An intact insect embryo as a test system for neurotoxicity and developmental neurotoxicity

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University of Veterinary Medicine Hannover Institute for Physiology and Cell Biology

An intact insect embryo

as a test system for neurotoxicity and developmental neurotoxicity

INAUGURAL – DISSERTATION

in partial fulfillment of the requirements for the degree of - Doctor rerum naturalium -

(Dr.rer.nat.)

submitted by

Karsten Bode, M.Sc.

Burg, Germany

Hannover 2020

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Scientific supervision

Prof. Dr. Gerd Bicker

Institute for Physiology and Cell Biology,

University of Veterinary Medicine Hannover, Germany

1

^st

evaluation

Prof. Dr. Gerd Bicker

Institute for Physiology and Cell Biology,

University of Veterinary Medicine Hannover, Germany

PD Dr. Michael Stern

Institute for Physiology and Cell Biology,

University of Veterinary Medicine Hannover, Germany

Prof. Dr. Stefanie Becker

Institute for Parasitology,

University of Veterinary Medicine Hannover, Germany

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^nd

evaluation

Prof. Dr. Thomas Roeder

Institute of Zoology, Kiel University

Day of the oral examination: June 9, 2020

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Dedicated to my family

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1. Zusammenfassung

Karsten Bode, „Ein intakter Insektenembryo als Testsystem für Neurotoxizität und Entwicklungsneurotoxizität“.

Das menschliche Gehirn durchläuft während seiner Entwicklung eine Vielzahl an strukturellen und funktionellen Veränderungen. Die Schädigung eines einzelnen Schlüsselprozesses kann in der Folge bereits eine normale Hirnentwicklung verhindern. Besonders verletzlich ist das Gehirn während bestimmter Schlüsselprozesse wie der Zellproliferation, der Zellmigration, der Zelldifferenzierung oder während der Bildung von Nervenzellfortsätzen (Axonen). Früheste Schädigungen sind nicht reversibel und bleiben für den Rest des Lebens erhalten. Demnach ist das sich entwickelnde kindliche Gehirn gegenüber toxischen Chemikalien in niedriger Konzentration empfindlicher als das eines Erwachsenen.

Verschiedenste Einflüsse von Umwelt- oder Industriechemikalien (u.a. Pestizide, Blei, Quecksilberverbindungen) aber auch Faktoren des Lebensstils (u.a. Rauchen, Medikamentenmissbrauch, Alkoholkonsum) können, bei den Nachkommen der Schwangeren oder Stillenden, für das vermehrte Auftreten von neurologischen Entwicklungsstörungen verantwortlich sein. Mittels epidemiologischer Studien konnte bisher lediglich eine Handvoll entwicklungsneurotoxischer Chemikalien identifiziert werden. Strikte Richtlinien der OECD und EPA für das Identifizieren solcher Chemikalien fordern die Verwendung großer Mengen an Nagetieren als Versuchsobjekte. Wegen der hohen finanziellen Kosten, den zeitintensiven Experimenten und dem hohen Verbrauch an Wirbeltieren, werden alternative Testsysteme benötigt.

Die meisten Ersatzmethoden basieren auf Zellkulturverfahren, die einfache Schlüsselprozesse (z.B.

Zellvermehrung oder Differenzierung) während der Hirnentwicklung in der Petrischale simulieren. Die Entwicklung eines funktionalen Gehirns erfordert jedoch die präzise, räumliche und zeitliche Verdrahtung von Nervenzellaxonen im Gewebe. Der Heuschreckenembryo als entwicklungsneurotoxisches intaktes Testsystem greift genau an diesem Punkt an und ist in der Lage, die komplexe Wegfindung von identifizierten Pionierneuronen innerhalb der metathorakalen Beinknospe darzustellen.

Die Pionierneurone des Heuschreckenembryos werden paarweise in der distalen Spitze des Sprungbeines geboren. Die davon auswachsenden Axone legen die ersten Bahnen von der Peripherie in das zentrale Nervensystem. Dabei orientieren sie sich an einer Reihe von Wegweisern wie z.B. den Semaphorinen. Semaphorine sind eine Klasse von sezernierten oder membranassoziierten Proteinen, die für die korrekte Navigation von axonalen Nerven innerhalb des Gewebes verantwortlich sind. Das Vorkommen dieser Proteine ist in der Evolution zwischen Evertebraten und Säugetieren konserviert.

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2 Durch die Inkubation der Embryonen mit Testchemikalien lässt sich deren Einfluss auf das axonale Längenwachstum, die korrekte Navigation sowie auf die Lebendigkeit genauestens untersuchen und in Konzentrations-Effekt-Kurven darstellen. Die Evaluation der Daten mittels konventioneller Fluoreszenzmikroskopie und eines geeigneten Prädiktionsmodells erlauben es präzise zwischen spezifischen entwicklungsneurotoxischen und unspezifischen zytotoxischen Effekten zu unterscheiden. Scanning laser optical tomography (SLOT) ist ein dreidimensionales Bildgebungsverfahren, das die Länge der Axone und die Wegfindungsfehler im „whole-mount“

Präparat auflösen kann. Dreidimensionale Rekonstruktionen von Pionierneuronen erweisen sich dabei als geeignetes Werkzeug, um klassische entwicklungsneurotoxische Chemikalien wie Methylquecksilber und Arsen zu identifizieren.

Das Heuschreckenembryo-Modell bietet daher eine wertvolle Ergänzung zu zellkulturbasierten Verfahren, um das axonale Auswachsen und die Navigation von Pionierneuronen im intakten Organismus zu quantifizieren und anschließend auf das entwicklungsneurotoxische Potential der Chemikalie zu schließen.

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2. Abstract

Karsten Bode, “An intact insect embryo as a test system for neurotoxicity and developmental neurotoxicity”.

The human brain has to undergo a variety of structural and functional changes throughout the development. At that point, developmental disturbances of single processes could inhibit normal brain formation. The developing brain is particularly vulnerable during certain key processes such as cell proliferation, cell migration, cell differentiation or the formation of axons. It should be emphasized that early changes in brain development are not reversible and persist for the rest of life. Accordingly, the developing infantile brain is much more susceptible towards lower toxic concentration of chemicals than the adult one.

Exposure to industrial or environmental compounds (e.g. pesticides, lead, methylmercury) or maternal lifestyle habits (e.g. smoking, alcohol consumption or drug usage) during pregnancy or lactation period can trigger developmental neurotoxic (DNT) diseases in the progeny. Based on epidemiological studies only a handful of chemicals have been identified as developmental neurotoxic agents. Currently, strict guidelines for DNT testing, released by the OECD and EPA, demand the usage of large numbers of rodents for animal experiments. According to the high financial burdens, the time-consuming experiments and the large number of animals, alternative DNT detection systems are required.

Most alternative methods are based on in vitro cell culture systems that can monitor mainly readily quantifiable key events such as proliferation or differentiation. However, the formation of a functional brain requires a precise, spatially and temporally coordinated navigation of axons within the tissue environment. The locust embryo system exactly addresses this issue and provides the opportunity to examine axon navigation of identified pioneers within the metathoracic limb bud.

Pioneers arise in the distal tip of the locust jumping leg and establish the first nerve pathway from the periphery to the central nervous system (CNS). On their way to the CNS, they orient on several guidance cues such as semaphorins. Semaphorins are membrane bound or secreted proteins, which are conserved between invertebrates and mammalians. They act as guidance molecules, which are mainly responsible for the correct navigation of axons to their predestined targets.

By incubating embryos with test chemicals, endpoints such as axonal elongation, navigation and viability are determined and then quantified in concentration-response curves. Evaluation of data by conventional fluorescence microscopy and an applicable prediction model allows the distinction between specific developmental neurotoxicity and unspecific general cytotoxicity. Scanning laser optical tomography (SLOT) is a three-dimensional imaging technique that resolves lengths of axons

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4 and navigation errors in whole mounts. Three-dimensional reconstructions of pioneer neurons prove as a suitable tool for identifying classic DNT compounds such as methylmercury and arsenic.

The locust embryo hence provides a valuable complement to cell culture-based systems, which quantifies axonal growth and guidance defects of pioneer neurons in an intact organism. The results allow the prediction of the developmental neurotoxic potential of test compounds.

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3. Introduction

3.1. Developmental neurotoxicity (DNT)

The Minamata disease was one of the most devastating environmental disasters worldwide. In the 1950s, a chemical factory discharged wastewater into the open water of the Minamata bay in Kumamoto, Japan. The wastewater contained a high amount of mercury, which was later metabolized into the more potent methylmercury by marine bacteria. The methylated form bioaccumulated in shellfish and fish, which were consumed by the local population. The poisoning with contaminated seafood resulted in several neurological disorders such as sensory disturbances in the distal parts of extremities, ataxia, muscle weakness, balance disorders, abnormal eye movement and other noticeable problems (Eto 2000). In 1965, scientists reported about cerebral palsy in children that had not eaten fish and shellfish and where their mothers showed no manifestation of neurological disorders. The authors concluded that the children were poisoned to methylmercury by prenatally exposure from the mother (Matsumoto et al. 1965). A reason could be that the placenta and the immature blood-brain barrier only offers little protection to most chemicals of toxicological interest (Needham et al. 2011). However, chemicals that can cross these barriers are difficult to identify because in most cases the consequences of toxicant exposure are not recognizable at the onset of exposure time. In some examples, the negative effect only becomes valid, when the exposure time has ended. This phenomenon is called “delayed consequence of early life exposure” (Aschner et al. 2017).

Developmental neurotoxicity (DNT) has been defined as any change in the structure or function of the nervous system, caused by exposure to chemicals during gestation or lactation period, when the formation of the nervous system has not completed (Mundy et al. 2015). Triggers for DNT could be exposure to industrial compounds or maternal lifestyle habits such as smoking, alcohol consumption or drug usage during pregnancy or postnatal period (Barenys et al. 2019; Chudley et al. 2005; Fried et al. 1992; Grandjean and Landrigan 2006).

3.2. The current state of DNT testing

The developing fetal and juvenile human brain is much more sensitive to chemicals than the adult one.

In cases of methylmercury poisoning, it was estimated that the vulnerability of a developing brain in utero is five times higher than in mature brains (Giordano and Costa 2012). The formation of a functional brain is characterized by different windows of vulnerability. Periods of vulnerability depend on the spatiotemporal occurrence of critical developmental processes such as migration, differentiation, proliferation, synaptogenesis, myelination or apoptosis (Rice and Barone 2000).

Alterations in these sensitive processes can lead to DNT, which can manifest in neurobehavioral

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6 diseases (e.g. autism, schizophrenia) or anatomical defects (e.g. spina bifida) (Mundy et al. 2015; Rice and Barone 2000).

Currently, only a small number of chemicals have been identified as DNT compounds for humans by epidemiological approaches. In contrast, tens of thousands industrial chemicals remain unclear for their potential to induce neurodevelopment disorders (Aschner et al. 2017; Grandjean and Landrigan 2006; Grandjean and Landrigan 2014) (Figure 1). The identification of DNT chemicals requires large number of vertebrates (e.g. rodents) that have to be exposed to chemicals, because of safety standards released by international authorities for in vivo experiments (US EPA 1998; OECD 2007). These internationally agreed guidelines demand three months of testing to evaluate only one chemical, by using 1000 rat pups, costing about $ 1.4 million. Due to the high costs of vertebrates, the time- consuming experiments and financial burdens, animal experiments are rather unsuitable for DNT studies (Smirnova et al. 2014).

Figure 1: DNT chemicals compared to industrial compounds (Aschner et al. 2017; illustration modified after Grandjean and Landrigan 2006)

Experts in developmental neurobiology have conceived strategies for future research that minimizes financial costs and ethical issues in consideration of the three Rs (reduction, replacement and refinement of animal experiments) (Russell et al. 1959). They recognized the requirement for fast, reliable alternative test systems maybe integrated in testing batteries, which simulate vulnerable

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7 windows of human brain development (Bal-Price et al. 2015; Bal-Price et al. 2010; Fritsche et al. 2017;

Li et al. 2019). Therefore, assays should be based on in vitro and non-mammalian model systems that reflect evolutionary conserved processes, which are key targets for DNT in humans (Bal-Price et al.

2012). Most in vitro assays, based on cell culture, monitor only readily accessible endpoints such as proliferation, migration, differentiation, neurite outgrowth, synaptogenesis or network formation (Breier et al. 2008; Frank et al. 2018; Fritsche et al. 2005; Harrill et al. 2010; Krug et al. 2013; Radio and Mundy 2008; Schmuck et al. 2017; Stern et al. 2014; Stiegler et al. 2011). However, none of these assays does recapitulate cell-cell and cell-matrix interactions or morphogen gradients that are essential for correct brain development (Lein et al. 2005). A recent review showed that non-mammalian organism-based models (e.g. zebrafish, C. elegans) provide an appropriate tool for toxicological analysis due to the conserved molecular and functional aspects across non-mammalians and mammalians during nervous system development (Li et al. 2019).

3.3. The intact locust embryo as assay for DNT detection

Non-mammalian, organism-based assays such as the zebrafish (Dach et al. 2019; Miller et al. 2018) or the chicken (Slotkin et al. 2008) are promising tools for DNT detection, but both classify as vertebrates.

Here we introduce a non-vertebrate assay, based on a pest insect, which can be used to quantify DNT effects in locust embryos. Therefore, an identifiable pair of pioneer neurons is visualized by antibodies, specific for a carbohydrate epitope on the surface of neurons (Haase et al. 2001; Jan and Jan 1982), and subsequently examined for their outgrowth behavior. This specialized set of pioneers arises in the distal tip of the metathoracic limb bud and establishes the first route to the central nervous system (CNS). These pioneer siblings have been investigated in remarkable detail for their growth cone navigation along the stereotyped pathway across the hind leg (Bate 1976; Bentley and O'Connor 1992).

Accurate neuronal connectivity requires directed axon initiation, growth cone pathfinding, target selection and synapse formation (Harrill et al. 2011; Isbister et al. 1999). In locust embryos, pathfinding substantially relies on the adhesion of growth cones to landmarks such as the basal lamina and guidepost cells. Apart from that, semaphorin signaling is essential for correct navigation through tissue environment as shown by antibody blocking experiments (Isbister et al. 1999; Kolodkin et al. 1992).

Semaphorins belong to a class of secreted or membrane bond proteins that mediate axon pathfinding and target selection in vertebrates, including mammalians, as well as in invertebrates (Culotti and Kolodkin 1996; Mark et al. 1997; Polleux et al. 2000). In the limb bud of locust embryos, epithelial cells express the transmembrane semaphorin 1, which allows axons to change the outgrowth direction from dorsal to the ventral side (Kolodkin et al. 1993; Kolodkin et al. 1992) (Figure 2). Two additional gradients of the diffusible semaphorins (sema 2a) repulse the axons of pioneers from distal to proximal, guiding their way to the CNS (Isbister et al. 1999). In combination with chemical exposure, this stereotypic

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8 outgrowth behavior provides a useful implement for toxicological studies. Our assay allows us to examine the axon outgrowth, the neuronal shape of cell bodies and the correct navigation of axons along their predefined pathway into the CNS. Comparisons with the viability of embryos, measured by a biochemical assay, enables us to differentiate between DNT and general cytotoxicity.

Figure 2: The stereotypic pathway of pioneer axons within the limb bud of a locust embryo (35 % of embryogenesis). Two pioneer neurons arise (cell body) in the distal tip and extend their axons (green) proximally. Guidepost cells (round-shaped, orange) and membrane bound proteins as semaphorin 1 (sema 1, orange transparent stripe) guide axons along their way to the CNS. Semaphorin 1 provides an access for pioneer axons for changing their outgrowth direction from the dorsal to the ventral side (Modified after Wolpert et al. 2002)

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4. Thesis outline

This thesis is compiled as a cumulative dissertation including three scientific publications, two of them as first author, ranked in chronological order.

The first part describes the introduction of a novel alternative DNT assay, based on intact locust embryos, which were cultivated for 24 hours in serum-free medium. As toxicological endpoint, we quantified axon outgrowth of pioneers in concentration-response curves by using an own-developed elongation score and compared it to general viability, which is essential for DNT measurements (Aschner et al. 2017). The assay was tested against selected compounds interfering with calcium signaling (diltiazem, verapamil), cytoskeletal organization (colchizin, cytochalasin D) and a reference compound (rotenone), which is known to cause DNT (Krug et al. 2013; Pamies et al. 2018).

Axon length quantification provides a suitable endpoint for detecting chemicals that can harm the nervous system before viability is affected. Our scoring scheme for pioneer length measurements is based on reaching visible landmarks along a stereotypical pathway within the limb buds. Evaluation of axon lengths by conventional fluorescence microscopy is sometimes limited in whole mounts due to torsion and positioning of the curved limb bud. Therefore, we applied a novel method for visualizing pioneers and their three-dimensional structure within the environment of surrounding tissue by using scanning laser optical tomography (SLOT). We compared both conventional fluorescence microscopy and SLOT measurements for axon outgrowth, correct navigation and neuron shape analysis after incubation with classical DNT chemicals such as MeHgCl, NaAsO3. Furthermore, we examined the potential candidate staurosporine for interfering with axonal elongation in our assay.

The last manuscript addresses the development of a suitable prediction model. The correct prediction of chemicals and their potential in affecting neuronal development requires a precise distinction between DNT positive compounds and chemicals that are either harmless or general cytotoxic.

Therefore, we challenged our locust assay with a training set of six DNT positive and six negative chemicals. Moreover, we included additional endpoints for viability (dead cell protease) and neurite outgrowth (correct navigation of pioneers), allowing for examination of four independent adverse outcomes that can be triggered by DNT positive chemicals.

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10 4.1. Author contributions

Publication 1: An intact insect embryo for developmental neurotoxicity testing of directed axonal elongation. GAB, SF, MS, KB and GB wrote the paper. GAB, KB and MS evaluated the data. GAB, SF, NJ 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.

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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 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,

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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

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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.

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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.

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7. References

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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

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8. Acknowledgements

First of all, I would like to thank my supervisor Prof. Dr. Gerd Bicker for giving me the opportunity to do research on this project. Thank you for your scientific support, your commitment in late shifts, your guidance and your patience in all areas of life. You are a great boss.

Moreover, my thank goes to Dr. Michael Stern, who was my personal point of contact in all scientific question. Michael, thank you for your support and for always having an open ear. I really appreciated your rationality on difficult issues. Stay as you are.

The next in line is technical assistant Saime Tan. Thank you for your help across-the-board. I really enjoyed the nice talks in the incubation periods apart from the day-to-day business. You are a great personality with an extremely big heart. Thank you very much.

I also want to thank all of the other laboratory members, students and interns I have met in the last three years. Thank you for the great time.

In the end, I want to say thank you to my parents, who always believed in me and consistently supported me throughout my whole life. It is good to know that you are by my side.

At last but not least, my special thank goes to my partner Claudia, thank you very much for your endless support whenever I needed help and for having my back all the time. I love you.

An intact insect embryo as a test system for neurotoxicity and developmental neurotoxicity

University of Veterinary Medicine Hannover Institute for Physiology and Cell Biology