Joshua A. Harrill

In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny.

Because the Developmental Neurotoxicity Testing Guidelines require large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemical or morphological, and do not assess function of neuronal networks. In this study, microelectrode arrays (MEAs) were used to determine if chemical-induced changes in function could be detected by assessing the development of spontaneous network activity. MEAs record individual action potential spikes as well as groups of spikes (bursts) in neuronal networks, and activity can be assessed repeatedly over days in vitro (DIV). Primary cultures of rat cortical neurons were prepared on MEAs and spontaneous activity was assessed on DIV 2, 6, 9, 13, and 20 to determine the in vitro developmental profile of spontaneous spiking and bursting in cortical networks. In addition, 5 μM of the protein kinase C inhibitor bisindolylmaleamide-1 (Bis-1) was added to MEAs (n = 9–18) on DIV 5 to determine if changes in spontaneous activity could be detected in response to inhibition of neurite outgrowth. A clear profile of in vitro activity development occurred in control MEAs, with the number of active channels increasing from 0/MEA on DIV 2 to 37 ± 5/MEA by DIV 13; the rate of increase was most rapid between DIV 6 and 9, and activity declined by DIV 20. A similar pattern was observed for the number of bursting channels, as well as the total number of bursts. Bis-1 decreased the number of active channels/MEA and the number of bursting channels/MEA. Burst characteristics, such as burst duration and the number of spikes in a burst, were unchanged by Bis-1. These results demonstrate that MEAs can be used to assess the development of functional neuronal networks in vitro, as well as chemical-induced dysfunction.

Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro.

Synaptogenesis is a critical process in nervous system development whereby neurons establish specialized contact sites which facilitate neurotransmission. Early life exposure to chemicals can result in persistent deficits in nervous system function at later life stages. These effects are often the result of abnormal development of synapses. Given the large number of chemicals in commerce with unknown potential to result in developmental neurotoxicity (DNT), the need exists for assays that can efficiently characterize and quantify chemical effects on brain development including synaptogenesis. The present study describes the application of automated high content image analysis (HCA) technology for examining synapse formation in rodent primary mixed cortical cultures. During the first 15 days in vitro (DIV) cortical neurons developed a network of polarized neurites (i.e., axons and dendrites) and expression of the pre-synaptic protein synapsin increased over time. The localization of punctate synapsin protein in close apposition to dendrites also increased, indicating an increase in synapse formation. Results demonstrated that: (1) punctate synapsin protein with a spatial orientation consistent with synaptic contact sites could be selectively measured, (2) the critical period for synaptogenesis in cortical cultures was consistent with previous reports, (3) chemicals known to inhibit synapse formation decreased automated measurements of synapse number and (4) parallel evaluation of neuron density, dendrite length and synapse number could distinguish frank cytotoxicity from specific effects on synapse formation or neuronal morphology. Collectively, these data demonstrate that automated image analysis can be used to efficiently assess synapse formation in primary cultures and that the resultant data is comparable to results obtained using lower throughput methods.

Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2 cells using automated high-content image analysis.

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and immortalized human and non-human clonal cell lines have been used to investigate the molecular mechanisms controlling neurite outgrowth and examine chemical effects on this process. The present study characterizes the molecular phenotype of hN2 human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24h post-plating hN2 cells express a number of protein markers indicative of a neuronal phenotype, including: nestin, beta(III)-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite outgrowth in hN2 cells proceeded rapidly, with a majority of cells extending one to three neurites by 48h in culture. In addition, concentration-dependent decreases in neurite outgrowth and ATP-content were observed following treatment of hN2 cells with either bisindolylmaleimide I, U0126, lithium chloride, sodium orthovanadate and brefeldin A, all of which have previously been shown to inhibit neurite outgrowth in primary rodent neural cultures. Overall, the molecular phenotype, rate of neurite outgrowth and sensitivity of hN2 cells to neurite outgrowth inhibitors were comparable to other in vitro models previously characterized in the literature. hN2 cells provide a model in which to investigate chemical effects on neurite outgrowth in a non-transformed human-derived cells and provide an alternative to the use of primary rodent neural cultures or immortalized clonal cell lines.

Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural cultures were more sensitive to neurite outgrowth inhibitors, they also had a lower dynamic range for detecting chemical-induced neurite outgrowth inhibition and greater variability from culture-to-culture as compared to rat primary cortical cultures.

Comparison of chemical-induced changes in proliferation and apoptosis in human and mouse neuroprogenitor cells

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 0.001-100 μM 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.

Use of high content image analyses to detect chemical-mediated effects on neurite sub-populations in primary rat cortical neurons

Traditional developmental neurotoxicity tests performed in vivo are costly, time-consuming and utilize a large number of animals. In order to address these inefficiencies, in vitro models of neuronal development have been used in a first tier screening approach for developmental neurotoxicity hazard identification. One commonly used endpoint for assessing developmental neurotoxicity in vitro is measurement of neurite outgrowth. This biological process is amenable to high-throughput measurement using high content imaging (HCI) based methodologies. To date, a majority of HCI studies of neurite outgrowth have focused on measurements of total neurite outgrowth without examining whether stereotypic neuronal growth patterns are disrupted or whether specific sub-populations of neurites (i.e. axons or dendrites) are selectively affected. The present study describes the development and implementation of two HCI based analysis methods for assessing chemical effects on neuronal maturation. These methods utilize the stereotypical growth pattern of primary rat cortical neurons in culture (i.e. the Staging Method), as well as the differential cytoplasmic distribution of β(III)-tubulin and MAP2 (i.e. the Subtraction Method), to quantify inhibition of neurite initiation, axon outgrowth and secondary neurite (or dendrite) outgrowth in response to chemical exposure. Results demonstrate that these distinct maturational processes are differentially affected by pharmacological compounds (K252a, Na(3)VO(4), Bis-1) known to inhibit neurite outgrowth. Furthermore, a group of known developmental neurotoxicants also differentially affected the growth of axons and secondary neurites in primary cortical culture. This work improves upon previous HCI methods by providing a means in which to rapidly and specifically quantify chemical effects on the growth of axons and dendrites in vitro.

Neurotrophic Effects of Leukemia Inhibitory Factor on Neural Cells Derived from Human Embryonic Stem Cells

Various growth factor cocktails have been used to proliferate and then differentiate human neural progenitor (NP) cells derived from embryonic stem cells (ESC) for in vitro and in vivo studies. However, the cytokine leukemia inhibitory factor (LIF) has been largely overlooked. Here, we demonstrate that LIF significantly enhanced in vitro survival and promoted differentiation of human ESC‐derived NP cells. In NP cells, as well as NP‐derived neurons, LIF reduced caspase‐mediated apoptosis and reduced both spontaneous and H2O2‐induced reactive oxygen species in culture. In vitro, NP cell proliferation and the yield of differentiated neurons were significantly higher in the presence of LIF. In NP cells, LIF enhanced cMyc phosphorylation, commonly associated with self‐renewal/proliferation. Also, in differentiating NP cells LIF activated the phosphoinositide 3‐kinase and signal transducer and activator of transcription 3 pathways, associated with cell survival and reduced apoptosis. When differentiated in LIF+ media, neurite outgrowth and ERK1/2 phosphorylation were potentiated together with increased expression of gp130, a component of the LIF receptor complex. NP cells, pretreated in vitro with LIF, were effective in reducing infarct volume in a model of focal ischemic stroke but LIF did not lead to significantly improved initial NP cell survival over nontreated NP cells. Our results show that LIF signaling significantly promotes human NP cell proliferation, survival, and differentiation in vitro. Activated LIF signaling should be considered in cell culture expansion systems for future human NP cell‐based therapeutic transplant studies. STEM CELLS2012;30:2387–2399

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.

Quantitative Assessment of Neurite Outgrowth in PC12 Cells

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity. In order to identify potential developmental neurotoxicants, assessment of critical neurodevelopmental processes, such as neuronal differentiation and growth has been proposed. PC12 cells have been widely used to study the neurotrophic factor-induced signaling pathways that control differentiation, and as in vitro models to detect the effect of chemicals on neurite outgrowth. Upon exposure to nerve growth factor (NGF), PC12 cells cease to proliferate, extend multiple neurites, and acquire the properties of sympathetic neurons. Measurement of the number and length of neurites during exposure to NGF provides a quantitative assessment of neuronal differentiation and growth. Differentiation and neurite outgrowth can be measured using simple contrast microscopy in live cells, or using automated imaging systems in cells prepared with immunocytochemistry.

Transcriptional response of rat frontal cortex following acute in vivo exposure to the pyrethroid insecticides permethrin and deltamethrin.

BackgroundPyrethroids are neurotoxic pesticides that interact with membrane bound ion channels in neurons and disrupt nerve function. The purpose of this study was to characterize and explore changes in gene expression that occur in the rat frontal cortex, an area of CNS affected by pyrethroids, following an acute low-dose exposure.ResultsRats were acutely exposed to either deltamethrin (0.3 – 3 mg/kg) or permethrin (1 – 100 mg/kg) followed by collection of cortical tissue at 6 hours. The doses used range from those that cause minimal signs of intoxication at the behavioral level to doses well below apparent no effect levels in the whole animal. A statistical framework based on parallel linear (SAM) and isotonic regression (PIR) methods identified 95 and 53 probe sets as dose-responsive. The PIR analysis was most sensitive for detecting transcripts with changes in expression at the NOAEL dose. A sub-set of genes (Camk1g, Ddc, Gpd3, c-fos and Egr1) was then confirmed by qRT-PCR and examined in a time course study. Changes in mRNA levels were typically less than 3-fold in magnitude across all components of the study. The responses observed are consistent with pyrethroids producing increased neuronal excitation in the cortex following a low-dose in vivo exposure. In addition, Significance Analysis of Function and Expression (SAFE) identified significantly enriched gene categories common for both pyrethroids, including some relating to branching morphogenesis. Exposure of primary cortical cell cultures to both compounds resulted in an increase (~25%) in the number of neurite branch points, supporting the results of the SAFE analysis.ConclusionIn the present study, pyrethroids induced changes in gene expression in the frontal cortex near the threshold for decreases in ambulatory motor activity in vivo. The penalized regression methods performed similarly in detecting dose-dependent changes in gene transcription. Finally, SAFE analysis of gene expression data identified branching morphogenesis as a biological process sensitive to pyrethroids and subsequent in vitro experiments confirmed this predicted effect. The novel findings regarding pyrethroid effects on branching morphogenesis indicate these compounds may act as developmental neurotoxicants that affect normal neuronal morphology.