and KB performed experiments and acquired the data. GAB, SF and MS designed the figures. GB conceived the project.
Publication 2: Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons. KB and LN provided the first draft of the manuscript and wrote the paper with help of GB. KB, LN, PB, GAB, AU, HK, MD and JR performed experiments. KB and LN analyzed the data. MS and TR contributed to discussions on theoretical feasibility and designed improvements. GB and HM conceived and supervised the research.
Publication 3: A locust embryo as predictive developmental neurotoxicity testing system for pioneer axon pathway formation (Submitted for publication in Archives of Toxicology). KB provided the first draft of the manuscript and wrote the paper with help of GB. KB, MB, JR and PB performed the experiments and acquired the data. KB evaluated the data and designed figures. MS contributed to discussions on theoretical feasibility and design improvements. GB conceived the research project, provided funding, and supervised the research.
11
5. Publications
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
6. Discussion
6.1. Cell culture systems and organism-based models
Human cell culture systems are the gold standard for in vitro DNT assessment. However, most human-based test systems pose several disadvantages. For instance, some of them are human-based on transformed and immortalized cell lines that are characterized by the expression of tumor growth-related genes.
These genes can alter the response of neurons after chemical exposure and make it difficult to interpret the results. On the other hand, different cell lines rely on human embryonic stem cells (hESCs), derived from fetal brain tissues, which pose ethical issues in their generation and application.
Further cell lines differentiated from human induced pluripotent stem cells (hiPSCs) are limited in the number of glia cells, which are involved in chemically-induced neurotoxicity (Bal-Price et al. 2018).
Invertebrates (e.g. planarian) and non-mammalian vertebrates (e.g. zebrafish) are often used for morphological and behavioral analysis as alternatives to traditional animal testing. Comparative analysis of different endpoints for both non-mammalian systems show a high concordance with mammalian outcomes (Hagstrom et al. 2019). Moreover, neuroanatomical and functional studies on Drosophila melanogaster revealed that many relevant molecular mechanisms for axonal outgrowth are conserved between invertebrates and higher organisms (Sánchez-Soriano et al. 2007). The main advantage of organism-based models is that they display the physiologically relevant microenvironment, which is essential for neurodevelopment (Li et al. 2019).
6.2. The locust embryo test system
This thesis presents an ex vivo embryo assay, based on a pest insect that displays the complexity of the in vivo situation for DNT screening. Up to 50 embryos of the same age can be obtained from one egg pod, which allows collecting for multiple replicates in a single experiment. Embryos can grow in multiwell plates due to their small size, short generation time and thus are easy to handle in laboratories. Subjects dissected out of their egg continue to develop for some time in serum-free medium without supplementation of animal serum, and thus are avoiding any ethical concerns.
Furthermore, the phylogenetic distance of an insect embryo to vertebrates minimizes further ethical issues and makes it valuable as a model system for DNT testing.
Here, we incubated locust embryos with a training set consisting of heavy metals, pesticides and medications that are well recognized as DNT-positive or general cytotoxic agents (Aschner et al. 2017;
Smirnova et al. 2014). Since molecules for axon guidance such as semaphorins, are conserved between invertebrates, vertebrates, and mammalians, navigation of pioneers could be representative for the wiring of pyramidal cells in the human cortex (Kolodkin et al. 1992; Polleux et al. 2000). In our study,
58 axon navigation proved to be the most sensitive endpoint (MSE) for DNT chemicals such as methylmerury(II)chloride, sodium(meta)arsenite, rotenone and valproic acid. Altogether 83 % of the test chemicals were correctly identified for their DNT potential. Only the two organophosphate insecticides (chlorpyrifos, chlorpyrifos-oxon) contradicted our assumption and classified as DNT-negatives. Similar to our results, other well-established in vitro assays such as LUHMES (Lund human mesencephalic neurons), hNPCs (human neural progenitor cells) and rNPCs (rat neural progenitor cells) failed to predict chlorpoyrifos and its liver metabolite as DNT chemicals (Baumann et al. 2016; Krug et al. 2013; Stiegler et al. 2011), which increases our predictability to 100 %. Chlorpyrifos mainly interferes with the acetylcholine-signaling cascade through binding to the acetylcholine esterase (AChE) (Grandjean and Landrigan 2006). Neuroanatomical analysis revealed the absence of AChE-enzymes on pioneer neurons, when embryos have reached 35 % of embryogenesis (Bicker et al. 2004). This could be a reason that both pesticides were not identified as DNT chemicals, by using our assay. Based on the results, the locust embryo assay provides a useful complementary method to cell culture systems.
In cases where other cells would be more susceptible to chemicals than neurons, general cytotoxicity could be over-interpreted. However, assessed pioneer neurons mostly had filopodia and non-fragmented axons, which led us to conclude that pioneers were still alive at the moment of fixation.
Another point for over-interpretation of general cytotoxicity is, when endpoints for viability or axonal growth and guidance defects were not inhibited up to 50 %. In these cases, the highest tested concentration was used as IC 50 as seen for example for chlorpyrifos and clorpyrifos-oxon evaluation.
6.3. Evaluation of DNT effects by scanning laser optical tomography (SLOT)
In contrast to cell cultures, most in vivo models suffer from undefined concentrations in the tissue of interest due to diffusion barriers or metabolism (Hagstrom et al. 2019). Since the locust embryo, staged to 32.5 % of embryogenesis, lacks a chitineous cuticle, our model system provides free access to small molecules and antibodies (Isbister et al. 1999; Kolodkin et al. 1992). Furthermore, the embryo is not protected against chemicals by mammalian homologues as the blood-brain barrier, placenta or maternal liver, which facilitates the diffusion of test compounds to their targets. In cooperation with the Laser Zentrum Hannover e.V. (LZH), we calculated the volume of three-dimensional reconstructions of locust embryos, obtained by SLOT measurements, which resulted in a total embryonic tissue of 0.066 µl (0.033µl / embryo) per well. Compared to the extensive incubation volume of 200 µl per test compound it is rather unlikely that embryos chelate or inactivate these chemicals through biotransformation. This allows us to assume that the concentration of the incubation volume coincides with the concentration close to pioneer neurons.
SLOT is a relatively fast 3D-imaging technique that tracks absorption and fluorescence signals in biological transparent organisms of mesoscopic size. It is an advancement of the optical projection
59 tomography (OPT) with enhanced photon collection efficiency (Lorbeer et al. 2011; Nolte et al. 2018).
Moreover, SLOT provides isotopic resolution and avoids shading effects, when compared to other 3D fluorescence based imaging techniques such as OPT, confocal laser scanning microscopy and light sheet fluorescence microscopy (Nolte et al. 2017). Therefore, locust embryos had to be optically cleared by a special embedding method called CRISTAL (Curing Resin-Infiltrated Sample for Transparent Analysis with Light), whereby structure of specimen remained unaffected and showed no biodegradation (Kellner et al. 2016). Our comparative studies between 2D conventional fluorescence microscopy and SLOT revealed that both techniques are suitable to detect outgrowth and navigation errors on pioneer neurons, induced by MeHgCl and arsenic. SLOT currently requires a manual adjustable threshold for the 3D segmentation step. This leads to a broader range of axon lengths, because in some cases faintly labeled axons dropped below this value, which resulted in shorter segmented axons. It has also been recognized that a single experiment (biological replicate) was not sufficient for DNT evaluation by both methods. To minimize the biological variability in independent experiments, we increased the number of biological replicates to n = 3, where both techniques recognized significant differences for axon elongation.
6.4. Cytostatic agents and calcium channel blocker
As shown by the experiments with cytostatic agents as cytochalasin D and colchicine, translation of in vitro findings to in vivo effects can be problematic. Our studies revealed a much higher DNT potential for the actin inhibitor cytochalasin D than reported in cell culture tests (Krug et al. 2013). In contrast, the microtubule inhibitor colchicine showed no effects in behavioral assays on zebrafish larvae (30 µM) (Dach et al. 2019) and reduced axon elongation in locust embryos only to some extent (up to 40 %) at the highest concentration (5 µM). In vitro assays reported about contradictory observations, where axon outgrowth was significantly inhibited at much lower concentrations (IC 50 = 4nM) (Krug et al.
2013). These differences could be due to the absence of guidance molecules in in vitro assays and indicate that axon movement in intact organisms is much more dependent on actin driven forces than in cell culture. Since calcium is essential for growth cone motility and cytoskeletal dynamics (Kater et al. 1988; Lau et al. 1999) we blocked L-type calcium channels with verapamil and diltiazem. Both chemicals reduced axon elongation before the viability curve decreased. IC 50 values of axon elongation were in the same range as reported for insect neurons and glia cells (Lohr et al. 2005).
Furthermore, in vitro preparations of young mouse spinal cord fibers were only 5 - 6.5 times more sensitive to L-type calcium channel blockers, than in locust embryos. (Martinez-Gomez and Lopez-Garcia 2007). Accordingly, verapamil as well as diltiazem displayed endpoint specific DNT effects for neurite elongation in our insect assay.
60 6.5. Outlook
Experiments with cytostatic agents on locusts revealed that in vitro assays are not sufficient to capture all possible health hazards that may attack intact organisms. Our studies exemplified that the locust embryo is a powerful tool for recognizing DNT defects in axon outgrowth and pathfinding with the potential to reduce and replace animal experiments. Thereby it provides a complement to cell culture based assays, which do not measure properties of axonal navigation.
The combination of insect embryo culture and 3D SLOT imaging, allows a semi-automated resolution of axonal navigation and formation of abnormal neurites in an alternative model system. The development of a fully automated recognition tool would be a desirable advancement to diminish biased scoring of developmental neurotoxic effects.
Currently, the assay is more adjusted to a rapid screening system than for mechanistic analysis. Despite the abundance of guidance cues (fasciclins, semaphorins, guidepost cells, basal lamina), the disruption of only one can lead to an error in neuronal pathfinding. In order to identify the specific mode of action (MoA) of DNT chemicals, systematic elimination of single components such as digestion of basal lamina, laser ablation of guidepost cells or antibody blocking of semaphorins would be favorable (Bentley and Caudy 1983; Bentley and O'Connor 1992; Condic and Bentley 1989; Isbister et al. 1999).
The determination of birth and death of transient pioneer neurons would allow us to incorporate additional endpoints such as apoptosis and neurogenesis for DNT quantification, which would enhance the possibility to examine six different endpoints by using only one test system.
Since no model is likely suitable to generate transferable results for all neurodevelopmental processes in humans, there is consensus to implement DNT models in a test battery for prioritization and identification of hazards on neuronal development (Bal-Price et al. 2018; Hagstrom et al. 2019).
Currently, there is no in vitro or in vivo non-mammalian assay, at least to my knowledge, that monitors the wiring of identified neurons in an intact non-vertebrate. The locust embryo exactly addresses this problem and provides the opportunity to investigate this novel endpoint without the use of animal experiments.
61
7. References
Aschner M, Ceccatelli S, Daneshian M, et al. (2017) Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use. ALTEX 34(1):49-74. https://doi.org/10.14573/altex.1604201
Bal-Price A, Crofton KM, Leist M, et al. (2015) International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes. Arch Toxicol 89(2):269-287. https://doi.org/10.1007/s00204-015-1464-2
Bal-Price A, Pistollato F, Sachana M, Bopp SK, Munn S, Worth A (2018) Strategies to improve the regulatory assessment of developmental neurotoxicity (DNT) using in vitro methods. Toxicology and applied pharmacology 354:7-18. https://doi.org/10.1016/j.taap.2018.02.008
Bal-Price AK, Coecke S, Costa L, et al. (2012) Advancing the science of developmental neurotoxicity (DNT): testing for better safety evaluation. ALTEX 29(2):202-215.
https://doi.org/10.14573/altex.2012.2.202
Bal-Price AK, Hogberg HT, Buzanska L, Coecke S (2010) Relevance of in vitro neurotoxicity testing for regulatory requirements: challenges to be considered. Neurotoxicology and teratology 32(1):36-41.
https://doi.org/10.1016/j.ntt.2008.12.003
Barenys M, Reverte I, Masjosthusmann S, Gomez-Catalan J, Fritsche E (2019) Developmental neurotoxicity of MDMA. A systematic literature review summarized in a putative adverse outcome pathway. Neurotoxicology. https://doi.org/10.1016/j.neuro.2019.12.007
Bate CM (1976) Pioneer neurones in an insect embryo. Nature 260(5546):54-56.
https://doi.org/10.1038/260054a0
Baumann J, Gassmann K, Masjosthusmann S, et al. (2016) Comparative human and rat neurospheres reveal species differences in chemical effects on neurodevelopmental key events. Arch Toxicol 90(6):1415-1427. https://doi.org/10.1007/s00204-015-1568-8
Bentley D, Caudy M (1983) Pioneer axons lose directed growth after selective killing of guidepost cells. Nature 304(5921):62-65. https://doi.org/10.1038/304062a0
Bentley D, O'Connor TP (1992) Guidance and steering of peripheral pioneer growth cones in grasshopper embryos. The nerve growth cone. Raven Press, Ltd, New York, pp 265-282
Bicker G, Naujock M, Haase A (2004) Cellular expression patterns of acetylcholinesterase activity during grasshopper development. Cell Tissue Res 317(2):207-220. https://doi.org/10.1007/s00441-004-0905-7
Breier JM, Radio NM, Mundy WR, Shafer TJ (2008) Development of a high-throughput screening assay for chemical effects on proliferation and viability of immortalized human neural progenitor cells. Toxicological Sciences 105(1):119-133. https://doi.org/10.1093/toxsci/kfn115
Chudley AE, Conry J, Cook JL, Loock C, Rosales T, LeBlanc N (2005) Fetal alcohol spectrum disorder:
Canadian guidelines for diagnosis. Cmaj 172(5 suppl):S1-S21. https://doi.org/10.1503/cmaj.1040302
62 Condic M, Bentley D (1989) Pioneer growth cone adhesion in vivo to boundary cells and neurons after enzymatic removal of basal lamina in grasshopper embryos. Journal of Neuroscience 9(8):2687-2696. https://doi.org/10.1523/JNEUROSCI.09-08-02687.1989
Culotti JG, Kolodkin AL (1996) Functions of netrins and semaphorins in axon guidance. Current Opinion in Neurobiology 6(1):81-88. https://doi.org/10.1016/S0959-4388(96)80012-2
Dach K, Yaghoobi B, Schmuck MR, Carty DR, Morales KM, Lein PJ (2019) Teratological and Behavioral Screening of the National Toxicology Program 91-Compound Library in Zebrafish (Danio rerio).
Toxicol Sci 167(1):77-91. https://doi.org/10.1093/toxsci/kfy266
Eto K (2000) Minamata disease. Neuropathology 20(s1):14-19. https://doi.org/10.1046/j.1440-1789.2000.00295.x
Frank CL, Brown JP, Wallace K, Wambaugh JF, Shah I, Shafer TJ (2018) Defining toxicological tipping points in neuronal network development. Toxicol Appl Pharmacol 354:81-93.
https://doi.org/10.1016/j.taap.2018.01.017
Fried PA, Watkinson B, Gray R (1992) A follow-up study of attentional behavior in 6-year-old children exposed prenatally to marihuana, cigarettes, and alcohol. Neurotoxicology and teratology 14(5):299-311. https://doi.org/10.1016/0892-0362(92)90036-A
Fritsche E, Cline JE, Nguyen NH, Scanlan TS, Abel J (2005) Polychlorinated biphenyls disturb differentiation of normal human neural progenitor cells: Clue for involvement of thyroid hormone receptors. Environ Health Persp 113(7):871-876. https://doi.org/10.1289/ehp.7793
Fritsche E, Crofton KM, Hernandez AF, et al. (2017) OECD/EFSA workshop on developmental
neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes. Altex-Altern Anim Ex 34(2):311-315. https://doi.org/10.14573/altex.1701171
Giordano G, Costa LG (2012) Developmental neurotoxicity: some old and new issues. ISRN Toxicol 2012:814795. https://doi.org/10.5402/2012/814795
Grandjean P, Landrigan PJ (2006) Developmental neurotoxicity of industrial chemicals. The Lancet 368(9553):2167-2178. https://doi.org/10.1016/S0140-6736(06)69665-7
Grandjean P, Landrigan PJ (2014) Neurobehavioural effects of developmental toxicity. Lancet Neurol 13(3):330-338. https://doi.org/10.1016/S1474-4422(13)70278-3
Haase A, Stern M, Wächtler K, Bicker G (2001) A tissue-specific marker of Ecdysozoa. Development genes and evolution 211(8-9):428-433. https://doi.org/10.1007/s004270100173
Hagstrom D, Truong L, Zhang S, Tanguay R, Collins E-MS (2019) Comparative analysis of zebrafish and planarian model systems for developmental neurotoxicity screens using an 87-compound library.
Toxicological Sciences 167(1):15-25. https://doi.org/10.1093/toxsci/kfy180
Harrill JA, Freudenrich TM, Machacek DW, Stice SL, Mundy WR (2010) Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2™ cells using automated high-content image analysis. Neurotoxicology 31(3):277-290. https://doi.org/10.1016/j.neuro.2010.02.003
63 Harrill JA, Robinette BL, Mundy WR (2011) Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro. Toxicology in Vitro 25(1):368-387.
https://doi.org/10.1016/j.tiv.2010.10.011
Isbister CM, Tsai A, Wong ST, Kolodkin AL, O'Connor TP (1999) Discrete roles for secreted and transmembrane semaphorins in neuronal growth cone guidance in vivo. Development 126(9):2007-2019.
Jan LY, Jan YN (1982) Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos. Proceedings of the National Academy of Sciences 79(8):2700-2704.
https://doi.org/10.1073/pnas.79.8.2700
Kater SB, Mattson MP, Cohan C, Connor J (1988) Calcium regulation of the neuronal growth cone.
Trends in neurosciences 11(7):315-321. https://doi.org/10.1016/0166-2236(88)90094-X
Kellner M, Heidrich M, Lorbeer R-A, et al. (2016) A combined method for correlative 3D imaging of biological samples from macro to nano scale. Scientific reports 6:35606.
https://doi.org/10.1038/srep35606
Kolodkin AL, Matthes DJ, Goodman CS (1993) The Semaphorin Genes Encode a Family of Transmembrane and Secreted Growth Cone Guidance Molecules. Cell 75(7):1389-1399.
https://doi.org/10.1016/0092-8674(93)90625-Z
Kolodkin AL, Matthes DJ, O'Connor TP, et al. (1992) Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo. Neuron 9(5):831-845.
https://doi.org/10.1016/0896-6273(92)90237-8
Krug AK, Balmer NV, Matt F, Schonenberger F, Merhof D, Leist M (2013) Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants. Arch Toxicol 87(12):2215-31. https://doi.org/10.1007/s00204-013-1072-y
Lau P-m, Zucker RS, Bentley D (1999) Induction of filopodia by direct local elevation of intracellular calcium ion concentration. The Journal of cell biology 145(6):1265-1276.
https://doi.org/10.1083/jcb.145.6.1265
Lein P, Silbergeld E, Locke P, Goldberg AM (2005) In vitro and other alternative approaches to developmental neurotoxicity testing (DNT). Environ Toxicol Pharmacol 19(3):735-744.
https://doi.org/10.1016/j.etap.2004.12.035
Li JP, Settivari R, LeBaron MJ, Marty MS (2019) An industry perspective: A streamlined screening strategy using alternative models for chemical assessment of developmental neurotoxicity.
Neurotoxicology 73:17-30. https://doi.org/10.1016/j.neuro.2019.02.010
Lohr C, Heil JE, Deitmer JW (2005) Blockage of voltage-gated calcium signaling impairs migration of glial cells in vivo. Glia 50(3):198-211. https://doi.org/10.1002/glia.20163
Lorbeer R-A, Heidrich M, Lorbeer C, et al. (2011) Highly efficient 3D fluorescence microscopy with a scanning laser optical tomograph. Optics express 19(6):5419-5430.
https://doi.org/10.1364/OE.19.005419
64 Mark MD, Lohrum M, Puschel AW (1997) Patterning neuronal connections by chemorepulsion: the semaphorins. Cell Tissue Res 290(2):299-306. https://doi.org/10.1007/s004410050934
Martinez-Gomez J, Lopez-Garcia J (2007) Simultaneous assessment of the effects of L-type current modulators on sensory and motor pathways of the mouse spinal cord in vitro. Neuropharmacology 53(3):464-471. https://doi.org/10.1016/j.neuropharm.2007.06.007
Matsumoto H, Koya G, Takeuchi T (1965) Fetal Minamata disease: a neuropathological study of two cases of intrauterine intoxication by a methyl mercury compound. Journal of Neuropathology and Experimental Neurology 24(4):563-574. PMID: 5890913
Miller GW, Chandrasekaran V, Yaghoobi B, Lein PJ (2018) Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity.
Neurotoxicology 67:102-111. https://doi.org/10.1016/j.neuro.2018.04.016
Mundy WR, Padilla S, Breier JM, et al. (2015) Expanding the test set: Chemicals with potential to disrupt mammalian brain development. Neurotoxicol Teratol 52(Pt A):25-35.
https://doi.org/10.1016/j.ntt.2015.10.001
Needham LL, Grandjean P, Heinzow B, et al. (2011) Partition of environmental chemicals between maternal and fetal blood and tissues. Environ Sci Technol 45(3):1121-1126.
https://doi.org/10.1021/es1019614
Nolte L, Antonopoulos GC, Rämisch L, Heisterkamp A, Ripken T, Meyer H (2018) Enabling second harmonic generation as a contrast mechanism for optical projection tomography (OPT) and scanning laser optical tomography (SLOT). Biomedical Optics Express 9(6):2627-2639.
https://doi.org/10.1364/BOE.9.002627
Nolte L, Tinne N, Schulze J, et al. (2017) Scanning laser optical tomography for in toto imaging of the murine cochlea. PloS one 12(4):e0175431. https://doi.org/10.1371/journal.pone.0175431
OECD, Organisation for Economic Co-operation and Development. (2007). Test No. 426:
Developmental Neurotoxicity Study. OECD Publishing.
https://www.oecd-ilibrary.org/environment/test-no-426-developmental-neurotoxicity-study_9789264067394-en.
Accessed 22 March 2020
Pamies D, Block K, Lau P, Gribaldo L et al. (2018) Rotenone exerts developmental neurotoxicity in a human brain spheroid model. Toxicology and applied pharmacology 354:101-114.
https://doi.org/10.1016/j.taap.2018.02.003
Polleux F, Morrow T, Ghosh A (2000) Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404(6778):567-573. https://doi.org/10.1038/35007001
Radio NM, Mundy WR (2008) Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth. Neurotoxicology 29(3):361-376.
https://doi.org/10.1016/j.neuro.2008.02.011
Rice D, Barone S, Jr. (2000) Critical periods of vulnerability for the developing nervous system:
evidence from humans and animal models. Environ Health Perspect 108 Suppl 3:511-533.
https://doi.org/10.1289/ehp.00108s3511
65 Russell WMS, Burch RL, Hume CW (1959) The principles of humane experimental technique, vol 238.
Methuen, London
Sánchez-Soriano N, Tear G, Whitington P, Prokop A (2007) Drosophila as a genetic and cellular model for studies on axonal growth. Neural development 2(1):9. https://doi.org/10.1186/1749-8104-2-9 Schmuck MR, Temme T, Dach K, et al. (2017) Omnisphero: a high-content image analysis (HCA) approach for phenotypic developmental neurotoxicity (DNT) screenings of organoid neurosphere cultures in vitro. Archives of toxicology 91(4). https://doi.org/10.1007/s00204-016-1852-2 Slotkin TA, Seidler FJ, Ryde IT, Yanai J (2008) Developmental neurotoxic effects of chlorpyrifos on acetylcholine and serotonin pathways in an avian model. Neurotoxicol Teratol 30(5):433-439.
https://doi.org/10.1016/j.ntt.2008.02.005
Smirnova L, Hogberg HT, Leist M, Hartung T (2014) Developmental neurotoxicity - challenges in the 21st century and in vitro opportunities. ALTEX 31(2):129-156.
Smirnova L, Hogberg HT, Leist M, Hartung T (2014) Developmental neurotoxicity - challenges in the 21st century and in vitro opportunities. ALTEX 31(2):129-156.