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High-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system. Therefore, chemicals that affect apoptosis during neuronal development can have deleterious effects on the developing brain. The present study examined the utility of a high-throughput assay to detect chemical-induced apoptosis in mouse or human neuroprogenitor cells, as well as differentiated human neurons derived from induced pluripotent stem cells. Apoptosis was assessed using an assay that measures enzymatic activity of caspase-3/7 in a rapid and cost efficient manner. The results show that all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format. There were differences in the response of rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro. Neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery. [ABSTRACT FROM AUTHOR]

Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characterize the ability of drugs, chemicals and particles to cause neurotoxicity. While compound effects on spontaneous network activity are easily determined by MEA recordings, compound cytotoxicity is not routinely assessed, particularly within the same network from which recordings are collected. With the advent of higher-throughput 48 and 96 well MEA systems, rapid and simple methods to measure compound effects on cell health are required to facilitate efficient compound screening using MEAs. The present experiments sought to develop a multiplexed approach that allows measurement of network activity and cell health in the same MEA well. Primary cultures from rat cortex were exposed to six different compounds (glyphosate, β-cyfluthrin, domoic acid, tributyltin, lindane and fipronil). Effects of these compounds (0.03–100 μM) on spontaneous network activity (mean firing rate; MFR), cellular metabolic activity (Cell Titer Blue™ (CTB) assay) and lactate dehydrogenase (LDH) release were determined in the same well following a 60-min exposure. Glyphosate elicited no effect on MFR, LDH release or CTB reduction. Tributyltin caused concomitant decreases in MFR and CTB reduction and increases LDH release, while domoic acid and β-cyfluthrin decreased MFR in a concentration-dependent manner without altering either LDH release or CTB reduction. By contrast, lindane and fipronil did not alter LDH release or CTB reduction, but caused biphasic alterations in MFR, with increases in MFR at lower concentrations followed by decreases at higher concentrations. These results demonstrate a simple and rapid method for the simultaneous determination of test compound effects on spontaneous electrical activity and cell health from the same network, and will facilitate rapid screening of compounds for potential neurotoxicity. [ABSTRACT FROM AUTHOR]

The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways. [ABSTRACT FROM AUTHOR]

Glufosinate (GLF) at high levels in mammals causes convulsions and amnesia through a mechanism that is not completely understood. The structural similarity of GLF to glutamate (GLU) implicates the glutamatergic system as a target for GLF neurotoxicity. The current work examined in vitro GLF interaction with N -methyl- d -aspartate subtype GLU receptors (NMDARs) and GLT-1 transporters via [ 3 H]CGP 39653 binding experiments and [ 3 H]GLU uptake assays, respectively. GLF effects on neuronal network activity were assessed using microelectrode array (MEA) recordings in primary cultures of cortical neurons. GLF and its primary metabolite N -acetylglufosinate (NAcGLF) bind to the NMDAR; the IC 50 value for GLF was 668 μM and for NAcGLF was about 100 μM. Concentrations of GLF greater than 1000 μM were needed to decrease GLU uptake through GLT-1. In MEA recordings from networks of rat primary cortical neurons, the concentration-responses for NMDA, GLF and NAcGLF on network mean firing rates (MFR) were biphasic, increasing at lower concentrations and decreasing below control levels at higher concentrations. Increases in MFR occurred between 3–10 μM NMDA (290% control, maximum), 100–300 μM NAcGLF (190% control, maximum) and 10–1000 μM GLF (340% control, maximum). The NMDAR antagonist MK801 attenuated both NMDA and GLF increases in MFR. The GLF concentration required to alter GLU transport through GLT-1 is not likely to be attained in vivo, and therefore not relevant to the neurotoxic mode of action. However, toxicokinetic data from reports of intentional human poisonings indicate that GLF concentrations in the CNS after acute exposure could reach levels high enough to lead to effects mediated via NMDARs. Furthermore, the newly characterized action of NAcGLF at the NMDAR suggests that both the parent compound and metabolite could contribute to neurotoxicity via this pathway. [ABSTRACT FROM AUTHOR]

DL-glufosinate ammonium (DL-GLF) is a registered herbicide for which a guideline Developmental Neurotoxicity (DNT) study has been conducted. Offspring effects included altered brain morphometrics, decreased body weight, and increased motor activity. Guideline DNT studies are not available for its enriched isomers L-GLF acid and L-GLF ammonium; conducting one would be time consuming, resource-intensive, and possibly redundant given the existing DL-GLF DNT. To support deciding whether to request a guideline DNT study for the L-GLF isomers, DL-GLF and the L-GLF isomers were screened using in vitro assays for network formation and neurite outgrowth. DL-GLF and L-GLF isomers were without effects in both assays. DL-GLF and L-GLF (1–100 μM) isomers increased mean firing rate of mature networks to 120–140% of baseline. In vitro toxicokinetic assessments were used to derive administered equivalent doses (AEDs) for the in vitro testing concentrations. The AED for L-GLF was ∼3X higher than the NOAEL from the DL-GLF DNT indicating that the available guideline study would be protective of potential DNT due to L-GLF exposure. Based in part on the results of these in vitro studies, EPA is not requiring L-GLF isomer guideline DNT studies, thereby providing a case study for a useful application of DNT screening assays. • A guideline developmental neurotoxicity (DNT) study exists for DL-Glufosinate. • L-glufosinate isomers lack DNT data. • In vitro DNT toxicity and toxicokinetics of DL and L-glufosinate were evaluated. • Weight-of-evidence (WOE) supports waiving the guideline DNT study for L-glufosinate. • This is a case-study for use of DNT new approach methodologies in WOE evaluations. [ABSTRACT FROM AUTHOR]

Spontaneous activity in neuronal cultures on microelectrode arrays (MEAs) is sensitive to effects of drugs, chemicals, and particles. Multi-well MEA (mwMEA) systems have increased throughput of MEAs, enabling their use for chemical screening. The present experiments examined a subset of EPA's ToxCast compounds for effects on spontaneous neuronal activity in primary cortical cultures using 48-well MEA plates. A first cohort of 68 compounds was selected from the ToxCast Phase I and II libraries; 37 were positive in one or more of 20 individual ToxCast Novascreen assays related to ion channels (NVS_IC), with the remainder selected based on known neuroactivity. A second cohort of 25 compounds was then tested with 20 originating from the ToxCast Phase I and II libraries (not hits in NVS_IC assays) and 5 known negatives from commercial vendors. Baseline activity (1 h) was recorded prior to exposing the networks to compounds for 1 h, and the weighted mean firing rate (wMFR) was determined in the absence and presence of each compound. Compounds that altered activity by greater than the weighted change of DMSO-treated wells plus 2SD were considered “hits”. Of the first set of 68 compounds, 54 altered wMFR by more than the threshold, while in the second set, 13/25 compounds were hits. MEAs detected 30 of 37 (81.1%) compounds that were hits in NVS_IC assays, as well as detected known neurotoxicants that were negative in NVS_IC assays, primarily pyrethroids and GABA A receptor antagonists. Conversely, wMFR of cortical neuronal networks on MEAs was insensitive to nicotinic compounds, as only one neonicotinoid was detected by MEAs; this accounts for the bulk of non-concordant compounds between MEA and NVS_IC assays. These data demonstrate that mwMEAs can be used to screen chemicals efficiently for potential neurotoxicity, and that the results are concordant with predictions from ToxCast NVS_IC assays for interactions with ion channels. [ABSTRACT FROM AUTHOR]

Highlights: [•] Spontaneous activity in cortical networks in vitro was recorded with microelectrode arrays. [•] Effects of 16 chemicals on firing rate, bursting parameters and network synchrony were determined. [•] Principal components analysis and support vector machine analysis of the data were conducted. [•] PCA separated some GABAA antagonists from other chemicals and SVM predicted three effects classes. [•] These data indicate that MEA data may be useful to separate compounds by class. [Copyright &y& Elsevier]

Abstract: The need to assess large numbers of chemicals for their potential toxicities has resulted in increased emphasis on medium- and high-throughput in vitro screening approaches. For such approaches to be useful, efficient and reliable data analysis and hit detection methods are also required. Assessment of chemical effects on neuronal network activity using microelectrode arrays (MEAs) has been proposed as a screening tool for neurotoxicity. The current study examined a Bayesian data analysis approach for assessing effects of a 30 chemical training set on activity of primary cortical neurons grown in multi-well MEA plates. Each well of the MEA plate contained 64 microelectrodes and the data set contains the number of electrical spikes registered by each electrode over the course of each experiment. A Bayesian data analysis approach was developed and then applied to several different parsings of the data set to produce probability determinations for hit selection and ranking. This methodology results in an approach that is approximately 74% sensitive in detecting chemicals in the training set known to alter neuronal function (23 expected positives) while being 100% specific in detecting chemicals expected to have no effect (7 expected negatives). Additionally, this manuscript demonstrates that the Bayesian approach may be combined with a previously published weighted mean firing rate approach in order to produce a more robust hit detection method. In particular, when combined with the weighted mean firing rate approach, the joint analysis produces a sensitivity of approximately 96% and a specificity of 100%. These results demonstrate the utility of a novel approach to analysis of MEA data and support the use of neuronal networks grown on MEAs as a for neurotoxicity screening approach. [Copyright &y& Elsevier]

Abstract: There is a need to develop rapid and efficient models to screen chemicals for their potential to cause developmental neurotoxicity. Use of in vitro neuronal models, including human cells, is one approach that allows for timely, cost-effective toxicity screening. The present study compares the sensitivity of human (ReN CX) and mouse (mCNS) neuroprogenitor cell lines to chemicals using a multiplex assay for proliferation and apoptosis, endpoints that are critical for neural development. Cells were exposed to 0.001–100μM concentrations of 11 chemicals (cadmium, chlorpyrifos oxon, dexamethasone, dieldrin, ketamine, lead, maneb, methylmercury, nicotine, trans-retinoic acid, and trimethyltin) reported in the literature to affect proliferation and/or apoptosis, and 5 chemicals (dimethyl pthalate, glyphosate, omeprazole, saccharin, and d-sorbitol) with no reports of effects on either endpoint. High-content screening of markers for proliferation (BrdU incorporation) and apoptosis (activated caspase 3 and p53) was used to assess the effect of chemicals in both cell lines. Of the chemicals tested, methylmercury, cadmium, dieldrin, chlorpyrifos oxon, trans-retinoic acid, and trimethyltin decreased proliferation by at least 50% of control in either the ReN CX or mCNS cells. None of the chemicals tested activated caspase 3 or p53 in the ReN CX cells, while methylmercury, cadmium, dieldrin, chlorpyrifos oxon, trimethyltin, and glyphosate all induced at least a doubling in these apoptotic markers in the mCNS cells. Compared to control, cadmium, trans-retinoic acid, and trimethyltin decreased cell viability (ATP levels) by at least 50% in the ReN CX cells, while cadmium, dieldrin, and methylmercury decreased viability by at least 50% in the mCNS cells. Based on these results, BrdU is an appropriate marker for assessing chemical effects on proliferation, and human cells are more sensitive than mouse cells for this endpoint. By contrast, caspase 3 and p53 were altered by environmental chemicals in mouse, but not in human cells. Therefore, these markers are not appropriate to assess the ability of environmental chemicals to induce apoptosis in the ReN CX cells. [Copyright &y& Elsevier]

Abstract: Microelectrode array (MEA) approaches have been proposed as a tool for detecting functional changes in electrically excitable cells, including neurons, exposed to drugs, chemicals or particles. However, conventional single well-MEA systems lack the throughput necessary for screening large numbers of uncharacterized compounds. Recently, multi-well MEA (mwMEA) formats have become available to address the need for increased throughput. The current experiments examined the effects of a training set of 30 chemicals on spontaneous activity in networks of cortical neurons grown on mwMEA plates. Each plate contained 12 wells with 64 microelectrodes/well, for a total of 768 channels. Of the 30 chemicals evaluated, 23 were known to alter neuronal function in vivo (“positives”), including 6 GABAergic and 3 glutamatergic antagonists/agonists, 4 pyrethroids, 3 metals, 2 cholinesterase inhibitors, 2 nicotinic acetylcholine receptor agonists, valproic acid, verapamil, and fluoxetine. Seven compounds expected to have no effect on neuronal function were tested as “negatives” (glyphosate, acetaminophen, salicylic acid, paraquat, saccharin, d-sorbitol and amoxicillin). Following collection of 33min of baseline activity, chemical effects (50μM or highest soluble concentration) were recorded for 33min. Twenty of the positives altered the mean network spike rate by more than the 14% threshold (two standard deviations from the mean for DMSO control). The three positives without effect were bifenthrin, nicotine and imidacloprid. None of the negative compounds caused a change in activity beyond the threshold. Based on these results, the mwMEA assay has both high sensitivity (87% identification of positive compounds) and specificity (100% identification of negative compounds). These experiments demonstrate the capacity of mwMEAs to screen compounds for neurotoxic effects mediated by a broad variety of mechanisms. [Copyright &y& Elsevier]

Abstract: The “Hot Topic Keynotes: Channelopathies” session of the 26th International Neurotoxicology Conference brought together toxicologists studying interactions of environmental toxicants with ion channels, to review the state of the science of channelopathies and to discuss the potential for interactions between environmental exposures and channelopathies. This session presented an overview of chemicals altering ion channel function and background about different channelopathy models. It then explored the available evidence that individuals with channelopathies may or may not be more sensitive to effects of chemicals. [Copyright &y& Elsevier]

Abstract: Microelectrode arrays (MEAs) have been in use over the past decade and a half to study multiple aspects of electrically excitable cells. In particular, MEAs have been applied to explore the pharmacological and toxicological effects of numerous compounds on spontaneous activity of neuronal and cardiac cell networks. The MEA system enables simultaneous extracellular recordings from multiple sites in the network in real time, increasing spatial resolution and thereby providing a robust measure of network activity. The simultaneous gathering of action potential and field potential data over long periods of time allows the monitoring of network functions that arise from the interaction of all cellular mechanisms responsible for spatio-temporal pattern generation. In these functional, dynamic systems, physical, chemical, and pharmacological perturbations are holistically reflected by the tissue responses. Such features make MEA technology well suited for the screening of compounds of interest, and also allow scaling to high throughput systems that can record from multiple, separate cell networks simultaneously in multi-well chips or plates. This article is designed to be useful to newcomers to this technology as well as those who are currently using MEAs in their research. It explains how MEA systems operate, summarizes what systems are available, and provides a discussion of emerging mathematical schemes that can be used for a rapid classification of drug or chemical effects. Current efforts that will expand this technology to an influential, high throughput, electrophysiological approach for reliable determinations of compound toxicity are also described and a comprehensive review of toxicological publications using MEAs is provided as an appendix to this publication. Overall, this article highlights the benefits and promise of MEA technology as a high throughput, rapid screening method for toxicity testing. [Copyright &y& Elsevier]

Abstract: Pyrethroid insecticides have potent actions on voltage-gated sodium channels (VGSC), inhibiting inactivation and increasing channel open times. These are thought to underlie, at least in part, the clinical symptoms of pyrethroid intoxication. However, disruption of neuronal activity at higher levels of organization is less well understood. In order to characterize pyrethroid effects on neurotransmitter release and neuronal excitability in glutamatergic networks, we examined the effects of deltamethrin (DM) and permethrin (PM) on neuronal activity in hippocampal neuronal cultures using patch-clamp and microelectrode array (MEA) recordings. In the presence of inhibitors of GABA receptors, spontaneous excitatory post-synaptic currents (sEPSCs) and spontaneous spike rates were reduced in a concentration-dependent manner by both DM and PM. IC50 values were 0.037 and 0.70μM for inhibition of sEPSCs and 0.60 and 21.8μM for inhibition of spontaneous spike rate by DM and PM, respectively. Both compounds altered burst activity by decreasing the number of spikes during spontaneous bursting, the number of sEPSCs within a bursting release event and the duration of sEPSC bursts while increasing both the interspike interval and the time between sEPSCs. Exposure of neurons to the VGSC-specific modulator veratridine had effects similar to both DM and PM, while inhibition of voltage-gated calcium channels had no effect on spontaneous spike rates. In the absence of GABA receptor antagonists, both DM and PM increased spontaneous spike rates. Altogether, these data demonstrate that DM and PM disrupt network activity in vitro, largely via a VGSC-dependent mechanism. [Copyright &y& Elsevier]

Abstract: Disruption of neuronal voltage-sensitive sodium channels (VSSCs) by pyrethroid insecticides such as deltamethrin (DLT) has been widely studied using Xenopus laevis oocytes transfected with VSSC. However, the extent of pyrethroid accumulation in VSSC-expressing oocytes is unknown. Therefore, accumulation of [3H]-DLT in non-transfected, sham (water)-transfected and VSSC (Nav1.2+β1)-transfected oocytes after a 1h exposure was measured using liquid scintillation counting. Successful transfection of Nav1.2+β1 VSSCs in X. laevis oocytes was confirmed by two-electrode voltage-clamp; inward, tetrodotoxin (TTX)-sensitive currents were obtained in 98% of all oocytes examined (n =60 in nine experiments). DLT (1.0μM) induced tail currents in all VSSC-transfected oocytes; TTX also blocked these DLT-induced tail currents. In 0.1μM DLT solution, non-transfected oocytes accumulated 0.098±0.01ppm [3H]-DLT, sham-transfected oocytes accumulated 0.06±0.01ppm DLT, and VSSC-transfected oocytes accumulated 0.050±0.009ppm DLT. In 1.0μM DLT solution, non-transfected oocytes accumulated 0.62±0.08ppm DLT, sham-transfected oocytes accumulated 0.60±0.09ppm DLT, and VSSC-transfected oocytes accumulated 0.51±0.07ppm DLT. There was a significant difference in DLT accumulation between VSSC-transfected oocytes and non-transfected controls, where the transfected oocytes consistently had less accumulation. [Copyright &y& Elsevier]

Immunohistochemical expression in the neocortex, hippocampus and cerebellum of the α1A or α1E subunit of the voltage-sensitive Ca2+ channel was examined in Long–Evans hooded rats on gestational day 18 and postnatal days 1, 4, 7, 10, 14, 21, 90, 360 and 720. On gestational day 18 and postnatal day 1, α1A immunoreactivity was more dense in the neocortex and hippocampus than the cerebellum. By postnatal day 7, levels of α1A immunoreactivity increased dramatically in the cerebellum, while in neocortex, α1A immunoreactivity became more sparse, which approached the more diffuse pattern of cellular staining in the mature brain. Expression of α1E in the neocortex, hippocampus and cerebellum was much less dense than α1A between gestational day 18 and postnatal day 4. There was also significant α1E immunoreactivity in the mossy fibers of the hippocampus and in dendrites of Purkinje cells of the cerebellum. Depolarization-dependent 45Ca2+ influx was examined in rat brain synaptosomes on postnatal days 4, 7, 10, 14, 21 and >60. In neocortical and hippocampal synaptosomes, 45Ca2+ influx increased steadily with age and reached adult levels by postnatal day 10. In cerebellar synaptosomes, 45Ca2+ influx was constant across all ages, except for a spike in activity which was observed on postnatal day 21. In neocortical and hippocampal synaptosomes, 100 nM ω-conotoxin MVIIC significantly inhibited 45Ca2+ influx on postnatal day 10 and 14, respectively, or after. In cerebellar synaptosomes, influx was inhibited by ω-conotoxin MVIIC only on postnatal day 10 or prior. On postnatal day 7, 45Ca2+ influx was not inhibited in neocortical and hippocampal synaptosomes by a combination of 10 μM nifedipine, 1 μM ω-conotoxin GVIA and 1 μM ω-conotoxin MVIIC, suggesting that an ‘insensitive’ flux predominates at this age. Overall, the results suggest that expression of voltage-sensitive Ca2+ channels during development is dynamic and is important in central nervous system development. [Copyright &y& Elsevier]

The US EPA's Toxicity Forecaster (ToxCast) is a suite of high-throughput in vitro assays to screen environmental toxicants and predict potential toxicity of uncharacterized chemicals. This work examines the relevance of ToxCast assay intended gene targets to putative molecular initiating events (MIEs) of neurotoxicants. This effort is needed as there is growing interest in the regulatory and scientific communities about developing new approach methodologies (NAMs) to screen large numbers of chemicals for neurotoxicity and developmental neurotoxicity. Assay gene function (GeneCards, NCBI-PUBMED) was used to categorize gene target neural relevance (1 = neural, 2 = neural development, 3 = general cellular process, 3 A = cellular process critical during neural development, 4 = unlikely significance). Of 481 unique gene targets, 80 = category 1 (16.6 %); 16 = category 2 (3.3 %); 303 = category 3 (63.0 %); 97 = category 3 A (20.2 %); 82 = category 4 (17.0 %). A representative list of neurotoxicants (548) was researched (ex. PUBMED, PubChem) for neurotoxicity associated MIEs/Key Events (KEs). MIEs were identified for 375 compounds, whereas only KEs for 173. ToxCast gene targets associated with MIEs were primarily neurotransmitter (ex. dopaminergic, GABA)receptors and ion channels (calcium, sodium, potassium). Conversely, numerous MIEs associated with neurotoxicity were absent. Oxidative stress (OS) mechanisms were 79.1 % of KEs. In summary, 40 % of ToxCast assay gene targets are relevant to neurotoxicity mechanisms. Additional receptor and ion channel subtypes and increased OS pathway coverage are identified for potential future assay inclusion to provide more complete coverage of neural and developmental neural targets in assessing neurotoxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

New approach methodologies (NAMs) can address information gaps on potential neurotoxicity or developmental neurotoxicity hazard for data-poor chemicals. Two assays have been previously developed using microelectrode arrays (MEA), a technology which measures neural activity. The MEA acute network function assay (AcN) uses dissociated rat cortical cells cultured at postnatal day 0 and evaluates network activity during a 40-minute chemical exposure on day in vitro (DIV)13 or 15. In contrast, the MEA network formation assay (NFA) uses a developmental exposure paradigm spanning DIV0 through DIV12. Measures of network activity over time at DIV5, 7, 9, and 12 in the NFA are reduced to an estimated area under the curve to facilitate concentration-response evaluation. Here, we evaluated the hypothesis that chemicals with effects in the AcN also perturb the NFA by examining quantitative and qualitative concordance between assays. Out of 243 chemicals screened in both assays, we observed 70.3% concordance between the AcN and NFA after eliminating activity inferred to be cytotoxic (selective activity), with the majority of discordance explained by chemicals that altered selective activity in the AcN but not NFA. The NFA detected more active chemicals when evaluating activity associated with cytotoxicity. Median potency values were lower in the NFA compared to the AcN, but within-chemical potency values were not uniformly lower in the NFA than the AcN. Lastly, the AcN and NFA captured unique bioactivity fingerprints; the AcN was more informative for identifying chemicals with a shared mode of action, while the NFA provided information relevant to developmental exposure. Taken together, this analysis provides a rationale for using both approaches for chemical evaluation with consideration of the context of use, such as screening/ prioritization, hazard identification, or to address questions regarding biological mechanism or function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Exposure to environmental chemicals can impair neurodevelopment, and oligodendrocytes may be particularly vulnerable, as their development extends from gestation into adulthood. However, few environmental chemicals have been assessed for potential risks to oligodendrocytes. Here, using a high-throughput developmental screen in cultured cells, we identified environmental chemicals in two classes that disrupt oligodendrocyte development through distinct mechanisms. Quaternary compounds, ubiquitous in disinfecting agents and personal care products, were potently and selectively cytotoxic to developing oligodendrocytes, whereas organophosphate flame retardants, commonly found in household items such as furniture and electronics, prematurely arrested oligodendrocyte maturation. Chemicals from each class impaired oligodendrocyte development postnatally in mice and in a human 3D organoid model of prenatal cortical development. Analysis of epidemiological data showed that adverse neurodevelopmental outcomes were associated with childhood exposure to the top organophosphate flame retardant identified by our screen. This work identifies toxicological vulnerabilities for oligodendrocyte development and highlights the need for deeper scrutiny of these compounds' impacts on human health., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Per- and polyfluoroalkyl substances (PFAS) are widely used, and their fluorinated state contributes to unique uses and stability but also long half-lives in the environment and humans. PFAS have been shown to be toxic, leading to immunosuppression, cancer, and other adverse health outcomes. Only a small fraction of the PFAS in commerce have been evaluated for toxicity using in vivo tests, which leads to a need to prioritize which compounds to examine further. Here, we demonstrate a prioritization approach that combines human biomonitoring data (blood concentrations) with bioactivity data (concentrations at which bioactivity is observed in vitro) for 31 PFAS. The in vitro data are taken from a battery of cell-based assays, mostly run on human cells. The result is a Bioactive Concentration to Blood Concentration Ratio (BCBCR), similar to a margin of exposure (MoE). Chemicals with low BCBCR values could then be prioritized for further risk assessment. Using this method, two of the PFAS, PFOA (Perfluorooctanoic Acid) and PFOS (Perfluorooctane Sulfonic Acid), have BCBCR values < 1 for some populations. An additional 9 PFAS have BCBCR values < 100 for some populations. This study shows a promising approach to screening level risk assessments of compounds such as PFAS that are long-lived in humans and other species.

There is a need to assess compounds reliably and quickly for neurotoxicity (NT) and developmental neurotoxicity (DNT). Adverse outcome pathways (AOPs) enable the mapping of molecular events to an apical endpoint in a chemical agnostic manner and have begun to be applied in NT and DNT testing frameworks. We assessed the status of NT/DNT AOPs in the AOP-Wiki (ca. 2/1/23; https://aopwiki.org/), to characterize the state of AOP development, identify strengths and knowledge gaps, elucidate areas for improvement, and describe areas for future focus. AOPs in the Wiki database were assessed for inclusion of NT/DNT molecular events and endpoints, AOP development and endorsement, as well as the linkages of key neurodevelopmental processes with in vitro new approach methods (NAMs). This review found that 41 AOPs have been proposed detailing NT/DNT, of which eight were endorsed by working parties in OECD. Further, this review determined that learning and memory is included as an adverse outcome in eight NT/DNT AOPS, often without distinction regarding the varying forms of learning and memory, regional specification, temporal dynamics, or acquisition mechanisms involved. There is also an overlap with key events (KEs) and in vitro NAMs, which synaptogenesis appeared as a common process. Overall, progress on NT/DNT AOPs could be expanded, adding in modes of action that are missing, improvement in defining apical endpoints, as well as utilizing NAMs further to develop AOPs and identify gaps in current knowledge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Per- and polyfluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors.

Microelectrode array (MEA) technology is a neurophysiological method that allows for the measurement of spontaneous or evoked neural activity to determine chemical effects thereon. Following assessment of compound effects on multiple endpoints that evaluate network function, a cell viability endpoint in the same well is determined using a multiplexed approach. Recently, it has become possible to measure electrical impedance of cells attached to the electrodes, where greater impedance indicates greater number of cells attached. This would allow rapid and repeated assessments of cell health as the neural network develops in longer exposure assays without impacting cell health. Typically, the lactate dehydrogenase (LDH) assay for cytotoxity and CellTiter-Blue® (CTB) assay for cell viability are only performed at the end of the chemical exposure period because these assays involve lysing of the cells. Procedures describing the multiplexed methods in acute and network formation screening are included in this chapter., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Neurotoxicology is the study of adverse effects on the structure or function of the developing or mature adult nervous system following exposure to chemical, biological, or physical agents. The development of more informative alternative methods to assess developmental (DNT) and adult (NT) neurotoxicity induced by xenobiotics is critically needed. The use of such alternative methods including in silico approaches that predict DNT or NT from chemical structure (e.g., statistical-based and expert rule-based systems) is ideally based on a comprehensive understanding of the relevant biological mechanisms. This paper discusses known mechanisms alongside the current state of the art in DNT/NT testing. In silico approaches available today that support the assessment of neurotoxicity based on knowledge of chemical structure are reviewed, and a conceptual framework for the integration of in silico methods with experimental information is presented. Establishing this framework is essential for the development of protocols, namely standardized approaches, to ensure that assessments of NT and DNT based on chemical structures are generated in a transparent, consistent, and defendable manner., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Myatt reports grants from NIH during the conduct of the study; and FDA’s Center for Drug Evaluation and Research (CDER) and Leadscope Inc. (an Instem company) are parties to a formal Research Collaboration Agreement (RCA).

Neurotoxicity can be defined by the ability of a drug or chemical to alter the physiology, biochemistry, or structure of the nervous system in a manner that may negatively impact the health or function of the individual. Electrophysiological approaches have been utilized to study the mechanisms underlying neurotoxic actions of drugs and chemicals for over 50 years, and in more recent decades, high-throughput patch-clamp approaches have been utilized by the pharmaceutical industry for drug development. The use of microelectrode array recordings to study neural network electrophysiology is a relatively newer approach, with commercially available systems becoming available only in the early 2000s. However, MEAs have been rapidly adopted as a useful approach for neurotoxicity testing. In this chapter, I will review the use of MEA approaches as they have been applied to the field of neurotoxicity testing, especially as they have been applied to the need to screen large numbers of chemicals for neurotoxicity and developmental neurotoxicity. In addition, I will also identify challenges for the field that when addressed will improve the utility of MEA approaches for toxicity testing.

Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).

Less than 1% of environmental chemicals have been evaluated for developmental neurotoxicity (DNT). Current guideline DNT studies are resource intensive and not amenable to screening large numbers of compounds for hazard. As part of evaluating a battery of more rapid and scalable in vitro assays for DNT hazard, 86 compounds were screened for their ability to alter function during cortical network development. Developing rat cortical networks were treated with a concentration series (usually 0.03-30 µM) of 86 compounds, 60 of which have known in vivo DNT effects ("DNT Reference Set"). Spontaneous network activity was monitored by microelectrode array recordings over 12 days in vitro, and 17 measures of network activity and synchrony were quantified. Following recordings on days in vitro 12, in-well cell assessment of metabolic activity (Alamar blue) and total cellular content (lactase dehydrogenase) were conducted. Of the 86 compounds tested, 64 perturbed cortical network function in a concentration-dependent manner; 49 of the 60 DNT Reference Set compounds (81.7%) altered network formation. Compounds were ranked by potency (network effect EC50) and selectivity (separation of network and cell viability EC50) for hazard prioritization. Machine learning indicates a combination of an overall network activity metric with a measure of network coordination is key in distinguishing network-disruptive from benign treatments. These data demonstrate that this microelectrode array-based assay for developing cortical network function is amenable to medium-throughput evaluation of environmental substances for DNT hazard and further prioritization. For comprehensive identification of compounds of concern, this assay will be a useful component of a battery of assays targeting independent neurodevelopmental processes., (Published by Oxford University Press on behalf of the Society of Toxicology 2017. This work is written by US Government employees and is in the public domain in the US.)

Microelectrode array (MEA) technology is a neurophysiological method that allows for the spontaneous measure of activity in neural cultures and determination of drug and chemical effects thereon. Recent introduction of multi-well MEA (mwMEA) formats have dramatically increased the throughput of this technology, allowing more efficient compound screening. Rapid characterization of compounds for neuroactivity or neurotoxicity hazard evaluation following acute, chronic, or developmental exposures ideally would also consider compound effects on cell health, and to do so in the same well requires a multiplexed approach. Procedures describing the multiplexed method to acute and developmental screening are described in this chapter.

We examined neural network ontogeny using microelectrode array (MEA) recordings made in multiwell MEA (mwMEA) plates over the first 12 days in vitro (DIV). In primary cortical cultures, action potential spiking activity developed rapidly between DIV 5 and 12. Spiking was sporadic and unorganized at early DIV, and became progressively more organized with time, with bursting parameters, synchrony, and network bursting increasing between DIV 5 and 12. We selected 12 features to describe network activity; principal components analysis using these features demonstrated segregation of data by age at both the well and plate levels. Using random forest classifiers and support vector machines, we demonstrated that four features (coefficient of variation [CV] of within-burst interspike interval, CV of interburst interval, network spike rate, and burst rate) could predict the age of each well recording with >65% accuracy. When restricting the classification to a binary decision, accuracy improved to as high as 95%. Further, we present a novel resampling approach to determine the number of wells needed for comparing different treatments. Overall, these results demonstrate that network development on mwMEA plates is similar to development in single-well MEAs. The increased throughput of mwMEAs will facilitate screening drugs, chemicals, or disease states for effects on neurodevelopment., (© 2016 Society for Laboratory Automation and Screening.)

Approaches for extrapolating in vitro toxicity testing results for prediction of human in vivo outcomes are needed. The purpose of this case study was to employ in vitro toxicokinetics and PBPK modeling to perform in vitro to in vivo extrapolation (IVIVE) of lindane neurotoxicity. Lindane cell and media concentrations in vitro, together with in vitro concentration-response data for lindane effects on neuronal network firing rates, were compared to in vivo data and model simulations as an exercise in extrapolation for chemical-induced neurotoxicity in rodents and humans. Time- and concentration-dependent lindane dosimetry was determined in primary cultures of rat cortical neurons in vitro using "faux" (without electrodes) microelectrode arrays (MEAs). In vivo data were derived from literature values, and physiologically based pharmacokinetic (PBPK) modeling was used to extrapolate from rat to human. The previously determined EC50 for increased firing rates in primary cultures of cortical neurons was 0.6μg/ml. Media and cell lindane concentrations at the EC50 were 0.4μg/ml and 7.1μg/ml, respectively, and cellular lindane accumulation was time- and concentration-dependent. Rat blood and brain lindane levels during seizures were 1.7-1.9μg/ml and 5-11μg/ml, respectively. Brain lindane levels associated with seizures in rats and those predicted for humans (average=7μg/ml) by PBPK modeling were very similar to in vitro concentrations detected in cortical cells at the EC50 dose. PBPK model predictions matched literature data and timing. These findings indicate that in vitro MEA results are predictive of in vivo responses to lindane and demonstrate a successful modeling approach for IVIVE of rat and human neurotoxicity., (Published by Elsevier Inc.)

Background: Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Although previous work has tested the additivity of pyrethroids in vivo, this has not been assessed directly at the primary molecular target using a functional measure., Objectives: We investigated the potency and efficacy of 11 structurally diverse food-use pyrethroids to evoke sodium (Na+) influx in neurons and tested the hypothesis of dose additivity for a mixture of these same 11 compounds., Methods: We determined pyrethroid-induced increases in Na+ influx in primary cultures of cerebrocortical neurons using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Concentration-dependent responses for 11 pyrethroids were determined, and the response to dilutions of a mixture of all 11 compounds at an equimolar mixing ratio was assessed. Additivity was tested assuming a dose-additive model., Results: Seven pyrethroids produced concentration-dependent, tetrodotoxin-sensitive Na+ influx. The rank order of potency was deltamethrin > S-bioallethrin > β-cyfluthrin > λ-cyhalothrin > esfenvalerate > tefluthrin > fenpropathrin. Cypermethrin and bifenthrin produced modest increases in Na+ influx, whereas permethrin and resmethrin were inactive. When all 11 pyrethroids were present at an equimolar mixing ratio, their actions on Na+ influx were consistent with a dose-additive model., Conclusions: These data provide in vitro relative potency and efficacy measurements for 7 pyrethroid compounds in intact mammalian neurons. Despite differences in individual compound potencies, we found the action of a mixture of all 11 pyrethroids to be additive when we used an appropriate statistical model. These results are consistent with a previous report of the additivity of pyrethroids in vivo.

Recent studies have demonstrated that lipophilic compounds (e.g., methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neurons have been widely utilized to study the actions of pyrethroids, yet pyrethroid accumulation in these cells has not been studied to date. To test the hypothesis that pyrethroids rapidly accumulate in neurons in vitro, the time (0-90 min) and concentration (0.05-10 μM) dependent accumulation of [(3)H]-deltamethrin (DM), [(3)H]-bifenthrin (BF) and [(14)C]-cis permethrin (PM) into primary cortical cultures was examined. Accumulation of all three pyrethroids was time- and concentration-dependent, with only small differences observed between the compounds. Concentration-dependent accumulation of PM and BF were similar, achieving a of total ∼0.25 nmol in cells after 30 min in a 10 μM solution. DM accumulation was lower, reaching a maximum of 0.14 nmol after 30 min in a 10 μM solution. In 1 μM solutions, DM and PM content in cells were 0.039 and 0.038 nmol after 90 min. At all concentrations and times, pyrethroid accumulation in cells was less than 5% of the total mass applied for DM and PM, and was less than 8% for BF. However, after 90 min, accumulation of all three compounds increased to as much as ∼30-50-fold higher than the surrounding medium. The amount of compound recovered in the media at the end of incubation ranged from ∼77% to 89%; the remainder (6-15%) was presumed to bind to the plastic of the culture plates. These results demonstrate rapid time- and concentration-dependent accumulation of pyrethroids in neurons in vitro. Further, for the three pyrethroids examined, there were not statistically significant differences in their accumulation. This data will be useful for making comparisons between in vivo and in vitro studies regarding effective concentrations of pyrethroids., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Pyrethroid insecticides have been used for more than 40 years and account for 25% of the worldwide insecticide market. Although their acute neurotoxicity to adults has been well characterized, information regarding the potential developmental neurotoxicity of this class of compounds is limited. There is a large age dependence to the acute toxicity of pyrethroids in which neonatal rats are at least an order of magnitude more sensitive than adults to two pyrethroids. There is no information on age-dependent toxicity for most pyrethroids. In the present review we examine the scientific data related to potential for age-dependent and developmental neurotoxicity of pyrethroids. As a basis for understanding this neurotoxicity, we discuss the heterogeneity and ontogeny of voltage-sensitive sodium channels, a primary neuronal target of pyrethroids. We also summarize 22 studies of the developmental neurotoxicity of pyrethroids and review the strengths and limitations of these studies. These studies examined numerous end points, with changes in motor activity and muscarinic acetylcholine receptor density the most common. Many of the developmental neurotoxicity studies suffer from inadequate study design, problematic statistical analyses, use of formulated products, and/or inadequate controls. These factors confound interpretation of results. To better understand the potential for developmental exposure to pyrethroids to cause neurotoxicity, additional, well-designed and well-executed developmental neurotoxicity studies are needed. These studies should employ state-of-the-science methods to promote a greater understanding of the mode of action of pyrethroids in the developing nervous system.

Immunohistochemical expression in the neocortex, hippocampus and cerebellum of the alpha(1A) or alpha(1E) subunit of the voltage-sensitive Ca(2+) channel was examined in Long-Evans hooded rats on gestational day 18 and postnatal days 1, 4, 7, 10, 14, 21, 90, 360 and 720. On gestational day 18 and postnatal day 1, alpha(1A) immunoreactivity was more dense in the neocortex and hippocampus than the cerebellum. By postnatal day 7, levels of alpha(1A) immunoreactivity increased dramatically in the cerebellum, while in neocortex, alpha(1A) immunoreactivity became more sparse, which approached the more diffuse pattern of cellular staining in the mature brain. Expression of alpha(1E) in the neocortex, hippocampus and cerebellum was much less dense than alpha(1A) between gestational day 18 and postnatal day 4. There was also significant alpha(1E) immunoreactivity in the mossy fibers of the hippocampus and in dendrites of Purkinje cells of the cerebellum. Depolarization-dependent 45Ca(2+) influx was examined in rat brain synaptosomes on postnatal days 4, 7, 10, 14, 21 and >60. In neocortical and hippocampal synaptosomes, 45Ca(2+) influx increased steadily with age and reached adult levels by postnatal day 10. In cerebellar synaptosomes, 45Ca(2+) influx was constant across all ages, except for a spike in activity which was observed on postnatal day 21. In neocortical and hippocampal synaptosomes, 100 nM omega-conotoxin MVIIC significantly inhibited 45Ca(2+) influx on postnatal day 10 and 14, respectively, or after. In cerebellar synaptosomes, influx was inhibited by omega-conotoxin MVIIC only on postnatal day 10 or prior. On postnatal day 7, 45Ca(2+) influx was not inhibited in neocortical and hippocampal synaptosomes by a combination of 10 microM nifedipine, 1 microM omega-conotoxin GVIA and 1 microM omega-conotoxin MVIIC, suggesting that an 'insensitive' flux predominates at this age. Overall, the results suggest that expression of voltage-sensitive Ca(2+) channels during development is dynamic and is important in central nervous system development.

The neurotoxicant methylmercury (CH(3)Hg(+)) inhibits voltage-sensitive Na(+) and Ca(2+) currents in neuronal preparations following acute, in vitro, exposure. In the present study, effects on voltage-sensitive Na(+) (I(Na)) and Ca(2+) (I(Ca)) currents in pheochromocytoma (PC12) cells were examined following prolonged exposure to CH(3)Hg(+). When PC12 cells cultured in the presence of nerve growth factor (NGF) for 7 days ('primed') were replated in the presence of NGF and 30 nM CH(3)Hg(+), I(Ca), but not I(Na), amplitude was reduced (29%) significantly approximately 24 h later. Quantitative assessment of morphology indicated that this approximately 24 h exposure to CH(3)Hg(+) significantly reduced neurite length. The N-type voltage-sensitive Ca(2+) channel (VSCC) antagonist omega-conotoxin GVIA (500 pM) was without significant effect on current amplitude or morphology in this exposure protocol. When undifferentiated cells were cultured in the presence of NGF and 10 nM CH(3)Hg(+) for 6 days, I(Ca) and I(Na) amplitude were reduced by 36 and 52%, respectively. I(Ca) at the end of a 150 ms test pulse was also reduced by 40% in CH(3)Hg(+)-treated cells. Thus, both inactivating and non-inactivating I(Ca) were reduced equally. There was no change in [(3)H]saxitoxin or omega-[(125)I]conotoxin GVIA binding, nor were there any morphological alterations in cells treated with CH(3)Hg(+) for 6 days. Omega-conotoxin GVIA (500 pM, 6 days), reduced significantly I(Ca), but not I(Na), but was without effect on morphology. These results demonstrate that prolonged exposure to low concentrations of CH(3)Hg(+) reduces cationic currents in differentiating PC12 cells, but that current reduction is not always associated with morphological alteration.