Upstream eventInhibition, NMDARs
Decreased, Calcium influx
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities||adjacent||Moderate|
|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||adjacent||High|
Life Stage Applicability
Key Event Relationship Description
The NMDA receptor is distinct in two ways: firstly, it is both ligand-gated and voltage-dependent and secondly, it requires co-activation by two ligands: glutamate and either D-serine or glycine.
NMDA receptor activation allows the influx of Ca2+ only when the receptor is occupied by L-glutamate or other agonists (and removal of Mg++ block) resulting in the postsynaptic membrane depolarization. In contrast, binding of antagonist to NMDA receptor decreases or eliminates Ca2+ influx and consequently dramatically decreases intracellular influx of Ca2+ levels (reviewed in Higley and Sabatini, 2012).
Evidence Supporting this KER
The relationship between KE (NMDARs, Inhibition) and KE (Calcium influx, Decreased) is plausible as the function evaluation of NMDA receptors is commonly carried out by measurement of intracellular influx of Ca2+ upon NMDA receptor stimulation by agonist. Calcium imaging techniques have been extensively utilized to investigate the relationship between these two KEs. Almost 15% of the current through NMDA receptors is mediated by Ca2+ under physiological conditions (Higley and Sabatini, 2012).
It has been shown that less than five and, occasionally only a single NMDA receptor opens under physiological conditions, causing a total Ca2+ influx of about 6000 ions into a dendritic spine head reaching a concentration of ∼10 µM (Higley and Sabatini, 2012). However, the majority of the ions are rapidly eliminated by binding Ca2+ proteins, reaching ∼1 µM of free Ca2+ concentration (Higley and Sabatini, 2012).
In rat primary forebrain cultures, the intracellular Ca2+ increases after activation of the NMDA receptor and this increase is blocked when the cells are cultured under Ca2+ free conditions, demonstrating that the NMDA-evoked increase in intracellular Ca2+ derives from extracellular and not intracellular sources (Liu et al., 2013).
Neurons in brain slices from wild-type (GluRε2+/+) mice showed increase of intracellular Ca2+ in the presence of 100 μM NMDA that was completely inhibited after exposure to 100 mM APV. In contrast, the NMDA-mediated increase in Ca2+ was absent in brain slices from GluRε2−/− mice that do not possess any functional NMDA receptors in the developing neocortex (Okada et al., 2003).
Include consideration of temporal concordance here
Pb2+: There are a few studies examining the effect of Pb2+ exposure on the changes in intracellular Ca2+. Incubation of rat synaptosomes with Pb2+ stimulates the activity of calmodulin reaching the higher effect at 30 µM, whereas higher concentrations of Pb2+ causes inhibition (Sandhir and Gill, 1994). Pb2+ exposure increases the activity of calmodulin by 45% in animal models. The IC50 values for inhibition of Ca2+ ATPase by Pb2+ has been found to be 13.34 and 16.69 µM in calmodulin-rich and calmodulin-depleted synaptic plasma membranes, respectively. Exposure of rats to Pb2+ has also inhibitory effect on Ca2+ ATPase activity, causing increase in intrasynaptosomal Ca2+ (Sandhir and Gill, 1994). In embryonic rat hippocampal neurons, exposure to 100 nM Pb2+ for periods from 1 hour to 2 days showes decrease of intracellular Ca2+ by a calmodulin-dependent mechanism (Ferguson et al., 2000).Calmodulin, as a calcium binding protein, has been strongly implicated in Pb toxicity. However, interactions between calcium and Pb on several binding sites suggest complex interactions (Kirberger, 2011).
There is evidence that Pb2+ exposure affects Ca2+ homeostasis causing alterations in the phosphorylation state of different kinases. For example, Pb2+ has been shown to interfere with MAPK signaling as it increases the phosphorylation of both ERK1/2 and p38(MAPK) (Cordova et al., 2004). However, the findings regarding calcium/calmodulin kinase II (CamKII) activity are not clear (Toscano and Guilarte, 2005). On one hand, Pb2+ has been found to cause reduction of CREB phosphorylation in the hippocampus of rats exposed during brain development (Toscano et al., 2003; Toscano et al., 2002). On the other hand, the levels of phosphorylation of CamKII have not been explored but only the mRNA expression levels have been studied in rat pups on PND 25 that received Pb2+ (180 and 375-ppm lead acetate in food for 30 days) and reached blood Pb2+ levels 5.8 to 10.3 μg/dl on PND 55 (Schneider et al., 2012). More specifically, CamKIIα gene expression has been found to be very sensitive to Pb2+ exposure in the frontal cortex but not in the hippocampus, whereas CamKIIβ gene expression in both brain structures remained unchanged (Schneider et al., 2012).
Acute Pb2+ (10μM) exposure impairs LTP (125.8% reduction of baseline) in CA1 region of hippocampus derived from Sprague-Dawley rats (15-18 PND) as it has been recorded by whole cell patch-clamp technique (Li et al., 2006). In the same study, through calcium imaging, it has been shown in the 10mM caffeine-perfused cultured hippocampal neurons that 10μM Pb2+ reduces intracellular Fluo-4 fluorescence ratio to 0.44 (Li et al., 2006).
Pb2+ chronically or acutely applied, significantly reduces LTP in CA1 region of hippocampus from Wistar or Sprague-Dawley rats (30 and 60 PND) (Carpenter et al., 2002). These animals were exposed to Pb2+ via the mother’s drinking water either through gestation and lactation (upto day 21) (perinatal), only by lactation through the mother’s drinking water and then in the pup’s drinking water (post) or from gestation (pre and post). The concentrations of Pb2+ used in the drinking water were 0.1 and 0.2%. In CA1, LTP has been reduced at both ages and Pb2+ concentrations or duration of exposure. In CA3, there have been no differences with time of exposure, but there was a dramatic difference in response as the age of animals increased. At 30 days LTP was significantly reduced, but at 60 days LTP was increased by about 30% (Carpenter et al., 2002). In the same brain structure and area (CA3) the effects of Pb2+ on LTP have been different in 30 PND and 60 PND rats after either acute perfusion of Pb2+ or from slices derived from rats after chronic developmental exposure to Pb2+, as inhibition of LTP has been recorded in 30 PND CA3, whereas potentiation has been measured in 60 PND CA3 with either exposure paradigm that have been attributed to possible involvement of protein kinase C (Hussain et al., 2000).
|Stressor||Experimental Model||Tested concentrations||Exposure route||Exposure duration||Inhibition of NMDAR (KE up) (measurements, quantitative if available)||Reduced Ca 2+ influx (KE down) (measurements, quantitative if available)||References|
|Lead||CA1 pyramidal neurons derived from Sprague-Dawley rats (15-18 PND)||5-20 µM||25 min||By bath application of 5µM lead for 15 min prior to and 10 min after the tetanus, the LTP was induced to 151.4±3.5% of baseline. In the presence of 10µM lead, the LTP was reduced significantly to 125.8±2.9% of baseline. The reduction of 20µM lead to LTP was not significant with that of 10µM.||In the 10mM caffeine-perfused cultured hippocampal neurons, 10µM lead reduced intracellular Fluo-4 fluorescence ratio to 0.44±0.08||Li et al., 2006|
|Lead||CA1 region of hippocampus from Wistar or Sprague-Dawley rats (30 and 60 PND)||Two concentrations of lead were used in the drinking water 0.1 and 0.2%.||in utero and per os||Rats exposed to lead via the mother’s drinking water either through gestation and lactation (to day 21) (perinatal), only by lactation through the mother’s drinking water and then in the pup’s drinking water until use (post) or from gestation until use (pre and post).||In CA1, LTP is reduced at both ages, and there were no significant differences in the effects of the two lead concentrations or with the duration of exposure. In CA3 there were no differences with time of exposure, but there was a dramatic difference in response as a function of age. At 30 days LTP was significantly reduced, but at 60 days LTP was increased by about 30%. As in the chronic exposure studies, lead reduced LTP in CA1 at both ages but reduced LTP in CA3 in 30-day animals while potentiating LTP in 60-day animals.||Carpenter et al., 2002|
Uncertainties and Inconsistencies
The structural diversity of NMDA subunits can influence the functionality of the receptors and their permeability to Ca2+. For example, NR2B subunits show higher affinity for glutamate binding and higher Ca2+ permeability (reviewed in Higley and Sabatini, 2012). But NMDA receptor subunit composition is not the only parameter that influences Ca2+ entrance in the cytosol. Membrane potential due to pore blockade by extracellular Mg2+ and receptor phosphorylation are two additional regulator of Ca2+ influx through NMDA receptors (reviewed in Higley and Sabatini, 2012).
Entrance of Ca2+ into neuronal cell can also happen through KA and AMPA receptors but to a smaller extent compared to NMDA receptors (reviewed in Higley and Sabatini, 2012). However, recent findings suggest that AMPA receptors may also contribute to Ca2+ signalling during CNS development (reviewed in Cohen and Greenberg, 2008). Early in development cortical pyramidal neurons express calcium-permeable, GluR2 subunit–lacking AMPA receptors. During postnatal development these neurons undergo a switch in the subunit composition of AMPA receptors, expressing instead GluR2-containing, calcium-impermeable AMPA receptor suggesting that the main point entrance of Ca2+ at this developmental stage are NMDA receptors.
Furthermore, Ca2+ entry occurs through L- and H-type voltage-dependent Ca2+channels (L-VDCCs) (Perez-Reyes and Schneider, 1994; Berridge, 1998; Felix, 2005) that are encountered in neurons, suggesting that there are more possible entrance sites for Ca2+ to get into the cytosol rather than only through NMDA receptors.
Interestingly, Pb2+ has the ability to mimic or even compete with Ca2+ in the CNS (Flora et al., 2006). Indeed, Pb2+ is accumulated in the same mitochondrial compartment as Ca2+ and it has been linked to disruptions in intracellular calcium metabolism (Bressler and Goldstein, 1991). So, it can be that the reduced levels of Ca2+ after Pb2+ exposure may not be attributed to NMDA receptor inhibition but also to the ability of this heavy metal to compete with Ca2+. To make things more complicated, recent findings suggest that BDNF can also acutely elicit an increase in intracellular Ca2+ concentration, which is attributed not only to the influx of extracellular Ca2+ but also to Ca2+ mobilization from intracellular calcium stores (Numakawa et al., 2002; He et al., 2005). These findings derive from primary cultures of cortical neurons (E18 or 2-3 PND), where BDNF-evoked Ca2+ signals have not been altered neither by tetrodotoxin nor by a cocktail of glutamate receptor blockers (CNQX and APV), pointing out the importance of BDNF in Ca2+ homeostasis (Numakawa et al., 2002; He et al., 2005).
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?
No enough data is available to address the questions above.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Besides the above studies described in rodents, intracellular Ca2+ regulation has been studied at the neuromuscular junction of larval Drosophila exposed to 0, 100 μM or 250 μM Pb2+ (He et al., 2009).
Berridge MJ. (1998) Neuronal calcium signaling. Neuron 21: 13-26.
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Carpenter DO, Hussain RJ, Berger DF, Lombardo JP, Park HY. (2002) Electrophysiologic and behavioral effects of perinatal and acute exposure of rats to lead and polychlorinated biphenyls. Environ Health Perspect. 110: 377-386.
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Schneider JS, Mettil W, Anderson DW. (2012) Differential Effect of Postnatal Lead Exposure on Gene Expression in the Hippocampus and Frontal Cortex. J Mol Neurosci. 47: 76-88.
Sinner B, Friedrich O, Zink W, Martin E, Fink RH, et al. (2005) Ketamine stereoselectively inhibits spontaneous Ca2+-oscillations in cultured hippocampal neurons. Anesthesia and analgesia 100: 1660-1666.
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