Toxicology in Vitro 28 (2014) 411–418

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A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against bIII-tubulin Dina Popova, Stig O.P. Jacobsson ⇑ Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden

a r t i c l e

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Article history: Received 22 May 2013 Accepted 21 December 2013 Available online 31 December 2013 Keywords: Neurotoxicity Cell culture bIII-tubulin Calcein-AM Fluorescence Microplate assay

a b s t r a c t The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-bIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-bIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-bIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of bIII-tubulinlabeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-bIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity. In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in bIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxininduced neurite toxicity. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Legislation in both the European Union and in the United States of America has called for increased testing of chemicals. According to current toxicological methodology, various in vivo animal models are used to evaluate parameters such as acute systemic toxicity, sub-chronic and chronic toxicity, and embryotoxic effects of chemicals. However, the screening of a large number of chemicals in animal models is limited by factors such as high cost, low throughput, and not least the suffering of the animals used. Moreover, animal models may not always be sensitive enough to predict human neurotoxicity. For that reason, a valid, reproducible, and not least freely available in vitro model for neurotoxicity testing would provide a much-needed means by which rapidly to identify potential neurotoxic substances. It is difficult to design an in vitro model or even a battery of in vitro tests to replace the complexity of the nervous system in in vivo animal model systems, but neuronal in vitro models may be well suited to elucidate mechanisms of toxicity and delineate the cellular changes induced by neurotoxicants in a more isolated ⇑ Corresponding author. Tel.: +46 90 785 2713; fax: +46 90 785 2752. E-mail address: [email protected] (S.O.P. Jacobsson). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.12.009

context. Both biochemical and morphological end points can be used, and assays of electrical activity, neurotransmitter release, and neurite outgrowth have been described as relevant to cellbased neurotoxicity (Bal-Price et al., 2008; Radio and Mundy, 2008). Neurite outgrowth is an important parameter that has been extensively studied in cell models of neurotoxicity (Geldof, 1995; Radio et al., 2008), since the development of axons and dendrites is crucial for the formation of a functional neural network in the brain, and disruption of this process may lead to cognitive abnormalities (Berger-Sweeney and Hohmann, 1997; Ramakers, 2002). Although, various in vitro cell models have demonstrated the ability to detect changes in neurite outgrowth as a response to chemical exposure, most studies have been performed using lowthroughput manual or semiautomatic techniques. For screening of a large number of chemicals, a rapid and simple high-throughput method to study chemical-induced effects on neurite development is needed. In this study, we have investigated if the immunochemical detection of anti-bIII-tubulin antibody is useful in a microplate assay of neurite outgrowth in neurons derived from mouse P19 embryonal carcinoma (EC) cells. Fluorescence microplate readers are common laboratory equipment in academic research and clinical diagnostic laboratories, and we have developed and

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validated a rapid method for quantitative measurements of neurite outgrowth in P19-derived neurons cultured in the 96-well plate format. Primary neural cell cultures provide a model of neurite development that closely corresponds to neurons in situ, but the need repeatedly to prepare new cultures and the inter-culture variability, make such primary cultures less suitable for screening. Neuronal cell lines, on the other hand, are widely available and can easily provide a homogenous population of cells. The P19 cell line, which in the presence of retinoic acid (RA) differentiates into neurons, astrocytes and fibroblast-like cells (Jones-Villeneuve et al., 1982), is a useful model for studying chemical-induced effects on neural cells in vitro. The RA-differentiated P19 cells possess a number of properties characteristic for neurons in the mammalian nervous system, including a morphology that resembles the structure of cultured brain cells with small cell bodies and long axons and dendrites (McBurney et al., 1988; Yao et al., 1995), and they are postmitotic as are neurons in the mammalian brain (McBurney et al., 1988). Pre- and post-synaptic morphological features of chemical synapses have been discovered in the RA-differentiated P19 cells (McBurney et al., 1988) and electrophysiological studies suggest that the synapses are functionally active (Morasutti et al., 1994; Magnuson et al., 1995). This model makes it possible rapidly to obtain a homogeneous and reproducible neural cell culture for screening studies. Class III b-tubulin is a cytoskeletal protein expressed almost exclusively in neurons (Katsetos et al., 2003a). One of the main components of the cytoskeleton is microtubule that contains the globular protein tubulin as a major structural element. Tubulin consists predominantly of two subunits, a and b, and each of those peptides exists in various isotypic forms. Class III b-tubulin is one of seven b-tubulin isotypes found in humans (Luduena, 1998). It is expressed in high levels in the brain where it shows distinctive neuronal specificity (Luduena, 1998; Katsetos et al., 2003b). The protein has been used as neuronal marker in a number of studies of neuronal development. We have quantified the time-dependent neurite outgrowth in differentiated P19-derived neurons labeled with bIII-tubulin antibodies in a fluorescence microplate reader. The results have been compared with those obtained by measuring the neurite length in a semiautomatic tracing method of individual neurons in fluorescence microscopy images using the computer software NeuronJ, and with a microplate assay employing the fluorophores calceinAM and propidium iodide (PI). Furthermore, the concentrationdependent effects of nocodazole, methylmercury (MeHg), okadaic acid, clomipramine, glutamate, and dimethyl sulfoxide (DMSO) on the bIII-tubulin immunoreactivity were measured in the fluorescence microplate reader, and compared to the results obtained by staining the cells with calcein-AM and PI to assess neurotoxicity. We found that the fluorescence microplate reader assay for detection of neurite outgrowth by measuring changes in bIII-tubulin immunoreactivity is a rapid and sensitive method to assess neurite outgrowth and chemical-induced neurite toxicity in neuronal cultures.

2. Materials and methods 2.1. Chemicals MEM-a medium containing deoxyribonucleosides and ribonucleosides, fetal bovine serum, penicillin–streptomycin, MEM non-essential amino acids (NEAA), Neurobasal medium, B27 supplement, goat anti-rabbit IgG antibody conjugated to fluorescent dye Alexa Fluor 488, DAPI nucleic acid stain, calcein-acetoxymethyl (AM), and propidium iodide were purchased from Invitro-

gen Life Technologies (Uppsala, Sweden). Cytotoxicity detection kits (LDH) were obtained from Roche Diagnostics (Mannheim, Germany). All-trans retinoic acid (atRA), poly-D-lysine hydrobromide, 96-well culture plates (COSTAR 3603), formaldehyde solution, Dulbecco’s phosphate buffered saline with CaCl2 and MgCl2 (DPBS), okadaic acid (95%, CAS No. 78111-17-8), methylmercury (II) chloride (CAS No. 115-09-3), nocodazole (CAS No. 31430-189), clomipramine hydrochloride (CAS No. 17321-77-6), L-glutamic acid (CAS No. 56-86-0), glycine (CAS No. 56-40-6), and dimethyl sulfoxide were all purchased from Sigma–Aldrich (Stockholm, Sweden). 2.2. Cell culture P19 cells (passage 9–27), obtained from European Collection of Cell Cultures (Porton Down, UK), were cultured in MEM-a medium containing deoxyribonucleosides and ribonucleosides, supplemented with 10% fetal bovine serum (FBS), 100 unit/ml penicillin, 100 lg/ml streptomycin and 1% (v/v) MEM non-essential amino acids (NEAA). The cells were maintained at 37 °C in a humidified incubator containing 5% CO2. The medium was replaced every 48 h and the cultures were split every 96 h. P19 cells were induced to neuronal differentiation essentially as described in Yao et al. (1995) and cultured in Neurobasal medium with B27 supplement according to Svensson et al. (2006). Briefly, the process of neuronal differentiation was initiated by allowing P19 cells to aggregate for 4 days on bacterial-grade Petri dishes (£92 mm; Sarstedt Inc., Newton, NC) in the presence of 1 lM all-trans retinoic acid (RA) in MEM-a medium (5% FBS, 1% PEST and 1% NEAA) at a density of 1  106 cells/dish. After 48 h, the culture medium was replaced with fresh RA-containing medium. After a 96 h exposure to RA, the aggregates were trypsinized for 10 min and dispersed in serum-free Neurobasal medium with 2% (v/v) B27 supplement, 1 mM L-glutamine (Biochrom, Berlin), and 1% PEST, into poly-D-lysine precoated (50 lg/ml) 96-well culture plates at a density of 500 cells/mm2. Half of the medium per well was replaced every 48 h. Analyses were performed at day two, four, six, eight, or 10 after plating, unless otherwise specified. The RA-differentiated P19 cells are termed ‘‘P19 neurons’’ hereafter. 2.3. Analyses of immunofluorescence P19 neurons were immunostained against bIII-tubulin and total amount of fluorescence was measured using a FLUOstar Galaxy plate reader (excitation/emission: 485/520 nm) (BMG Labtechnologies GmbH, Offenburg, Germany). The P19-derived neurons were fixed with 3.7% formaldehyde solution in Dulbecco’s phosphate buffered saline with CaCl2 and MgCl2 (DPBS) for 30 min. The samples were washed with DPBS, permeabilized with 0.1% Trition X100 solution in DPBS for 5 min, washed two times and blocked with 3% fetal bovine serum in DPBS for 30 min. Primary polyclonal antibodies against bIII-tubulin (rabbit anti-mammalian) (Convance, Princeton, NJ) diluted 1:500 in the blocking buffer were applied for 1 h and the samples were washed three times. Secondary goat anti-rabbit IgG antibodies conjugated to the fluorescent dye Alexa Fluor 488 were diluted 1:250 in the blocking buffer and applied for 1 h 20 min. The samples were washed three times and analyzed. Samples incubated only with secondary antibodies served as controls for background bindings. 2.4. Neurite tracing with NeuronJ The cell samples immunostained against bIII-tubulin were visualized using a Nikon Eclipse TE2000-U inverted microscope with a Plan Fluor ELWD 20/0.45 objective with a Nikon Digital

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Sight DS-5Mc camera (Tekno Optik AB, Skärholmen, Sweden). Three images per well were acquired (left side, middle and right side of a well). Using Photoshop Elements 8.0 (Adobe, San Jose, CA), the size of the images was converted to 800  600 pixels and saved in the graphics interchange format (GIF). The processed pictures of two cell samples (three images per sample) were analyzed with a semiautomatic neurite tracing technique, the NeuronJ (1.4.2) plug-in to ImageJ (1.44) software (Meijering et al., 2004). The number of cell bodies was counted and the neurite length was measured in pixels (line width 5) from which the total length (sum of the length of all neurites in an image) and the total neurite length per cell containing neurites (positive neurons) were counted. At least two cell samples of each experimental condition marked against bIII-tubulin were co-stained with DAPI nucleic acid stain (0.3 mM/well) for 3 min, and the DAPI-stained nuclei were manually counted.

2.5. Assays of neuronal viability Cell viability in P19 neurons was measured with calcein-AM assay in the above-mentioned fluorescence plate reader. Calcein-AM, a non-fluorescent cell-permeant compound that is hydrolized by intracellular esterases to a fluorescent green calcein, was dissolved in DMSO (4 mM) and stored for 1 h at 25 °C followed by dilution in DPBS to the final concentration of 1 lM in the cell culture. The cells were incubated with calcein-AM for 40–60 min (with or without PI staining, see below), washed with DPBS and analyzed in the plate reader (excitation/emission filter: 485 nm/520 nm). A PI assay was applied to detect total nuclear content in the cell samples and a general cell death during the process of neuronal differentiation. PI is a red fluorescent dye that penetrates only damaged cellular membranes and undergoes a 20- to 30-fold fluorescence enhancement upon binding to double-stranded DNA. To determine a total nuclear content, cell samples were treated with 0.2% Triton X-100 in DPBS for 10 min prior to PI application. For estimation of cell death during the process of neuronal differentiation, non-treated cells samples were used. PI was diluted in DPBS to the final concentration of 10 lg/ml in the cell culture. The samples were incubated for 40–60 min with calcein-AM, washed once with DPBS and analyzed in the plate reader (excitations/emissions filter: 544 nm/620 nm). P19-derived neurons that had been allowed to differentiate for 6 days in the serum-free medium were exposed to different concentrations of nocodazole, MeHg, okadaic acid, clomipramine, glutamate (with 10 lM glycine), and DMSO diluted in culture medium. Stock solutions of okadaic acid, nocodazole and MeHg were prepared in DMSO, and the final concentration of DMSO never exceeded 0.5%. Culture wells incubated with 2% Triton X100 served as controls for maximal cell death. After 24 h or 48 h, the neuronal cultures were analyzed for total amount of bIII-tubulin fluorescence and cell viability (calcein-AM assay) in the FLUOstar Galaxy microplate reader. Data are presented as either untransformed fluorescence units or percentage of untreated controls. Statistically significant differences between data from bIIItubulin fluorescence and calcein-AM fluorescence in the neurotoxicity studies were examined using unpaired t-tests. The LDH activity in each well was measured by transferring 100 ll aliquots of the culture medium to an optically clear 96-well flat bottom microtiter plate. To each well, 100 ll of a Cytotoxicity Detection Kit assay mixture was added and the samples were measured spectrophotometrically at 490 nm (reference wavelength 650 nm) in the computer-operated SPECTROstar Nano absorbance microplate reader (BMG LABTECH GmbH, Offenburg, Germany). To determine the total LDH content (cell LDH activity) in each well, the cells were lysed with 2% Triton X-100 solution.

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3. Results 3.1. Time-dependent increase in bIII-tubulin immunoreactivity in P19derived neuronal cultures Neurite outgrowth in RA-induced P19 cells progressed rapidly on poly-D-lysine-coated 96-well plates in serum-free B27-supplemented Neurobasal medium. Within hours after plating, thin neurites began to emerge from the cell body on a small proportion of cells (data not shown), and by 48 h over 80% of the cells immunopositive for bIII-tubulin showed neurite outgrowth. Analysis of fluorescence microscopy images of neuronal cultures stained against bIII-tubulin, showed that this microtubule protein was present in both the cell bodies and all neurites of viable cells (Fig. 1A). The semi-automatic morphometrical analyses of the fluorescence microscopy images using the NeuronJ software showed that both the neurite length per neurite-expressing cell and the total length of neurites in the images increased almost linearly with time up to 10 days in culture (Fig. 1B and C). At the same time, the total number of cells did not increase (Fig. 1E; 129 ± 11 cells per image at day 2, and 113 ± 15 cells per image at day 10), as assessed by manual counting of DAPI-stained cells in the microscopy images; nor did the fraction of dead cells, as assessed by measuring the fluorescence of PI using a microplate reader (Fig. 1D). However, the fraction of cell bodies without neurites increased from 30 ± 4 cells per image on day 2, to 47 ± 13 cells per image on day 10. The time-dependent increase in anti-bIII-tubulin immunofluorescence was also observed by measuring the fluorescence in a microplate reader, and the relative fluorescence units increased 4.3-fold from 2 to 10 days in culture. The results obtained in the plate reader corresponded well with those obtained by the much more time-consuming method of semi-automatic tracing of neurites in fluorescence microscopy images (Fig. 1B). The results obtained by measuring bIII-tubulin immunoreactivity in the microplate reader were compared to those obtained in parallel cultures assayed for fluorescence of calcein-AM. Cultures incubated with calcein-AM showed mainly cytosolic fluorescence of calcein in live cells (Fig. 1A), with relatively weak labeling of the fine branching neurites that were clearly visualized in the anti-bIII-tubulin-stained cultures. The time-dependent increase in fluorescence of calcein obtained in the microplate reader were more variable compared to the bIII-tubulin immunoreactivity, and a major increase in calcein fluorescence was seen between day 4 and day 6 followed by a plateau (Fig. 1D). The results suggest that measuring anti-bIII-tubulin immunofluorescence in the microplate reader is a superior method to assess developmental increase in neurite expression, compared to both semi-automatic analysis of fluorescence microscope images and measurement of intracellular fluorescence of calcein.

3.2. Measurement of bIII-tubulin immunoreactivity to detect concentration-dependent neurite toxicity of toxins and chemicals To examine if the bIII-tubulin immunoreactivity assay using the microplate reader is sensitive and specific enough to detect concentration-dependent chemical-induced neurite toxicity, P19 neurons cultured for 6 days were exposed to nocodazole, MeHg, okadaic acid, clomipramine, glutamate, and DMSO. The chosen timing for the exposure was based on the previous results (Fig. 1A–C) showing that after 6 days in culture, P19 neurons produce an extensive network of neurites that are still under development. The fluorescence in cultures incubated with anti-bIII-tubulin antibodies or calcein-AM and PI were measured in the microplate reader, and the results are shown in Fig. 2. Following 24 h of incubation with nocodazole, a broad-spectrum anthelmintic drug with

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Fig. 1. Development of neurons derived from RA-treated mouse P19 EC cells (‘‘P19 neurons’’) up to 10 days after plating on poly-D-lysine-coated 96-well plates in serum-free B27-supplemented Neurobasal medium. (A) representative fluorescence microscopy images of neurons labeled with the neuron specific bIII-tubulin antibody (upper row) or incubated with calcein-AM and PI (lower row). (B) Assessment of the time-dependent neurite outgrowth by measuring fluorescence of the bIII-tubulin antibody in a microplate reader (green circles corresponds to relative fluorescence units, RFU, on the left y-axis) or by semi-automatic tracing of neurites in fluorescence microscopy images of bIII-tubulin-labeled neurons using the NeuronJ software (triangles corresponds to arbitrary units, AU, on the right y-axis). (C) Time-dependent increase in total neurite length per fluorescence microscopy images divided by number of outgrowth positive neurons, as assessed by the NeuronJ image analysis. (D) Fluorescence in cultures stained with calcein-AM and PI (with or without pretreatment with 0.2% Triton X-100 to determine the total amount of nuclei). (E) Manual counting of neuronal cell bodies (with or without neurites) labeled with the nuclear stain DAPI in fluorescence microscopy images. Data are means ± SEM of 3–4 independent experiments.

antineoplastic properties that interfere with the polymerization of microtubules (De Brabander et al., 1977), a reduction in anti-bIIItubulin immunofluorescence was observed at nanomolar concentrations (Fig. 2B and G). This was not seen when measuring cytosolic fluorescence of calcein. Thus, for example, the fluorescence (as % of controls) seen following treatment with 1 nM of nocodazole was 85 ± 2% and 106 ± 2% for the bIII-tubulin and calcein-AM methodologies, respectively. The lowest concentration required for a statistically significant increase in extracellular LDH activity was 1 lM nocodazole (Fig. 3A). Similar results were obtained when the neurons were exposed to MeHg (Fig. 2C and G), with statistically significant differences between the bIII-tubulin assay and the calcein-AM assay being found at 50 nM (83 ± 6% vs. 107 ± 6% fluorescence of controls, respectively), and 100 nM (64 ± 2% of controls vs. 85 ± 5%, respectively); no difference were seen at 0.5 or 1 lM. The potent cytotoxin okadaic acid, originally derived from marine dinoflagellates (An et al., 2010), produced a significant reduction in both antibIII-tubulin immunofluorescence and cytosolic fluorescence of calcein (Fig. 2D and G), but the neurite assay proved to be more sensitive to detect neurotoxicity at low concentrations (59 ± 7% of

untreated control cultures at 1 nM okadaic acid) compared to the calcein-AM assay (86 ± 7% of controls). However, at higher concentrations of okadaic acid (>5 nM) there was no statistically significant difference between the assays. The tricyclic antidepressant clomipramine produced a dose-dependent cytotoxicity (Fig. 2E and G), and at the highest concentration examined (30 lM) the calcein-AM assay showed a fluorescence of 19 ± 3% of controls, that was statistically significant less than the reduction in anti-bIIItubulin immunofluorescence (62 ± 14% of controls; P < 0.05). At this concentration, the extracellular activity of LDH was 42 ± 1% of Triton X-100-treated control wells (Fig. 2B; P < 0.0001 compared to untreated control cultures). On the other hand, none of the microplate-based fluorescence assays were able to detect significant cytotoxicity in cultures exposed to the excitatory amino acid glutamate (Fig. 2F and G) for 48 h, whereas the LDH activity assay revealed a small but statistically significant cytotoxicity at 100 lM (11 ± 1% of total; P < 0.05) and 0.5 mM (13 ± 0.5%; P < 0.01) glutamate. The organosulfur solvent DMSO, at concentrations between 0.1% and 1% was without effect on either neurite expression or intracellular calein fluorescence, but at 5% both assays indicated a major neurotoxicity (Fig. 2A).

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Fig. 2. Concentration-dependent effects of (A) DMSO, (B) the antineoplastic agent nocodazole, (C) the heavy metal MeHg, (D) the neurotoxin okadaic acid, (E) the tricyclic antidepressant clomipramine, and (F) the excitatory amino acid glutamate on the immunofluorescence of neuron specific bIII-tubulin antibodies (green circles) and the fluorescence of calcein-AM (red triangles) in cultures of P19 neurons as measured in a fluorescence microplate reader. The cultures were exposed to the test compounds for 48 h (24 h for the nocodazole exposure). (G) Representative fluorescence microscopy images of untreated control cultures, and P19 neurons exposed to 0.1 lM nocodazole, 0.5 lM MeHg, 1 nM okadaic acid, 30 lM clomipramine or 0.5 mM glutamate, followed by staining with the neuron specific bIII-tubulin antibody and the nuclear stain DAPI (upper panels) or incubation with calcein-AM and PI (lower panels). Data (means ± SEM of 3–5 independent experiments) are plotted as percentage change in fluorescence units from untreated control cultures. Statistical treatment of data was undertaken using two-tailed t-test: P < 0.05, P < 0.01, and P < 0.001 (comparisons between fluorescence values of bIII-tubulin and fluorescence values of calcein-AM at each concentration).

Taken together, these data suggest that anti-bIII-tubulin immunoflureoscence in the microplate reader is a more sensitive method

to assess chemical-induced neurite toxicity compared to the calcein-AM assay and the LDH activity assay.

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Fig. 3. Concentration-dependent effects of 24 h exposure to (A) nocodazole, or 48 h exposure to (B) clomipramine, and (C) glutamate upon the release of LDH from P19 neurons. Data are means ± SEM of 5–8 experiments. Statistically significant differences (one-way ANOVA with Dunnet’s multiple comparison test) are indicated: P < 0.05,  P < 0.01, P < 0.001, and P < 0.0001 when compared with control release.

4. Discussion In vitro models and methods to measure neuronal cell viability and neurite outgrowth are valuable tools to study neurotoxic effects of drug compounds and environmental toxicants. Since neuronal cultures are heterogeneous by nature, robust and reproducible analysis techniques are required to extract meaningful information. In the present study, the time-dependent neurite outgrowth in differentiated P19-derived neurons labeled with bIII-tubulin antibodies was quantified by using a fluorescence microplate reader, and the results were compared to those obtained by using calcein-AM/PI staining, or by measuring the neurite length in a semiautomatic tracing method of individual neurons in fluorescence microscopy images. The mouse EC cell line P19 has been characterised as an useful model to study neuronal processes and the neuronal differentiation, since the P19-derived neurons bear a strong morphological and electrophysiological resemblance to normal mammalian neurons (Seeley and Faustman, 1998). The P19 cells were isolated by McBurney and Rogers (1982), and are derived from a teratocarcinoma in C3H/HeHa mice. The P19 cell line is multipotent and can differentiate into derivates from all three germ layers; endoderm, mesoderm and ectoderm (McBurney, 1993). The differentiation of EC cells in culture closely resembles what occurs in the embryo (Lehtonen et al., 1989), and RA-treated cultures of P19 cells are heterogenous in morphology and expression of neurotransmitters. The RA-induced P19 cultures contain mainly neurons, although glial cells and a population of fibroblast-like cells have also been identified (Jones-Villeneuve et al., 1982), and the P19-derived neurons express a variety of neuronal proteins including functional proteins for GABA and glutamate transmission as well as functional synapses (MacPherson et al., 1997). Neuronal bIII-tubulin is a protein expressed almost exclusively by neurons following early differentiation in the CNS (Lee and Pixley, 1994), and neuron specific anti-bIII-tubulin antibodies have become invaluable tools for identifying neuronal differentiation and neurite outgrowth. The results of the present study suggest that measuring anti-bIII-tubulin immunoflureoscence in the microplate reader is a superior method to assess developmental increase in neurite expression, compared to both semi-automatic analysis of fluorescence microscope images and measurement of intracellular fluorescence of calcein. The morphological changes observed in the differentiating P19 cells confirm the previous studies (Jones-Villeneuve et al., 1982; McBurney et al., 1988) indicating that the cells develop toward a neuronal phenotype. At 48 h after initial plating, the cells expressed bIII-tubulin characteristic of maturing neurons and a large proportion of cells had developed one to three neurites. The neurite outgrowth in the differentiating P19-derived neuronal cultures

was comparable to what has been described for primary neuronal cultures (Neumann et al., 2002). The time-dependent increase in bIII-tubulin fluorescence was not due to cell proliferation since the average number of cell nuclei did not increase over time, as assessed both by measuring fluorescence of PI and manual counting of cells in the images. The results obtained when the bIII-tubulin immunofluorescence was measured in the microplate reader were almost identical to those obtained by the method of semi-automatic tracing of neurites in fluorescence microscopy images. In the microplate reader method, the measured fluorescence signal consists of bIII-tubulin in both neurites and the cell bodies. However, the area of the extensive neurite network, particularly at the later stage of neural differentiation (days 6–10 after plating), are considerably larger than the total area of the non-dividing cell bodies. This suggests that even if there may be variations in fluorescence intensity between different parts of the cell, the majority of the fluorescence originates from the neurites and the microplate reader thus primarily measures neurite outgrowth. The NeuronJ protocol requires that users can unmistakably recognize and assign neurite structures, and the measurements can therefore be subject to user bias. On the other hand, the microplate reader method provides a rapid and reliable screen of neurite outgrowth without the risk of bias. After the staining procedure, the neurite outgrowth in each well in a 96-well microplate can be quantified within a couple of minutes using the microplate reader, whereas the same amount of data obtained by using the semiautomatic tracing method will take many hours to collect. Fluorometric assays for high throughput screening of cell viability and cytoxicity commonly involve fluorogenic esterase substrates such as calcein-AM and various fluorescein diacetate derivatives. Calcein-AM is cell-permeable and as such measures both cell-membrane integrity and enzymatic activity and we have found it to be a sensitive probe to detect chemical-induced cytotoxicity in various cell lines (Jacobsson et al., 2001; Gustafsson et al., 2008, 2013). In the present study, calein-AM produced fluorescence mainly in the cytosol of live cells and did not label the thinnest neurites that were visualized by anti-bIII-tubulin antibodies. The time-dependent increase in fluorescence of calcein reached a plateau between 6–10 days in culture. We have validated the specificity and the selectivity of the method by analyzing the concentration-dependent effects of chemicals known to affect the outgrowth of neurites in neural cultures. Our findings indicate that the fluorescence plate reader method was sensitive enough to detect chemical-induced changes in neurite outgrowth in P19 neurons marked with the anti-bIIItubulin antibody. Nocodazole is a broad-spectrum anthelmintic drug with antineoplastic properties that interfere with the polymerization of microtubules, and as such induces substantial morphological alteration of developing neurons and inhibits neurite

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growth (Solomon, 1980). Okadaic acid has been shown to potently inhibit neurite outgrowth in PC12 cells (Chiou and Westhead, 1992; Radio et al., 2008) and dorsal root ganglion neurons (Giasson and Mushynski, 1997). MeHg possess high affinity for tubulin sulfhydryl groups (Vogel et al., 1985) and target cytoskeletal components and especially microtubules. Accordingly, MeHg has been reported to promote microtubule disruption in several cell models (Castoldi et al., 2001) and induce neurite degeneration in PC12 cells (Parran et al., 2000; Radio et al., 2008) and primary cultures of mouse dopaminergic mesencephalic cells (Götz et al., 2002). The method of measuring bIII-tubulin immunoreactivity in a fluorescence microplate reader proved to be more sensitive than the calcein-AM assay to detect specific concentration-dependent neuritotoxic effects of nocodazole, MeHg and okadaic acid at low (nanomolar) concentrations. In order to assess the specificity of the fluorescence microplate screen assay using anti-bIII-tubulin antibodies, the effects of clomipramine, glutamate and DMSO were examined. Clomipramine, a tricyclic antidepressant, has shown to induce apoptosis in cancer cells by increasing the production of reactive oxygen species (ROS) with subsequent loss of mitochondrial membrane potential (for a review, see Pilkington et al., 2006). Clomipramine is also toxic to differentiated cells in culture in the low micromolar range. High concentrations of glutamate, the major excitatory amino acid in the CNS, can produce excitotoxicity in neurons by a glutamate receptor-mediated mechanism. The solvent DMSO was included in the study since it is a commonly used vehicle in vitro to solubilize compounds with low water solubility. When exposing the P19-derived neurons to clomipramine, glutamate or DMSO, no major differences in the sensitivity to detect concentration-dependent cytotoxicity between the microplatebased fluorescence assays were found. When assessing the toxicity produced by compounds that produce general cytotoxicity, such as clomipramine, or with a specific mechanism of action that do not primarily target neurite outgrowth, such as glutamate-induced neurotoxicity, other methods such as the extracellular LDH activity assay may be more appropriate to employ. In conclusion, the current study shows that neuron specific bIIItubulin fluorescence can be used to screen for chemical- or toxininduced effects on neurite outgrowth in a rapid and efficient manner using a standard fluorescence microplate reader. This method could serve as part of a test battery for identification of neurotoxic chemicals, but confirmation of the neurotoxic potential will require further assessment using in vivo methods. In the development of in vitro systems for chemical screening of neurite outgrowth, other cell culture models may be considered. With the increasing availability of human neural stem cell lines, it will hopefully be possible to assess neurite outgrowth in neuronal cultures that are potentially almost identical to neurons found in the human nervous system. Conflict of interest These authors declare that there are no conflicts of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tiv.2013.12.009. References An, T., Winshell, J., Scorzetti, G., Fell, J.W., Rein, K.S., 2010. Identification of okadaic acid production in the marine dinoflagellate Prorocentrum rhathymum from Florida Bay. Toxicon 55, 653–657.

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