmacological agents (<span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Hammarlund-Udenaes 2000)</span></span>. Older alternative <em>in vivo</em> methods for the study of neurotransmitter release are the push–pull technique used in the brain, (Singewald and Philippu 1998) spinal cord, (Zachariou and Goldstein 1997) and intrathecal space (Yaksh and Tyce 1980).</p>
</div>
<h4>References</h4>
<div>
<p>Benveniste H, Huttemeier PC. Microdialysis: theory and application. Progr Neurobiol. 1990;35:195.</p>
<p>Benveniste H. Brain microdialysis. J Neurochem. 1989;52:1667.</p>
<p>Chandra, Satya V., et al. "Behavioral and neurochemical changes in rats simultaneously exposed to manganese and lead." Archives of Toxicology 49 (1981): 49-56.</p>
<p>Di Chiara G. In vivo brain dialysis of neurotransmitters. Trends Pharmacol Sci. 1990;11:116.</p>
<p>Hammarlund-Udenaes M. The use of microdialysis in CNS drug delivery studies: pharmacokinetic perspectives and results with analgesics and antiepileptics. Adv Drug Deliv Rev. 2000;45:283.</p>
<p>Kirstie H. Stansfield and others, Dysregulation of BDNF-TrkB Signaling in Developing Hippocampal Neurons by Pb2+: Implications for an Environmental Basis of Neurodevelopmental Disorders, Toxicological Sciences, Volume 127, Issue 1, May 2012, Pages 277–295, https://doi.org/10.1093/toxsci/kfs090</p>
<p>Robinson TJ. Microdialysis in the Neurosciences. Vol. 7. Elsevier; Amsterdam: 1991. Techniques in the behavioral and neural sciences.</p>
<p>Singewald N, Philippu A. Release of neurotransmitters in the locus coeruleus. Progr Neurobiol. 1998;56:237.</p>
<p>Ungerstedt U. Microdialysis: principles and applications for studies in animals and man. J Intern Med. 1991;230:365</p>
<p>Westerink BH. Brain microdialysis and its application for the study of animal behaviour. Behav Brain Res. 1995;70:103.</p>
<p>Yaksh TL, Tyce GM. Resting and K+-evoked release of serotonin and norephinephrine in vivo from the rat and cat spinal cord. Brain Res. 1980;192:133.</p>
<p>Zachariou V, Goldstein BD. Dynorphin-(1-8)inhibits the release of substance P-like immunoreactivity in the spinal cord of rats following a noxious mechanical stimulus. Eur J Pharmacol. 1997;323:159.</p>
</div>
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/341">Event: 341: Impairment, Learning and memory</a></h4>
<h5>Short Name: Impairment, Learning and memory</h5>
<td><a href="/aops/13">Aop:13 - Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities</a></td>
<td>AdverseOutcome</td>
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<td><a href="/aops/48">Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.</a></td>
<td>AdverseOutcome</td>
</tr>
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<td><a href="/aops/54">Aop:54 - Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment </a></td>
<td>AdverseOutcome</td>
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<td><a href="/aops/77">Aop:77 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony death/failure 1</a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/78">Aop:78 - Nicotinic acetylcholine receptor activation contributes to abnormal role change within the worker bee caste leading to colony death failure 1</a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/87">Aop:87 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure </a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/88">Aop:88 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure via abnormal role change within caste</a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/89">Aop:89 - Nicotinic acetylcholine receptor activation followed by desensitization contributes to abnormal foraging and directly leads to colony loss/failure</a></td>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/90">Aop:90 - Nicotinic acetylcholine receptor activation contributes to abnormal roll change within the worker bee caste leading to colony loss/failure 2</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/12">Aop:12 - Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development leads to neurodegeneration with impairment in learning and memory in aging</a></td>
<td>AdverseOutcome</td>
</tr>
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<td><a href="/aops/99">Aop:99 - Histamine (H2) receptor antagonism leading to reduced survival</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/17">Aop:17 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/442">Aop:442 - Binding to voltage gate sodium channels during development leads to cognitive impairment </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/475">Aop:475 - Binding of chemicals to ionotropic glutamate receptors leads to impairment of learning and memory via loss of drebrin from dendritic spines of neurons</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/483">Aop:483 - Deposition of Energy Leading to Learning and Memory Impairment</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/490">Aop:490 - Co-activation of IP3R and RyR leads to socio-economic burden through reduced IQ and non-cholinergic mechanisms</a></td>
<td><a href="/aops/490">Aop:490 - Co-activation of IP3R and RyR leads to reduced IQ and increased socio-economic burden through non-cholinergic mechanisms</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/499">Aop:499 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via disrupted neurotransmitter release</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/520">Aop:520 - Retinoic acid receptor agonism during neurodevelopment leading to impaired learning and memory</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/525">Aop:525 - Reduced oligodendrocyte differentiation during neurodevelopment leading to impaired learning and memory</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/533">Aop:533 - Retinoic acid receptor antagonism during neurodevelopment leading to impaired learning and memory</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/610">Aop:610 - Decreased thyroid hormone levels in the brain regulated via transport, metabolism and TR activation leading to decreased cognition and motor function</a></td>
<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>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><strong>Life stage applicability</strong>: This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). </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><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). </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>
<p><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). </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><span style="color:#3498db">Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non-associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behavior. On the other hand, non-associative learning can be defined as an alteration in the behavioral response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning. </span></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><span style="color:#3498db">The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterized by the non-conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer). </span></p>
<p><span style="color:#3498db">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 </span></p>
<p> </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><span style="color:#3498db">brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).While the prefrontal cortex and frontostriatal neural circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014). </span></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><span style="color:#3498db">For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioral tests described below. </span></p>
<h4>How it is Measured or Detected</h4>
<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>In laboratory animals: in rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, Hebb-Williams maze, passive avoidance and Spontaneous alternation and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows. </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>RAM, Barnes, MWM,Hebb-Williams mazeare examples of spatial tasks, animals are required to learn the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze), or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). The Hebb-Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004). </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>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>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>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>Operant Responding.Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). </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>In humans: A variety of standardized learning and memory tests have been developed for human neuropsychological testing, including children (Rohlman et al., 2008). These include episodic autobiographical memory, perceptual motor tests, short and long term memory tests, working memory tasks, word pair recognition memory; object location recognition memory. Some have been incorporated in general tests of intelligence (IQ) such as the Wechsler Adult Intelligence Scale (WAIS) and the Wechsler. </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>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>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>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>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>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>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>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>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>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>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>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><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> </p>
<p>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>Attentional set-shifting (ATSET) task.Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015). </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>In Honey Bees: For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012; LaLone et al., 2017). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012). After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to </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>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>
<h4>Regulatory Significance of the AO</h4>
<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>A prime example of impairments in learning and memory as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD TG 426) as well as OECD TG 443 (OECD, 2018) both require testing of learning and memory (USEPA, 1998; OECD, 2007) advising to use the following tests passive avoidance, delayed-matching-to-position for the adult rat and for the infant rat, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and acquisition and retention of schedule-controlled behavior. These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009). </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>
<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>
<p> </p>
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<p> </p>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2942">Relationship: 2942: Activation of MEK, ERK1/2 leads to Increase, intracellular calcium</a></h4>
<p>Astrocytes are networked together by a series of gap junctions permitting to propagate Ca<sup>2+</sup> waves through the linked network (Lobsiger and Cleveland 2007), and Ca2+-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells (Verderio et al., 2001). Metal mixture (MM) induced alteration in astrocyte morphology may influence [Ca<sup>2+</sup>]i (Barres et al., 1989); in contrast, an increase in [Ca<sup>2+</sup>]<sub>i</sub> may also play a key role in altering astrocyte cytoskeleton, affecting the glia-neuron interaction (Shelton et al., 2000).</p>
<p>Inhibition of GFAP immunoreactivity by MM in developing brain appears to be caused by astrocyte apoptosis. In primary cultures of astrocytes, our data show that MM synergistically induced apoptosis (Rai and others 2010). This was manifested by the activation of MEK/ERK, followed by the activation of JNK pathways, which then enhanced intracellular Ca2+ levels and subsequently ROS generation.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Empirical Evidence</strong>
<p>We treated the astrocytes with a metal-mixture (MM) of arsenic, cadmium, and lead and observed that the MM triggered [Ca<sup>2+</sup>]<sub>i</sub> release (Rai and others 2010). The [Ca2+]<sub>i</sub> release reached its peak after 30 min of MM treatment. Similarly, MM triggered ROS generation, and the ROS generation reached its peak after 1 h of MM treatment. To investigate whether the [Ca<sup>2+</sup>]<sub>i</sub> release was ROS, ERK1/2, or JNK1/2 –dependent, we incubated the MM-treated astrocytes with an antioxidant (a-tocopherol, 200 lg/ml), PD98059 (10lM), or SP600125 (10lM). a-Tocopherol itself was nontoxic. We observed that PD98059 (10lM) or SP600125 (10lM) suppressed [Ca<sup>2+</sup>]<sub>i</sub> release, but a-tocopherol (200 lg/ml) did not. This suggested that [Ca<sup>2+</sup>]<sub>i</sub> release in MM-treated astrocytes was ERK1/2 and JNK1/2 dependent (Rai and others 2010).</p>
<p>Yael and Breitbart (2015) demonstrated for the first time that mouse sperm ERK1/2 is activated upon ZP addition, and that ERK1/2 mediates the elevation of intracellular Ca2+ in the sperm cell prior to the occurrence of the acrosome reaction. The fact that the acrosome reaction, induced by the Ca<sup>2+</sup>-ionophore A23187, was not inhibited by U0126 suggests that ERK1/2 mediates the acrosome reaction by activating Ca2+ transport into the cell. Direct determination of intracellular [Ca<sup>2+</sup>] revealed that Ca2+ influx induced by EGF or ZP was completely blocked by U0126. Thus, it has been established that the increase in ERK1/2 phosphorylation/activation in response to ZP or by activation of the EGF receptor (EGFR) by EGF, is a key event for intracellular Ca<sup>2+</sup> elevation and the subsequent occurrence of the acrosome reaction (Jaldety et al., 2015).</p>
<p>To examine the relationship between Ca<sup>2+</sup> and Erk1/2 signaling, Levin and Borodinsky (2022) inhibited Mek1/2 with PD0325901 and found that this prevents the injury-induced increase in Ca<sup>2+</sup> activity in cells lateral to the axial musculature across the entire 800 µm-wide region measured. This suggests that injury-induced Erk1/2 activation recruits Ca<sup>2+</sup> activity to promote regeneration of the larval tail. Consistent with recruitment of Ca<sup>2+</sup> activity across a wide region of tail, activated Erk1/2 is also present in at least the posterior 800 µm of stump (Levin et al., 2022). However, unlike Ca<sup>2+</sup> activity, Erk1/2 signaling at 20 mpa is activated in a gradient. This could mean that even the lowest level of Erk1/2 signal measured in 800 µm of amputated tail is sufficient to induce the Ca<sup>2+</sup> response, or that a signal is propagated anteriorly from the cells adjacent to the amputation where injury induces high Erk1/2 activation (Levin et al., 2022).</p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Time-scale</strong>
<p>Exposures were conducted for 2 min, 5 min, 10 min, 30 min, 1 h, 2 h, and 24 h. The [Ca2+]<sub>i</sub> release reached its peak after 30 min of MM treatment (Rai and others 2010).</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>The activity of many protein kinases is modulated by Ca2+ and/or Ca2+/calmodulin either directly (PKC, CaM kinase II) or indirectly (PKA via stimulation of adenylyl cyclase and phosphodiesterase by Ca2+/calmodulin) (Kern et al., 1995). Therefore, the effects of Ca2+ and protein kinases on cytoskeletal proteins and neurite initiation are likely to be mediated, at least in part, by changes in protein phosphorylation (Kern et al., 1995).</p>
<h4>References</h4>
<p>Asit Rai and others, Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions, Toxicological Sciences, Volume 118, Issue 2, December 2010, Pages 586–601, https://doi.org/10.1093/toxsci/kfq266</p>
<p>Barres, B. A., L. L. Chun, and Corey. "Calcium current in cortical astrocytes: induction by cAMP and neurotransmitters and permissive effect of serum factors." Journal of Neuroscience 9.9 (1989): 3169-3175.</p>
<p>Jaldety, Yael, and Haim Breitbart. "ERK1/2 mediates sperm acrosome reaction through elevation of intracellular calcium concentration." Zygote 23.5 (2015): 652-661.</p>
<p>Kern, Marcey, and Gerald Audesirk. "Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation." Toxicology and applied pharmacology 134.1 (1995): 111-123.</p>
<p>Levin, Jacqueline B., and Laura N. Borodinsky. "Injury-induced Erk1/2 signaling tissue-specifically interacts with Ca2+ activity and is necessary for regeneration of spinal cord and skeletal muscle." Cell calcium 102 (2022): 102540.</p>
<p>Lobsiger, C. S., and Cleveland, D. W. (2007). Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat. Neuro-sci. 10, 1355–1360.</p>
<p>Shelton, Marilee K., and Ken D. McCarthy. "Hippocampal astrocytes exhibit Ca2+‐elevating muscarinic cholinergic and histaminergic receptors in situ." Journal of neurochemistry 74.2 (2000): 555-563.</p>
<p>Verderio, Claudia, and Michela Matteoli. "ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-γ." The Journal of Immunology 166.10 (2001): 6383-6391.</p>
<p>While intracellular Ca regulation is an important aspect of a number of processes in a variety of cells, it is particularly critical in nerve cell terminals where Ca mediates transmitter release (Augustine et al., 1987). Many synaptic connections during brain development involve calcium signaling, which directs structural as well as functional adaptation in neurons (Lohmann 2009; Michaelson and Lohmann 2010) and astrocytes (Navarette et al., 2013) to establish synaptic selectivity in the developing brain (Katherine von Stackelberg 2015). While astrocytes have long been known to support neuronal signaling, there is increasing evidence that astrocytes detect synaptic activity and engage in reciprocal signaling with neurons, again based on variations in intracellular Ca<sup>2+</sup> (Volterra et al., 2014; Barkera and Ullian 2008).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Lead (1-30 uM) was also observed to induce a concentration-dependent release of dopamine from striatal synaptosomes under conditions of spontaneous release (Minnema et al., 1986). Similar lead induced neurotransmitter release has been demonstrated for acetylcholine at the neuromuscular junction, as reflected by increase miniature-end-plate potentials (Cooper and Manalis 1983), and in cortical synaptosomes (Suszkiw et al., 1984). Although the mechanisms by which lead induces transmitter release are unresolved, the increased release may result from an increase in intrasynaptosomal free calcium which has been shown to increase release (Katz 1969).</p>
<strong>Empirical Evidence</strong>
<p>Lead could act to increase spontaneous transmitter release by increasing the intraneuronal ionized Ca concentration (Kolton and Yarri 1982). One means by which the intraneuronal free Ca could be elevated is by inhibition of Ca extrusion; specifically, inhibition of the Mg2+-dependent Ca-ATPase (Minnema et al., 1988). The extrusion of Ca by Ca-ATPase at the plasma membrane is the dominant means by which the intraneuronal Ca concentration is maintained during “resting” conditions (Snelling and Nicholls 1985). Although Pb has been reported to be a weak inhibitor of this enzyme (Thompson and Nechay 1981), the Pb-induced increase in 45Ca efflux observed in the current study (Minnema et al., 1988) would not be expected if Ca-ATPase inhibition is the mechanism by which Pb increases transmitter release. The similar concentration/release effects and temporal relationships between transmitter release and 45Ca efflux suggest that Pb may displace bound Ca from intraneuronal Ca sources (Minnema et al., 1988). The slight temporal differences in onset and peak effects (i.e., the effect of Pb on transmitter release precedes its effect on 45Ca efflux) are consistent with the view that Pb increases the intraneuronal ionized Ca concentration, which would first interact at the intraneuronal site mediating transmitter release, and subsequently this Ca would be extruded from the nerve ending (Minnema et al., 1988).</p>
<p>We next investigated the consequences of astrocyte Ca<sup>2+</sup> signal on human neurons. In hippocampal slices, local application of ATP evoked astrocyte Ca2+ elevations that propagated as a wave throughout the Stratum radiatum reaching the Stratum pyramidale, and then evoking Ca<sup>2+ </sup>elevations in pyramidal neurons after a conspicuous delay from the initial astrocyte Ca<sup>2+</sup> elevations, suggesting that astrocyte Ca<sup>2+</sup> stimulates the release of gliotransmitters that acting on transmitter receptors affect the intracellular Ca<sup>2+</sup> levels in human neurons (Navarette et al., 2013).</p>
<p>Local application of ATP, which elevated Ca<sup>2+</sup> levels in astrocytes, also increased the frequency of slow inward currents (SIC) in both hippocampal and cortical neurons. While SIC frequency was insensitive to TTX (n = 3 neurons), SICs were abolished by 50 µM AP5, indicating that they were independent of action potential-evoked neurotransmitter release and that they were mediated by NMDARs. Therefore, in agreement with compelling evidence obtained in rodents (Parri et al., 2001; Fellin, Tommaso, et al. 2004; Gertrudis and Araque 2005; Navarrete et al., 2008; Shigetomi, Eiji, et al. 2008; Bardoni, Rita, et al., 2010; Sasaki, Takuya, et al. 2011), Ca<sup>2+</sup> elevations in human astrocytes stimulate the release of glutamate that activates NMDARs in neurons, indicating the existence of gliotransmission and astrocyte-to-neuron communication in human brain tissue (Navarette et al., 2013).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Synaptotagmin I (Syt) is a Ca<sup>2+</sup> -sensing protein found in neurotransmitter vesicles and is responsible for promoting vesicular fusion in the presence of Ca<sup>2+</sup> signaling (Chicka et al., 2008). Pb<sup>2+</sup> bound Syt with 1000-fold higher affinity than Ca<sup>2+</sup>, which may prevent detection of Ca<sup>2+</sup> signaling essential to neurotransmission (Bouton et al., 2001). Although Pb<sup>2+</sup> exposure did not affect Syt protein expression in cultured hippocampal neurons (Neal et al., 2010), it is possible that Pb<sup>2+</sup> may interfere with the Ca<sup>2+</sup>-sensing ability of Syt in neurons, thus masking the cellular signal for Ca<sup>2+</sup>-dependent vesicular release (Neal and Guilarte 2010).</p>
<p>Pb<sup>2+</sup> interactions with Syt may be related to the ability of Pb<sup>2+</sup> to mimic Ca<sup>2+ </sup>(Neal and Guilarte 2010). Pb<sup>2+</sup> has an ionic radius of 1.2 Å, which is similar to the ionic radius of Ca<sup>2+</sup> (0.99 Å) (Chao et al., 1984; Garza et al., 2006). The positive charges and high electronegativity (2.33 on the Pauling scale) of Pb<sup>2+</sup> may allow it to interact with the same residues on Ca<sup>2+</sup> binding sites that interact with Ca<sup>2+</sup> ions (Garza et al., 2006). Pb<sup>2+</sup> has been shown to interact with several neuronal intracellular Ca<sup>2+</sup>-binding proteins in addition to Syt (described above), such as the Ca<sup>2+</sup>-binding protein calmodulin (CaM) (Chao et al., 1984; Habermann et al., 1983; Kern et al., 2000), the CaM/Ca<sup>2+</sup>-dependent phosphatase calcineurin (Kern and Audesirk 2000), CaMKII (Toscano et al., 2005), and protein kinase C (Simons 1993; Sun et al., 1999; Toscano and Schanne 2000; Long et al., 1994), suggesting that Ca<sup>2+</sup> mimicry may be a common characteristic of Pb<sup>2+</sup> toxicity (Bressler et al., 1999; Marchetti 2003; Richardt et al., 1986). Thus, the ability of Pb<sup>2+</sup> to mimic Ca<sup>2+</sup> may interfere with normal synaptic signaling events (Neal and Guilarte 2010).</p>
<p>Another hypothesis regarding the disruption of neurotransmission is that Pb<sup>2+</sup> may interfere with Ca<sup>2+</sup> signals by inhibiting Ca<sup>2+</sup> channels (Xiao et al., 2006; Braga et al., 1999; <sup>35)</sup>. Neurotransmission relies on the influx of Ca<sup>2+</sup> from P/Q-, N-, and to some extent R-type voltage-gated Ca<sup>2+</sup> channels (VGCCs) (Xu et al., 2007). Pb<sup>2+</sup> has been shown to inhibit VGCCs in recombinant systems with high affinity (Peng et al., 2002). Furthermore, removal of extracellular Ca<sup>2+</sup> resulted in identical effects on IPSC frequency as Pb<sup>2+</sup> exposure, suggesting that the Pb<sup>2+</sup>-induced inhibition of IPSC frequency is via reduction of Ca<sup>2+</sup> influx through VGCCs (Xiao et al., 2006). Inhibition of presynaptic VGCCs may prevent the necessary rise in internal Ca<sup>2+</sup> required for fast, Ca<sup>2+</sup>-dependent vesicular release, thus interfering with neurotransmission (Neal and Guilarte 2010).</p>
<p>Cadmium may block the influx of Ca<sup>2+</sup> through membrane channels into the nerve terminal following the action potential, these decrease in calcium influx caused by Cd would be associated with an altered transmitter release (Antonio et al., 1999).</p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Time-scale</strong>
<p>Calcium efflux and induced spontaneous transmitter release occur on a seconds to minutes time-scale (Minnema et al., 1988).</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>It has been clear for quite some time that influx of calcium at the synapse mediates synaptic plasticity in adult as well as developing neurons (Malenka et al., 1988). Despite this long-standing appreciation of the importance of calcium signaling for synaptic plasticity, it is virtually unknown what the properties of calcium transients are that determine whether a synapse becomes potentiated or depressed (Malenka and Bear 2004). Some models suggest that moderate increases in calcium may activate primarily phosphatases (e.g., calcineurin and protein phosphatase-1) that in turn facilitate synaptic depression (Mansuy and Shenolikar 2006). In contrast, the activation of kinases (e.g., calcium/calmodulin-dependent protein kinase II, CaMKII) by high-amplitude calcium transients may favor potentiation (Lisman et al., 2002). This is in fact an interesting parallel to the regulation of attractive vs. repulsive axon guidance by calcium: larger calcium transients can activate CaMKII and induce turns toward the side of calcium elevation, whereas smaller calcium increases activate the phosphatases calcineurin and phosphatase-1 and trigger repulsive turns (Wen et al., 2004; Zheng and Poo 2007).</p>
<h4>References</h4>
<div>
<p>Augustine, George J., Milton P. Charlton, and Stephen J. Smith. "Calcium action in synaptic transmitter release." Annual review of neuroscience 10.1 (1987): 633-693.</p>
<p>Bardoni, Rita, et al. "Glutamate‐mediated astrocyte‐to‐neuron signalling in the rat dorsal horn." The Journal of physiology 588.5 (2010): 831-846.</p>
<p>Barker AJ, Ullian EM. New roles for astrocytes in developing synaptic circuits. Communicative & Integrative Biology, 2008; 1(2):207–211.</p>
<p>Bouton CMLS, Frelin LP, Forde CE, Godwin HA, Pevsner J (2001) Synaptotagmin i is a molecular target for lead. J Neurochem 76:1724–1735</p>
<p>Braga MFM, Pereira EFR, Albuquerque EX (1999) Nanomolar concentrations of lead inhibit glutamatergic and GABAergic transmission in hippocampal neurons. Brain Res 826:22–34</p>
<p>Bressler J, Kim KA, Chakraborti T, Goldstein G (1999) Molecular mechanisms of lead neurotoxicity. Neurochem Res 24:595–600</p>
<p>Chao SH, Suzuki Y, Zysk JR, Cheung WY (1984) Activation of calmodulin by various metal cations as a function of ionic radius. Mol Pharmacol 26:75–82</p>
<p>Chicka MC, Hui E, Lui H, Chapman ER (2008) Synaptotagmin arrests the snare complex before triggering fast, efficient membrane fusion in response to Ca2+. Nat Struct Mol Biol 15:827–835</p>
<p>Cooper, G., and Manalis, R. (1983). Influence of heavy metals on synaptic transmission: A review. Neurotoxicology 4, 69-84.</p>
<p>Fellin, Tommaso, et al. "Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors." Neuron 43.5 (2004): 729-743.</p>
<p>Garza A, Vega R, Soto E (2006) Cellular mechanisms of lead neurotoxicity. Med Sci Monit 12:RA57–RA65</p>
<p>Habermann E, Crowell K, Janicki P (1983) Lead and other metals can substitute for Ca2+ in calmodulin. Arch Toxicol 54:61–70</p>
<p>Katherine von Stackelberg & Elizabeth Guzy & Tian Chu & Birgit Claus Henn, 2015. Exposure to Mixtures of Metals and Neurodevelopmental Outcomes: A Multidisciplinary Review Using an Adverse Outcome Pathway Framework, Risk Analysis, John Wiley & Sons, vol. 35(6), pages 971-1016, June.</p>
<p>Katz, B. (1969). The Release of Neural Transmitter Substances. Thomas, Springfield, Ill.</p>
<p>Kern M, Audesirk G (2000) Stimulatory and inhibitory effects of inorganic lead on calcineurin. Toxicology 150:171–178</p>
<p>Kern M, Wisniewski M, Cabell L, Audesirk G (2000) Inorganic lead and calcium interact positively in activation of calmodulin. Neurotoxicology 3:353–363</p>
<p>Kolton, L., and Yarri, Y. (1982). Sites of action of lead on spontaneous transmitter release from motor nerve terminals. Isr. J. Med. Sci. 18, 165-17</p>
<p>Lisman, J., Schulman, H., & Cline, H. (2002). The molecular basis of CaMKII function in synaptic and behavioural memory. Nature Reviews. Neuroscience, 3, 175–190.</p>
<p>Lohmann C. Calcium signaling and the development of specific neuronal connections. Progress in Brain Research, 2009; 175:443–452.</p>
<p>Lohmann, Christian. "Calcium signaling and the development of specific neuronal connections." Progress in brain research 175 (2009): 443-452.</p>
<p>Long GJ, Rosen JF, Schanne FAX (1994) Lead activation of protein kinase C from rat brain. J Biol Chem 269:834–837</p>
<p>M.T. Antonio, I. Corpas, M.L. Leret Neurochemical changes in newborn rat's brain after gestational cadmium and lead exposure Toxicol. Lett., 104 (1999), pp. 1-9</p>
<p>Malenka, R. C., & Bear, M. F. (2004). LTP and LTD: an embarrassment of riches. Neuron, 44, 5–21.</p>
<p>Malenka, R. C., Kauer, J. A., Zucker, R. S., & Nicoll, R. A. (1988). Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission. Science, 242, 81–84.</p>
<p>Mansuy, I. M., & Shenolikar, S. (2006). Protein serine/threonine phosphatases in neuronal plasticity and disorders of learning and memory. Trends in Neurosciences, 29, 679–686.</p>
<p>Marchetti C (2003) Molecular targets of lead in brain neurotoxicity. Neurotox Res 5:221–236</p>
<p>Michaelson K, Lohmann C. Calcium dynamics at developing synapses: Mechanisms and functions. European Journal of Neuroscience, 2010; 32:218–223.</p>
<p>Minnema, Daniel J., I. A. Michaelson, and G. P. Cooper. "Calcium efflux and neurotransmitter release from rat hippocampal synaptosomes exposed to lead." Toxicology and applied pharmacology 92.3 (1988): 351-357.</p>
<p>Minnema, Daniel J., Robert D. Greenland, and I. Arthur Michaelson. "Effect of in vitro inorganic lead on dopamine release from superfused rat striatal synaptosomes." Toxicology and applied pharmacology 84.2 (1986): 400-411.</p>
<p>Navarette M, Perea G, Maglio L, Pastor J, de Sola RG, Araque A. Astrocyte calcium signal and gliotransmission in human brain tissue. Cerebral Cortex, 2013; 23:1240–1246.</p>
<p>Navarrete, Marta, and Alfonso Araque. "Endocannabinoids mediate neuron-astrocyte communication." Neuron 57.6 (2008): 883-893.</p>
<p>Neal AP, Stansfield KH, Worley PF, Thompson RE, Guilarte TR (2010) Lead exposure during synaptogenesis alters vesicular proteins and impairs vesicular release: potential role of NMDA receptor-dependent BDNF signaling. Toxicol Sci 116:249–263</p>
<p>Neal, A.P., Guilarte, T.R. Molecular Neurobiology of Lead (Pb2+): Effects on Synaptic Function. Mol Neurobiol 42, 151–160 (2010). https://doi.org/10.1007/s12035-010-8146-0</p>
<p>Parri, H. Rheinallt, Timothy M. Gould, and Vincenzo Crunelli. "Spontaneous astrocytic Ca2+ oscillations in situ drive NMDAR-mediated neuronal excitation." Nature neuroscience 4.8 (2001): 803-812.</p>
<p>Peng S, Hajela RK, Atchison WD (2002) Characteristics of block by Pb2+ of function of human neuronal L-, N-, and R-type Ca2+ channels transiently expressed in human embryonic kidney 293 cells. Mol Pharmacol 62:1418–1430</p>
<p>Perea, Gertrudis, and Alfonso Araque. "Properties of synaptically evoked astrocyte calcium signal reveal synaptic information processing by astrocytes." Journal of Neuroscience 25.9 (2005): 2192-2203.</p>
<p>Richardt G, Federolf G, Habermann E (1986) Affinity of heavy metal ions to intracellular Ca2+-binding proteins. Biochem Pharmacol 35:1331–1335</p>
<p>Shigetomi, Eiji, et al. "Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons." Journal of Neuroscience 28.26 (2008): 6659-6663.</p>
<p>Simons TJB (1993) Lead-calcium interactions in cellular lead toxicity. Neurotoxicology 14:77–86</p>
<p>Snelling, R., and Nicholls, D. (1985). Calcium efflux and cycling across the synaptosomal plasma membrane. Biochem. J. 226,225-23 1</p>
<p>Sun X, Tian X, Tomsig JL, Suszkiw JB (1999) Analysis of differential effects of Pb2+ on protein kinase C isozymes. Toxicol Appl Pharmacol 156:40–45</p>
<p>Suszkiw et al., (1984) Effects of Pb2+ and Cd2+ on acetylcholine release and Ca2+ movements in synaptosomes subcellular fractions from rat brain and torpedo electric organ. Brain Res. 323, 3 1-46.</p>
<p>Thompson, J., and Nechay, B. (1981). Inhibition by metals of canine renal calcium, magnesium-activated adenosinetriphosphatase. J. Toxicol. Environ. Health 7, 901-908</p>
<p>Toscano CD, O’Callaghan JP, Guilarte TR (2005) Calcium/calmodulin-dependent protein kinase II activity and expression are altered in the hippocampus of Pb2+-exposed rats. Brain Res 1044:51–58</p>
<p>Toscano CD, Schanne FAX (2000) Lead-induced activation of protein kinase C in rat brain cortical synaptosomes. Ann NY Acad Sci 919:307–311</p>
<p>Volterra A, Liaudet N, Savtchouk I. Astrocyte Ca2+ signalling: An unexpected complexity. Nature Reviews Neuroscience, 2014; 15:327–335,</p>
<p>Wen, Z., Guirland, C., Ming, G. L., & Zheng, J. Q. (2004). A CaMKII/calcineurin switch controls the direction of Ca(2+)-dependent growth cone guidance. Neuron, 43, 835–846.</p>
<p>Xiao C, Gu Y, Zhou CY, Wang L, Zhang MM, Ruan DY (2006) Pb2+ impairs GABAergic synaptic transmission in rat hippocampal slices: a possible involvement of presynaptic calcium channels. Brain Res 1088:93–100</p>
<p>Xu J, He L, Wu LG (2007) Role of Ca2+ channels in short-term synaptic plasticity. Curr Opin Neurobiol 17:352–35</p>
<p>Zheng, J. Q., & Poo, M. M. (2007). Calcium signaling in neuronal motility. Annual Review of Cell and Developmental Biology, 23, 375–404.</p>
</div>
</div>
<div>
<h4><a href="/relationships/2955">Relationship: 2955: Disruption, neurotransmitter release leads to Impairment, Learning and memory</a></h4>
<p>Neurotransmitters and their receptors are essential for brain functioning, learning, and memory. Catecholamines, including dopamine and norepinephrine, are the main neurotransmitters that mediate a variety of central nervous system (CNS) functions, such as motor control, cognition, emotion, memory processing, and endocrine modulation, determined by recent molecular genetic approaches in mice (Handra et al., 2019).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The N-methyl-D-aspartate receptor (NMDAR) plays an essential role in hippocampus-mediated learning and memory, based on studies showing that intra-ventricular administration of an NMDAR antagonist (aminophosphonovaleric acid (APV)) in rats resulted in spatial learning impairments similar to those encountered with hippocampal lesions (Morris et al., 1982; Morris et al., 1986).</p>
<p>Memory acquisition is considered to involve both short-term changes in electrical properties and long-term structural alterations in synapses. Short-term changes may include LTP and LTD whereas long-term morphological alterations may involve synaptogenesis and neuropil growth (Burns and Augustine 1995; Edwards 1995). Since BDNF significantly modulates both forms of synaptic changes and the expression is upregulated during memory acquisition, as described above, it may play a role in learning and memory (Lo DC 1995; Thoenen 1995; McAllister et al., 1999).</p>
<p>Cortical acetylcholine release increases (1) during acquisition but not during recall of a rewarded operant behavior (Orsetti et al., 1996), (2) during acquisition of operant tasks when demands on attentional processing are high (Muir et al., 1996),<sup> </sup>(3) during conditioned taste aversion (Miranda et al., 2003), and (4) during performance of visual attentional tasks (Himmelheber et al., 2001). It has been also related to attentional effort (Himmelheber et al., 2001). Furthermore, in the hippocampus, ACh release increases during the performance of a learned spatial memory task (Ragozzino et al., 1996; Stancampiano R, et al., 1999) and the increase is positively correlated to performance improvement during task learning (Fadda et al., 2000), showing that cholinergic neurons are modified functionally during learning and become progressively more active. Also, the initial use of a place strategy coincided with an immediate increase in hippocampus ACh release (Chang and Gold 2003). Furthermore, as rewarded spontaneous alternation testing progressed, a switch to a repetitive response strategy accompanied an increase in striatum ACh release (Pych JC et al., 2005).</p>
<p>The release of acetylcholine in different brain areas appears to be involved in processes of attention (Marrosu et al., 1995), detection of novelty or saliency (Baxter et al., 1999), and during the consolidation of different types of long-term memory (Power 2004; McIntyre et al., 2003; Hasselmo 1999).</p>
<p> </p>
<p> </p>
<strong>Empirical Evidence</strong>
<p>Miranda 2007 reviews many studies which demonstrate the activation of neurotransmitters such as glutamate, noradrenaline, and dopamine in several types of learning and during several stages of memory formation. The results of innumerable studies indicate that during memory formation different regions of the brain act in coordinated fashion through different neurotransmission systems (Miranda 2007).</p>
<p>Targeted knockout of the NMDAR in the hippocampus impairs spatial learning (Neal and Guilarte 2010), lending further support to the role of the NMDAR in hippocampus-mediated learning processes.</p>
<p>A neurotransmitter system that has been previously linked with the cognitive functions is the glutamate NMDA receptor system (May Simera and Levin 2003; Li et al., 2013). In 1991, Izquierdo, with the help of NMDA receptor antagonists (which impaired spatial working memory), concluded that if repeatedly stimulated, this system can regulate cognition (Izquierdo 1991). What is more, it was observed that blocking the NMDA receptor induces a resembling degree of memory impairment as the excision of the hippocampus (Lupușoru et al., 2017).</p>
<p>Learning-induced increases of ACh in the hippocampus and cortex have two important characteristics that strongly suggest that these increases are involved in memory consolidation (Power et al., 2003). First, the ACh increases induced during acquisition persist for at least 15 min after the end of the task (Orsetti et al., 1996; Ragozzino et al., 1996; Kopf et al., 2001; Miranda and Bermúdez-Rattoni 1999; Toumane et al., 1988). It is well established that during this posttraining period memory consolidation is strongly influenced by endogenous hormones and is highly susceptible to disruption and modulation by pharmacological interventions (McGaugh and Izquierdo 2000; McGaugh 2000). Furthermore, the persistence of the ACh levels in the hippocampus and cortex is correlated with the duration of these structures’ involvement in memory consolidation (Power et al., 2003).</p>
<h4>References</h4>
<p>A. Toumane, T. Durkin, A. Marighetto, D. Galey, R. Jaffard Differential hippocampal and cortical cholinergic activation during the acquisition, retention, reversal and extinction of a spatial discrimination in an 8-arm radial maze by mice Behavioural Brain Research, 30 (1988), pp. 225-234</p>
<p>Ann E. Power, Almira Vazdarjanova, James L. McGaugh, Muscarinic cholinergic influences in memory consolidation, Neurobiology of Learning and Memory, Volume 80, Issue 3, 2003, Pages 178-193, ISSN 1074-7427</p>
<p>Baxter MG, et al. Impairments in conditioned stimulus processing and conditioned responding after combined selective removal of hippocampal and neocortical cholinergic input. Behav Neurosci. 1999;113:486.</p>
<p>Chang Q, Gold PE. Switching memory systems during learning: changes in patterns of brain acetylcholine release in the hippocampus and striatum in rats. J Neurosci. 2003;23:3001.</p>
<p>Dalley JW, et al. Distinct changes in cortical acetylcholine and noradrenaline efflux during contingent and noncontingent performance of a visual attentional task. J Neurosci. 2001;21:4908.</p>
<p>Edwards FA. Anatomy and electrophysiology of fast central synapses lead to a structural model for long-term potentiation. Physiological Reviews 1995; 75: 759–87.</p>
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