Brian L. Robinette

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.

Comparison of PC12 and cerebellar granule cell cultures for evaluating neurite outgrowth using high content analysis

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process underlying nervous system development that can be quantified using automated microscopy and image analysis (high content analysis). The present study characterized and compared the PC-12 cell line (NS-1) and primary cultures of cerebellar granular cells (CGC), as models for assessing chemical effects on neurite outgrowth. High content analysis of neurite outgrowth was performed using the Cellomics ArrayScan V(Ti) automated epifluorescent imaging system to acquire and analyze images of beta-tubulin immunostained cells in 96-well plates. Cell viability was assessed using the CellTiter-Glo assay. Culture of NS-1 or CGC in nerve growth factor or serum respectively, rapidly induced neurite outgrowth that increased over four days in vitro. Seven compounds previously shown to affect neurite outgrowth in vitro were tested in both models for changes in total neurite length and cell viability. In NS-1 cells, four chemicals (PKC inhibitor Bis-I, MEK inhibitor U0126, trans-Retinoic acid, methylmercury) inhibited neurite outgrowth, while lead, amphetamine and valproic acid had no effect. In CGC, five chemicals inhibited neurite outgrowth (Bis-I, U0126, lead, methylmercury, and amphetamine), while trans-Retinoic acid decreased cell viability but not neurite outgrowth. Valproic acid was without effect. The sensitivity of the two models was chemical specific: NS-1 cells were more sensitive to Bis-I, methylmercury and trans-Retinoic acid, while CGC were more sensitive to U0126, lead, and amphetamine. For every chemical (except trans-Retinoic acid), neurite outgrowth was equal to or more sensitive than cell viability. In comparison, out of seven chemicals without prior evidence for effects on neurite outgrowth, only one decreased neurite outgrowth (diphenhydramine in CGC). These findings demonstrate that the effects of chemicals on neurite outgrowth may be cell type specific.

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.

Protein biomarkers associated with growth and synaptogenesis in a cell culture model of neuronal development

Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens for developmental neurotoxicity. The present study examined morphologic and biochemical markers of CGC neurite outgrowth and synaptogenesis in vitro using both qualitative and quantitative approaches. CGC exhibit a rapid outgrowth of neurites over 14 days in vitro, concomitant with the expression of the synaptic protein Synapsin 1 that was observed as puncta associated with cell bodies and neurites. The expression of neurotypic proteins associated with the cytoskeleton (NF68, MAP2), growth cones (GAP-43) and the synapse (Synapsin I) present an ontogeny that reflects the morphological growth of CGC. The utility of these neurotypic proteins as biomarkers was examined by inhibiting CGC growth using pharmacologic inhibitors of PKC activity and the MAP kinase pathway. Quantitative analysis of neurite outgrowth was performed using an automated image acquisition and analysis system. Treatment of CGC with the MAP kinase pathway inhibitor U0126 significantly decreased total neurite outgrowth, while the inhibitor of classic PKC isoforms Bis I had no effect on this measure. The ontogenetic expression of neurotypic proteins was reduced after treatment with both inhibitors. In particular, Synapsin 1 and GAP-43 expression were both significantly reduced by chemical treatment. These data demonstrate that neurotypic proteins can be used as biomarkers of neuronal development in vitro, and in some cases, may detect changes that are not apparent using morphologic measures.

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.

Metabolomic effects in HepG2 cells exposed to four TiO2 and two CeO2 nanomaterials

It is difficult to evaluate nanomaterials potential toxicity and to make science-based societal choices. To better assess potential hepatotoxicity issues, human liver HepG2 cells were exposed to four TiO2 and two CeO2 nanomaterials at 30 ug ml−1 for three days with dry mean primary particle sizes ranging from 8 to 142 nm. Two nanomaterials were also run at 3 ug ml−1. A metabolomics study was then performed using three mass spectroscopy dependent platforms (LC and GC). Five of the six nanomaterials strongly reduced glutathione concentration. The two strongest effects were from exposures to a TiO2 (59 nm) and a CeO2 (8 nm), both from NanoAmor. The decreases in the GSH system were observed in (a) GSH precursors (glutamate and cysteine), (b) GSH itself and (c) GSH metabolites (the gamma-glutamyl condensation products with glutamate, glutamine, alanine, valine and also 5-oxoproline and cysteine–GSH). The glutathione decreases were the largest decreases seen among the 265 biochemical metabolites determined and is consistent with nanomaterials acting via an oxidative stress mode of action. CeO2, but not TiO2, increased asymmetric dimethylarginine concentration and thus possible decreases in iNOS activity and NO concentration could result. One CeO2 (8 nm from NanoAmor) increased concentrations of many lipids, particularly fatty acids. Similar statistically significant elevations were seen in several other classes of lipids (lysolipid, monoacylglycerol, diacylglycerol and sphingolipid) but not in all classes of lipids (glycerolipid, carnitine and fatty acid dicarboxylate). None of the other 5 nanomaterials had this lipid effect. Thus, metabolomic analysis of nanomaterial treated HepG2 cells revealed several previously unknown biochemical effects.

Media formulation influences chemical effects on neuronal growth and morphology.

Screening for developmental neurotoxicity using in vitro, cell-based systems has been proposed as an efficient alternative to performing in vivo studies. One tool currently used for developmental neurotoxicity screening is automated high-content imaging of neuronal morphology. While high-content imaging (HCI) has been demonstrated to be useful in detection of potential developmental neurotoxicants, comparison of results between laboratories or assays can be complicated due to methodological differences. In order to determine whether high-content imaging-based developmental neurotoxicity assays can be affected by differences in media formulation, a systematic comparison of serum-supplemented (Dulbecco’s modified Eagle’s media (DMEM) + 10% serum) and serum-free (Neurobasal A + B27) culture media on neuronal morphology was performed using primary rat cortical neurons. Concentration–response assays for neuritogenesis, axon and dendrite outgrowth, and synaptogenesis were performed in each media type using chemicals with previously demonstrated effects. Marked qualitative and quantitative differences in the characteristics of neurons cultured in the two media types were observed, with increased neuronal growth and less basal cell death in Neurobasal A + B27. Media formulation also affected assay sensitivity and selectivity. Increases in assay sensitivity were observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. In some instances, a greater difference between effective concentrations for cell death and neurodevelopmental-specific endpoints was also observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. These data show that media formulation must be considered when comparing data for similar endpoints between studies. Neuronal culture maintained in Neurobasal A + B27 media had several features advantageous for HCI applications including less basal cell death, less cell clustering and neurite fasciculation, and a tendency towards increased sensitivity and selectivity in chemical concentration–response studies.