Neurotoxicity of tri-cresyl phosphate - Impairment of glutamate signaling in mouse central nervous system neurons in vitro
Dissertation to obtain the degree Doctor Rerum Naturalium (Dr. rer. nat.) at the Faculty of Biology and Biotechnology Ruhr-University Bochum
Leibniz Research Centre for Working Environment and Human Factors Neurotoxicology and Chemosensation
submitted by Vanessa Hausherr
from Sprockhövel, Germany
Bochum October 2015
1. Supervisor: PD. Dr. C. van Thriel 2. Supervisor: Prof. Dr. H. Lübbert
Neurotoxizität von Tri-cresylphosphaten - Beeinflussung der glutamatergen Signaltransduktion in murinen Neuronen des zentralen Nervensystems in vitro
Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und Biotechnologie an der Internationalen Graduiertenschule Biowissenschaften an der Ruhr-Universität Bochum
angefertigt im Leibniz-Institut für Arbeitsforschung Neurotoxikologie und Chemosensorik
vorgelegt von Vanessa Hausherr
aus Sprockhövel, Deutschland
Bochum Oktober 2015
1. Betreuer: PD. Dr. C. van Thriel 2. Betreuer: Prof. Dr. H. Lübbert
Index of Contents
Summary ______IV
Zusammenfassung ______VI
Abbreviations ______VIII
1 Introduction______1
1.1 Central and peripheral nervous system ______5
1.2 Neurite outgrowth and neurite degeneration ______6
1.3 Neurotransmitter systems and ion channels ______8 1.3.1 Glutamate receptors ______8 1.3.2 GABA receptors ______13 1.3.3 Voltage-gated calcium channels ______14 1.3.4 TRP Channels ______15
1.4 Neurotransmission and activity-dependent plasticity ______16
1.5 Organophosphate-induced delayed neuropathy ______18 1.5.1 Chemistry of OPIDN inducing compounds ______19 1.5.2 Neuropathology and clinical signs of OPIDN ______19
1.6 Tri-cresyl phosphate ______20 1.6.1 Structure of TCPs ______20 1.6.2 Metabolism of TCPs ______21 1.6.3 Toxicity of TCPs ______23 1.6.4 Human TCP poisoning ______24
1.7 The aerotoxic syndrome ______24
1.8 Objectives ______26
2 Material and methods ______27
2.1 Material ______27 2.1.1 Chemical Reagents and Kits ______27 2.1.2 Primary Antibodies ______29 2.1.3 Secondary Antibodies ______29 2.1.4 Consumables ______30 2.1.5 Technical Equipment ______31
2.2 Methods ______32 2.2.1 Animals ______32 2.2.2 Preparation and cell culture of primary mouse cortical neurons ______32
I Index of Contents
2.2.3 Preparation and cell culture of primary rat cortical neurons ______33 2.2.4 Preparation and cell culture of mouse dorsal root ganglia neurons ______33 2.2.5 Treatment conditions ______34 2.2.6 CellTiter-Blue® cell viability assay ______34 2.2.7 Immunocytochemistry ______35 2.2.8 Quantitative analysis of neurite outgrowth and neurite degeneration ______36 2.2.9 Quantitative analysis of neurite morphology ______36 2.2.10 Network Formation Assay ______37 2.2.11 Calcium Imaging ______38 2.2.12 RNA Isolation ______39 2.2.13 cDNA Synthesis ______39 2.2.14 Quantitative Real-Time PCR ______40 2.2.15 Statistical Analysis ______41
3 Results ______42
3.1 Morphology of mouse primary cortical neurons at different stages of the in vitro culture ______42
3.2 Impairment of cell viability ______43 3.2.1 TCP isomers and TCP mixture impair cell viability ______43 3.2.2 ToCP impairs cell viability ______44 3.2.3 CBDP impairs cell viability ______45
3.3 Impairment of neurite outgrowth and neurite degeneration ______46 3.3.1 Inhibition and degeneration of neuronal networks by TCP isomers ______46 3.3.2 Inhibition and degeneration of neuronal networks by ToCP ______47 3.3.2.1 Time course of neurite outgrowth inhibition – Network Formation Assay ______50 3.3.3 Inhibition and degeneration of neuronal networks CBDP ______51
3.4 Effects of TCPs on neurochemical processes ______52 3.4.1 TCP isomers impair glutamate signaling ______53 3.4.2 ToCP impairs glutamate signaling ______57 3.4.2.1 ToCP affects glutamate receptor expression ______65 3.4.3 Recovery of the glutamate sensitivity after ToCP exposure ______66 3.4.4 Effects of the ToCP metabolite CBDP on glutamate signaling ______70 3.4.5 ToCP induced effects on glutamate signaling in rat cortical neurons ______71 3.4.6 ToCP effects on non-glutamatergic signaling ______72 3.4.7 Direct receptor-mediated action of tri-cresyl phosphates and the metabolite CBDP ____ 74 3.4.8 Mechanism and target of ToCP-induced block of glutamate responses ______78 3.4.8.1 ToCP affects NMDA receptor-mediated responses ______79 3.4.8.2 ToCP affects AMPA receptor-mediated responses ______81 II Index of Contents
4 Discussion ______82
4.1 Impairment of glutamate signaling ______83 4.1.1 Direct receptor-mediated action of ToCP ______83 4.1.2 ToCP impairs glutamate sensitivity in a time- and concentration dependent manner ___ 86 4.1.3 Regeneration capacity of pCNs after ToCP treatment ______89 4.1.4 Is the reduced glutamate sensitivity a specific mode of action of ToCP? ______90 4.1.5 Impairment of glutamate signaling by CBDP ______92 4.1.6 Impairment of glutamate signaling by TCP isomers ______92 4.1.7 Increased basal calcium levels as an endpoint of TCP-induced neurotoxicity ______93
4.2 Impairment of neurite outgrowth and neurite degeneration ______94
4.3 ToCP, TCPs and the aerotoxic syndrome ______97
4.4 Conclusion ______99
5 List of Figures ______102
6 List of Tables ______104
7 References ______105
8 Publication List ______1
9 Curriculum Vitae ______4
10 Danksagung ______5
III Summary
Summary
The nervous system is characterized by its ability to transmit and process incoming information from extrinsic and intrinsic stimuli via specialized neuronal networks consisting of interconnecting neurites and synapses. The developing as well as the adult brain had an astonishing capacity to reorganize its connectivity on structural and functional levels to adapt to changing environmental conditions and based on behaviorally experience, which is called activity-dependent plasticity. As a result of incoming information and learning and memory mechanisms, synapses are strengthened and weakened during life changes. Synaptic strength is based on changes in trafficking, subunit composition, and signaling of AMPAR and NMDAR as fundamental processes. The nervous system with its strictly regulated processes is a highly sensitive target structure of a variety of toxins. Neurotoxins can cause alterations in a variety of pathways, which lead to adverse outcomes when sufficiently perturbed. Tri-ortho cresylphosphate is a well-known neurotoxin, which cause after high dose exposure organophosphate-induced delayed neuropathy. The main objective of the present thesis was the detection of new modes of action of tri-cresyl phosphate in the central nervous system. During this thesis a multifaceted approach with standardized structural and functional in vitro endpoint were implemented to discover all levels of neuronal impairment. The endpoints cell viability, alteration of neurite growth and impairment of neurochemical processes were in vitro endpoints were investigated. The results of the study at hand show that TCPs affected mouse pCNs in vitro in the range from nanomolar to micromolar concentrations and affected the tested endpoints in a concentration-dependent manner. Reduction of cell viability was observed in the high micromolar and millimolar concentration range for all tested TCP isomers. Neurite outgrowth and neurite degeneration were affected at non-cytotoxic concentrations of the micromolar concentration range. In the study at hand, it is for first time showed that low concentrations of ToCP that do not affect cell viability or neurite morphology and complexity, impair glutamate-related postsynaptic signaling in mouse primary cortical neurons in vitro. The reduction in glutamate sensitivity is a completely new mode of action of ToCP. ToCP impaired the glutamate signaling time- and concentration dependent with concentrations ≥ 1 nM. The AMPAR endocytosis and the recycling back in the membrane might act as a protective mechanism against ToCP. PCNs were able to recover their glutamate sensitivity after treatment with ToCP concentrations of the nanomolar range. Beside the reduced glutamate sensitivity after short- and long-term
IV Summary treatment, ToCP affected glutamate-induced responses in acute co-application experiments. ToCP had a direct receptor-mediated action and reduced specifically the AMPAR-mediated part of the overall glutamate response. The most sensitive endpoint of TCP-induced neurotoxicity was the perturbation of the glutamate signaling and was affected by nanomolar concentrations. The TCP isomers had a different potential to impair neurite morphology and glutamate signaling. The results of the present study showed that ToCP was the most potent isomer and that might be based on the structural differences, the position of the alkyl substitution, between ToCP and the other isomers. TpCP and TmCP were, in contrast to ToCP, steric hindered phenyl phosphates with lower neurotoxic potential. The ortho-position of the methyl group at the aromatic ring system seems to be more reactive compared to the other isomers with methyl groups in the meta- or para-position. The difference in the position of the methyl group might determine the activity/reactivity with the glutamate receptors. The present thesis, is one of the few studies in the context of developmental toxicity and neurotoxicology that researched the underlying mechanisms of new mode of actions of neurotoxins. The new functional based approach is particularly well-suited and highly sensitive and reflect the functional nervous system. The investigation of functional endpoints in the field of neurotoxicology is a relatively new approach, but important to investigate critical processes for the formation of functional neuronal networks. The different structural and functional endpoints show a clear hierarchy in their sensitivity. The investigation of only structural endpoints in developmental toxicology and neurotoxicology might underestimate the hazardousness of chemicals.
V Zusammenfassung
Zusammenfassung
Das Nervensystem ist charakterisiert durch seine Fähigkeit eingehende extrinsische und intrinsische Informationen zu verarbeiten und zu übertragen. Dazu sind die Neurone in speziellen Netzwerken organisiert und über Neurite und Synapsen miteinander verknüpft. Sowohl das sich entwickelnde Gehirn als auch das ausgereifte Gehirn hat eine erstaunliche Fähigkeit ihre Konnektivität auf struktureller und funktionaler Ebene zu reorganisieren, um sich damit auf veränderte Umgebungsbedingungen einzustellen. Dieser Prozess basiert auf Erfahrungen und wird als aktivitätsabhängige Plastizität bezeichnet. Synapsen werden durch eingehende Informationen und verschiedene Lern- und Gedächtnisprozesse stabilisiert oder geschwächt. Die synaptische Stärke basiert auf Veränderungen in der Rezeptoruntereinheitenzusammensetzung und des Rezeptortransportes sowie der gezielten Signaltransduktion durch AMPA und NMDA Rezeptoren. Das Nervensystem mit seiner feinstrukturierten Organisation stellt eine hochsensitive Zielstruktur für eine Vielzahl von Toxinen dar. Neurotoxine können Änderungen in einer Vielzahl von Signalwegen hervorrufen die zu negativen Folgen führen, wenn sie beeinträchtigt werden. Tri-ortho cresylphosphat ist ein bekanntes Neurotoxin das nach einer Exposition in hoher Dosis zu Organophosphat induzierter verzögerter Neuropathie führt. Das Ziel dieser Arbeit war der Nachweis und die Untersuchung neuer Wirkmechanismen von TCPs. In der Arbeit wurde ein vielseitiges Konzept mit strukturbedingten und funktionellen in vitro Endpunkten realisiert, um alle Ebenen der Neurotoxizität festzustellen. Dabei wurden Zellvitalität, Beeinflussung des Neuritenwachstums und die Beeinträchtigung von neurochemischen Prozessen untersucht. Die vorliegende Arbeit zeigt, dass TCPs Kortexneurone der Maus im millimolaren bis nanomolaren Konzentrationsbereich beeinflussen. Eine Reduktion in der Zellvitalität war im millimolaren und hohen mikromolaren Konzentrationsbereich zu beobachten. Die Beeinflussung der Neuritenmorphologie war nach Behandlung mit nicht zytotoxischen Konzentrationen des millimolaren Bereichs zu erkennen. Der sensitivste Endpunkt der TCP-induzierten Neurotoxizität war die Störung der glutamatergen Signaltransduktion, welche durch nanomolare TCP Konzentrationen beeinträchtigt wurde. Die verschiedenen Isomere des Tri-cresylphosphates hatten ein unterschiedliches Potential die Neuritenmorphologie und die Glutamat-induzierte Signalweiterleitung zu beeinträchtigen.
VI Zusammenfassung
In der vorliegenden Arbeit konnte das erste Mal gezeigt werden, dass geringe ToCP Konzentrationen, die weder die Zellvitalität noch die Neuritenmorphologie beeinflussen, das Glutamat-induzierte Antwortverhalten in pCNs in vitro verändern. Die Verringerung der Glutamat-induzierte Antwortamplitude ist ein zeit- und konzentrationsabhängiger Prozess und erfolgt mit Konzentrationen ≥ 1 nM. Die AMPAR Endozytose und der Wiedereinbau der Rezeptoren in die Membran könnten dabei als Schutzmechanismus gegen ToCP wirken. PCNs haben die Fähigkeit ihr Glutamat-induziertes Antwortverhalten nach der ToCP Behandlung mit nanomolaren Konzentrationen zu regenerieren. ToCP hat eine direkte Rezeptorwirkung und modifiziert den AMPAR-vermittelten Part der Glutamat-induzierten Antwort. Die Ergebnisse dieser Arbeit zeigen, dass ToCP das potenteste Isomer ist, wofür die unterschiedliche Anordnung der Methylgruppen am aromatischen Ringsystem verantwortlich ist. TpCP und TmCP sind im Gegensatz sterisch gehinderte Phosphate, wodurch sich auch das geringere neurotoxische Potenzial erklären lässt. Der ortho-ständige Methylsubstituent am aromatischen Ring ist reaktiver als meta- oder para- ständige Methylsubstituenten. Der Unterschied in der Position des Methylsubstituenten bestimmt wahrscheinlich die Reaktivität mit den Glutamatrezeptoren. Die vorliegende Arbeit ist eine der wenigen Studien die zugrunde liegende Wirkmechanismen von neuen Wirkungsweisen von Neurotoxinen untersucht. Der neue funktionelle Ansatz ist für diese Untersuchungen besonders geeignet und hoch sensitiv. Die Untersuchung der funktionalen Endpunkte ist besonders wichtig um die kritischen Prozesse zu untersuchen die der Bildung des funktionalen neuronalen Netzwerkes zugrunde liegen. Die verschiedenen strukturellen und funktionalen Endpunkte zeigen eine klare Hierarchie bezüglich ihrer Sensitivität. Die bisher im Gebiet der Neurotoxikologie verwendeten in vitro Verfahren begrenzen sich meist auf Untersuchungen der Zellvitalität oder morphologischer Veränderungen. Die Untersuchung nur auf diese Endpunkte zu begrenzen führt eventuell zu einer Unterschätzung der Gefährlichkeit von Chemikalien.
VII Abbreviations
Abbreviations
ACh Acetylcholine AChE Acetylcholine esterase AMPA α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid AMPAR α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor AOP Adverse outcome pathway AS Aerotoxic syndrome ATP Adenosine-triphosphate Ca Calcium
2+ [Ca ]i Intracellular calcium concentration
CaCl2 Calcium chloride CaMKII calcium\calmodulin-dependent protein kinase II CBDP Cyclic salignin-o-cresylphosphate Cl Chloride
- [Cl ]i Intracellular chloride concentration CNQ 6-cyano-7-nitroquinoxaline-2, 3- dione CNS Central nervous system
CO2 Carbon dioxide cpn Connections per node CTB CellTiter-Blue® Assay CTD Carboxyl-terminus DAPI 4’, 6-diamidino-2-phenylindole DIV Days in vitro DMSO Dimethyl sulfoxide DNAse Deoxyribonuclease I DNQX 6, 7-dinitro-quinoxaline-2, 3-dione DRG Dorsal root ganglia EPSCs Excitatory postsynaptic potentials FBS Fetal bovine serum GABA γ-amino butyric acid
VIII Abbreviations
GABAR GABA receptors gapdh glyceraldehyde-3-phosphate dehydrogenase gria1 α-amino-3-hydroxy-5-methy-4-isoxazolepropionic acid receptor subunit 1 grin2b N-methyl-D-aspartic acid receptor subunit 2B h Hour HBSS Hanks Balanced Salt Solution
IC50 Half maximal inhibitory concentration K Potassium KCC2 K+-Cl- co-transporter KCl Potassium chloride KE Key event LBD Ligand binding domain LTD Long-term depression LTP Long-term potentiation MAPs Microtubule-associated proteins Mg Magnesium
MgCl2 Magnesium chloride mGluR Metabotropic glutamate receptor MIE Molecular initiating event min Minute MK801 Dizocilpine Na Sodium
Na2HPO4 Disodium hydrogen phosphate NaCl Sodium chloride NaOH Sodium hydroxide NCSs Neuronal cellular structure NFA Network Formation Assay NKCC1 Na2+-K+-Cl- co-transporter 1 NMDA N-methyl-D-aspartate NMDAR N-methyl-D-aspartate receptor NTE Neuropathy target esterase OECD Organization of Economic Cooperation and Development OPIDN Organophosphate-induced delayed neuropathy OPs Organophosphates
IX Abbreviations
PBS Phosphate buffered Saline pCNs Primary cortical neurons PCP Phencyclidine PDL Poly-D-lysine PEG Polyethylenglycole PFA Paraformaldehyde PKA protein kinase A PKC protein kinase C PLL Poly-L-lysine PNS Peripheral nervous system PTFE Polytetrafluoroethylene REACH Registration, Evaluation, Authorization and Restriction of Chemicals RT Room temperature s Second TCP Tri-cresyl phosphate TCP mixture Tri-cresyl phosphate mixture TG Trigeminal ganglia TmCP Tri-meta-cresyl phosphate TMD Transmembrane domain ToCP Tri-ortho-cresyl phosphate TpCP Tri-para-cresyl phosphate TRP channels Transient receptor potential channels TRPA Transient receptor potential, ankyrin TRPM Transient receptor potential, melastatin TRPML Transient receptor potential, mucolipin TRPN Transient receptor potential, NOMPC-like TRPP Transient receptor potential, polycystin TRPV Transient receptor potential, vanilloid VCSA Virtual cell soma VGCC Voltage-gated calcium channels ZK200775 [1, 2, 3, 4-tetrahydr-7-morpholinyl-2, 3-dioxo-6-(trifluoromethyl) quinoxalin-1-yl] methylphosphonate
X Introduction
1 Introduction
“All things are poison, there is nothing without poison; only the dose makes a thing non- poisonous.” Paracelsus, 1530.
During the human lifetime there is a potential risk for exposure to various environmental chemicals or other hazardous substances via oral, dermal, or inhalative uptake routes. When possible chemicals are absorbed into the blood, they distributs to further body fluids and spreads to all tissues and organs (Ruiz-Garcia, Bermejo et al. 2008). Protoxicants are metabolized in the liver or in local compartments (e. g. in astrocytes) and become more toxic than the original compound. Some neurotoxins are able to cross the blood-brain barrier and can cause severe effects on a structural and functional level that leads to cognitive deficits or neurodegenerative disease. In addition to toxins occurring in nature with the origin in plants, animals and fungi or metals many synthetically produced chemicals with toxic effects are known (Davis, Otto et al. 1990, Wright and Baccarelli 2007). Possible endogenous neurotoxins are used as pesticides, plasticizer in plastics, lacquers, paints and glues, flame retardants or additives in lubricants and oils (Falck, Mooney et al. 2015, Mamane, Raherison et al. 2015, Lyche, Gutleb et al. 2009, Costa, de Laat et al. 2014, Dishaw, J Macaulay et al. 2014, Mallow and Fox 2014). For a whole variety of these and other substances no data concerning their ability to cause neurotoxicity or toxic outcomes are available.
Neurotoxicology occurs when exposure to a chemical results in persistent deficits in the function of the nervous system. Neurotoxicology and developmental neurotoxicology investigate the life stage specific susceptibility against chemically induced alterations of the nervous system (childhood, adulthood, and elderly) (Rice and Barone 2000, Landrigan, Rauh et al. 2010). Neurotoxicity is defined in accordance with the Organization of Economic Cooperation and Development (OECD) Guideline 424 as an adverse change in the structures or function of the nervous system that results from exposure to a chemical compound. ”Neurotoxicity” is a broad term describing a multitude of effects triggered by hazardous substances, which act (1) directly on the central nervous system, (2) and/or directly on the peripheral nervous system, (3) and/or indirectly via peripheral organs, where abnormal function can trigger brain activity (Chepelev, Moffat et al. 2015). Exposure to chemical compounds can result in neurotoxic alterations of neurons or the nervous system and lead to adverse effects.
1 Introduction
Neurotoxicology has recently become a clear focus of attention in science. Based on increased public interest and the awareness that the (developmental) nervous system is highly vulnerable to chemical influence, research efforts have increased. In this connection it is important to note that minor changes, alterations, or disturbances of the nervous system can lead to neurobehavioral alterations that affect the quality of the entire life. It is known that even subtle perturbation of neurite growth can result in neurobehavioral deficits (Rice and Barone 2000). Possibly hazardous compounds are detected and an assessment of health risk takes place, particularly because low-level and/or long-term exposure with environmental chemicals like pesticides, phthalates, metals, or volatile organic compounds often lead to intoxication events in humans. To avoid neurotoxic effects more research is needed to identify compounds as neurotoxins and to characterize their modes of action.
Over recent decades, standardized neurotoxic testing based on laboratory animal studies and neurobehavioral readouts like motor activity, learning and memory and auditory startle behavior was used to identify the neurotoxic potential of compounds (van Thriel, Westerink et al. 2012). These neurobehavioral readouts are an integrative approach for investigating nervous system integrity as a whole (van Thriel, Westerink et al. 2012). Toxicologists have evaluated human standards based on the results of animal testing for more than 50 years (Schmidt 2009). The gold standard of toxicity testing methods has remained largely unchanged over the past five decades. Organism-level responses (e.g. hepatotoxicity, cancer, reproductive/developmental toxicity, and neurotoxicity) deemed to serve as measures of adverse response in high-dose studies with homogeneous groups of laboratory animals (Bhattacharya, Zhang et al. 2011). Based on these high-dose animal studies human exposure limits of low-level (environmental) exposure are extrapolated. There has been broad dissatisfaction with this approach from both regulatory agencies and regulated communities (Bhattacharya, Zhang et al. 2011).
The new guidelines of the European Union called REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) regulate the production and use of chemical compounds and address their potential impact on health. Many not sufficiently tested compounds should be evaluated and an adequate exposure assessment is of particular importance. The estimates vary, but tens of thousands of industrial chemicals are used in consumer products without any knowledge of their potential toxicity (Schmidt 2009). In vitro approaches are seen as best hope for evaluating the enormous backlog of these untested chemicals (Schmidt 2009). Along with the new toxicity testing program called “Toxicity Testing in the 21st- century: A Vision and a Strategy” from the U.S. National Research Council, REACH lead to a paradigm shift in toxicology from neurobehavioral readouts to in vitro based test systems. Behavioral animal testing provides no or only limited mechanistic insights compared to in vitro testing (van Thriel, 2 Introduction
Westerink et al. 2012). The main objectives of the 21st- century toxicology program are the identification of environmental chemicals, which lead to biological responses, and the analysis of their underlying mechanism of action. Neurotoxins can cause alterations in a variety of pathways. These toxicity pathways are normal biological pathways, which lead to adverse outcomes when sufficiently perturbed. Toxicity pathways are aligned with adverse outcome pathways (AOPs), which is a concept used as a novel tool in toxicology (Vinken 2013). AOPs are designed to provide a clear mechanistic representation of a critical toxicological effect that spans over different layers of biological organization relevant to risk assessment (Ankley, Bennett et al. 2010). The AOP framework applies mechanistic understanding into regulatory decisions, and serves as a toolbox for consolidating, managing, and exchanging knowledge within the research community (Bal-Price, Crofton et al. 2015). In general, an AOP includes three consecutive steps: (1) the molecular initiating event (MIE), which is, for example, a ligand-receptor interaction or a protein binding event, (2) a series of intermediate steps and (3) the adverse outcome (Vinken 2013). AOPs can vary in resolution and expanse and can include qualitative and quantitative descriptions of key events (KE) and their interlinking causal relationships (Bal-Price, Crofton et al. 2015). There are a number of challenges in implementing the AOP framework in the field of neurotoxicology and developmental neurotoxicology. In this context, often the MIEs that trigger the KEs are not be identified. The functional and structural heterogeneity of the nervous system suggest that a broad array of MIEs may be involved in adverse neurological outcomes. However, a limited number of neurotoxic outcomes that have well-defined pathophysiological outcomes and MIEs, like “Binding of certain organophosphates to NTE results in delayed neuropathy” or “Impairment of learning and memory induced by binding of electrophilic chemicals to the SH(thiol)-group of proteins and non-protein molecules in neuronal and glial cells during development” are described. The advantage of understanding (neurotoxic) AOPs is that the knowledge can be used in a predictive manner (Bal-Price, Crofton et al. 2015). For that issue, tools and approaches are developed and applied to evaluate possible effects of chemical compounds on human health. To predict possible modes of actions and AOPs, and identify disrupted biological pathways, new tools like high-throughput in vitro techniques, approaches of biochemistry and system biology as well as computer-modeling technologies were utilized. The development of screening methods for potential developmental neurotoxic chemicals is critical. In vitro endpoints should fulfill several requirements like exposure paradigms and exposure concentration (relevant concentration range in the human situation). Additionally, the lack of metabolism in the in vitro system should be considered (Westerink 2013). The implementation of in vitro assays is important to identify and prioritize specific cellular mechanisms, which are induced by neurotoxic chemicals in the brain. Especially the inclusion of
3 Introduction functional parameters is important to investigate critical processes for the formation of functional neuronal networks that receive, conduct, and transmit signals via chemical or electrical synapses, and relay information between specific brain regions for information processing as well as learning and memory (de Groot, Westerink et al. 2013). Functional in vitro endpoints, like chemical-induced changes in intracellular calcium dynamics, including calcium homeostasis and neurotransmitter release, and intracellular communication, which relies on the function of neurotransmitter receptors, reflect the functional nervous system (de Groot, Westerink et al. 2013). Several chemicals have been shown to modulate neurotransmitter receptor function (Atchison 1988). These endpoints should be used in addition to biochemical, structural and morphological endpoints like neurite outgrowth inhibition and neurite morphology. In vitro assays are useful regulators involved in risk assessment and legislation of chemicals (de Groot, Westerink et al. 2013) and the investigations of the affected pathways result in a priorization of chemicals for which more extensive testing is necessary. This prioritization of test compounds based on biological approaches is more decisive than other criteria such as production volume, likelihood for human exposure, or structural similarity to other known chemicals (Schmidt 2009). The shift from classical animal testing to cell-based systems (Leist, Kadereit et al. 2008, Hartung 2010, van Thriel, Westerink et al. 2012) is more efficient and less animal-, cost- and time-consuming. Neurotoxins can be detected and neurotoxic pathways and modes of actions can be investigated (Bal-Price, Hogberg et al. 2010, van Thriel, Westerink et al. 2012). Concurrently, the 3R principle, which described the reduction, refinement and replacement of animal testing is compiled (Flecknell 2002). The 3R principle was developed by Russel and Burch in 1959 and first explained in their book “The Principles of Human Experimental Technique” and should minimize the suffering of laboratory animals. The new approaches will create a new database with knowledge of the potential risk by exposure to environmental chemicals and agents, and help to assess the potentially hazardous compounds efficiently. However, with respect to the complexity of the brain and the nervous system, in vitro models are not likely to completely replace in vivo testing. The advantage of in vitro assays is the higher testing capacity (de Groot, Westerink et al. 2013) but they should represent the in vivo situation as closely as possible (Westerink 2013). One of the key objectives is the implementation of a multifaceted approach in neurotoxicological studies to recognize all levels of neuronal impairment by neurotoxins. The following part gives an introduction into the basics of the nervous system, neurite outgrowth, and neurotransmission, including the different neurotransmitter systems and pathways, which are important in connection with neurotoxicology and the investigation of adverse effects of neurotoxins.
4 Introduction
1.1 Central and peripheral nervous system
The nervous system can be divided into the central and the peripheral nervous system. The central nervous system (CNS) is a bilateral structure, which consists of the brain and the spinal cord. The brain can be divided into six main parts. Sensory information from all parts of the body (e. g. skin, trunk, or muscles of the limbs) is received and processed by the spinal cord, which continues as the brain stem (medulla, pons, and midbrain). The brain stem receives sensory information from the head, and processes information between the spinal cord and the brain. Autonomic functions like breathing and control of the heart rate is mediated by the medulla oblongata. The pons transfers information about movement from the hemispheres to the cerebellum. Force and range of movements are modified by the cerebellum, which is also involved in learning of motor skills. Sensory and motor function, as well as visual and auditory reflexes, are controlled by the midbrain. The diencephalon contains the thalamus, which processes information from the cerebral cortex, and the hypothalamus, which regulates endocrine and visceral function. The cerebral hemispheres are composed of one outer layer, the cerebral cortex, and three inner structures, the basal ganglia, the hippocampus, and the amygdala. Cognitive abilities occur primarily in the cerebral cortex (Kandel, Schwartz et al. 2000). The mammalian neocortex is a six layer structure, which is highly organized and complex. This brain region is responsible for cognitive function and sensory perception (Finlay and Darlington 1995). The key feature of the CNS is the capacity to reorganize structures depending on the environmental surroundings and the ability to modify synaptic connections. The nervous system consists of different cell types. Glia cells, which have a supportive and protective function in the nervous system, and neurons, which enables the processing of information via the interconnection of the cells. Neurons can be classified as central or peripheral by their morphology, their function, and their origin. Neurons consist of three different structures: the soma or cell body, one single axon, and several dendrites (collectively called neurites). Neurons assemble into functional networks by growing neurites to connect with each other. Neurons are highly specialized cells that are electrically excitable and transmit information by electrical and chemical signals. The communication between neurons plays an important role in the propagation of information in the brain and requires a finely organized connectivity among these cells. In the process of neuronal signaling, dendrites receive signals from at least one other neuron. The electric signal then propagates via the soma along the axon to the presynaptic terminals, where the axon forms synapses with the dendrites of other neurons (Stiess and Bradke 2011). The cerebral cortex is, evolutionarily the youngest and the most complex region of the brain. Neurons of the cortex are defined either as excitatory glutamatergic projection neurons, which have typically pyramidal shaped cell bodies, or as a smaller fraction of inhibitory,
5 Introduction
GABAergic local interneurons (Molyneaux, Arlotta et al. 2007, Gelman and Marín 2010). Cortical neurons are responsible for cognitive features. In vitro cultures of cortical neurons are used to study questions of cellular biology and the function of central nervous system processes. Neuron cultures can also be used to analyze neurotoxic effects, study underlying mechanisms and identify new neurotoxic pathways (Suñol, Babot et al. 2008).
The peripheral nervous system (PNS) is divided into the somatic and the autonomic part. The somatic division contains sensory neurons. The cell bodies of these neurons are located in the dorsal root ganglia or the trigeminal ganglia and their neurite structures innervate a variety of body parts. The trigeminal system innervates the eye, the nose, the skin, and the mouth and throat region. The neurons provide sensory information about muscle and limb position, touch, pain, and temperature at the skin surface to the CNS. The autonomic division mediates visceral sensation/information such as cardiac and smooth muscles and reflexes (Kandel et al., 2000). Primary afferent sensory neurons, including trigeminal ganglia (TG) neurons and dorsal root ganglia (DRG) neurons, are responsible for processing important sensory information (e. g. temperature, pain, and touch). The cell bodies of DRG neurons are clustered in the dorsal root ganglia within the vertebral column adjacent to the spinal cord. The neurons are pseudo- unipolar with a bifurcated axon with central and peripheral branches. DRG neurons are well suited to fulfill two principal functions: on the one hand stimulus transduction and on the other hand transmission of encoded stimulus information to the CNS. DRG neurons contain serotonin- (Wu, Zhu et al. 2001), glycine- (Schlösser, Barthel et al. 2015), glutamate- (Marvizon, McRoberts et al. 2002, Kung, Gong et al. 2013), and GABA-receptors (Lee and Gold 2012, Mao, Garzon- Muvdi et al. 2012) as well as transient receptor potential channels (TRP channels) (Schoenen, Delree et al. 1989, Delrée, Ribbens et al. 1993).
1.2 Neurite outgrowth and neurite degeneration
Cellular morphogenesis is the ability of neurons to change their shape. Neurons extended a long axon and several shorter dendrites to transmit signals in the nervous system (Dotti, Sullivan et al. 1988). The formation of neuronal networks which connected the neurons to each other is essential for neurotransmission. The process of neuronal polarization is essential for the correct function of the CNS and is driven by the cytoskeleton (Craig and Banker 1994, Arimura and Kaibuchi 2007, Lalli 2014). The cytoskeleton is a very dynamic system that determines cell polarity by forming the structural scaffold for the neuronal shape, and also organizing intracellular transport (Stiess and Bradke 2011). Three major classes of cytoskeletal proteins are responsible for these morphological changes: microtubules, microfilaments, and intermediate filaments (Dehmelt, Smart et al. 2003, Howard and Hyman 2003, Pollard and Cooper 2009, Flynn
6 Introduction
2013, Shirao and Gonzalez-Billault 2013). Cytoskeletal dynamics underlie the different growth activities of neurites (Gartner, Fornasiero et al. 2012). Neurite outgrowth, and especially axon growth, takes place at the tip of the neurite, the so-called growth cone (Letourneau 1979, Bray and Gilbert 1981, Smith 1988, Rehder and Kater 1992, Lowery and Van Vactor 2009). Cytoskeletal dynamics underlie the mortality and the forward movement of the growth cone that determines the direction and elongation of the process (Howard and Hyman 2003, Dent, Gupton et al. 2011). Actin filaments regulate the shape and the directed progress of the growth cone, whereas microtubules give structure and are essential for extension (Ono 2007, Pollard and Cooper 2009, Stiess and Bradke 2011). Neurofilaments are the matrix of the cytoskeleton. The architecture of the neurites is important for connectivity, plasticity, and synaptic transmission of the neurons as well as for proper wiring in the CNS.
Figure 1.1 Neuronal development and neurite outgrowth. Neuronal outgrowth can be divided into a series of stages which were initially characterized in culture (Dotti et al., 1988), but also seem to occur in vivo. First neurons begin extending lamellipodia and filopodia (Stage 1). In stage 2 the neurite initiation takes place and each neuron typically exhibits multiple minor neurites. In the next step, one neurite with a large and dynamic growth cone becomes the axon (Stage 3). The axon continues to grow and differentiate while remaining processes become dendrites (Stage 4). Neurons then begin to form synapses, develop dendritic spines, and form neuronal circuits (Stage 5). Modified from Flynn, 2013. The process of neurite outgrowth has been well-studied in primary cultures of rodents. Primary cortical and hippocampal neurons from late embryonic or early postnatal tissue rapidly extend neurites when prepared in a dissociated culture. They develop axonal and dendritic structures with the morphology of neurons observed in the brain in vivo (De Lima, Merten et al. 1997). In vitro, primary cortical neurons follow a stereotypic pattern of neuronal maturation (Dotti, Banker et al. 1987, De Lima, Merten et al. 1997). The study by Dotti et al., indicates that growth and maturation of neurons can be characterized by different developmental stages. At stage 1, cells were rounded and do not extend neurites but may extend lamelliopodial protrusions. Neurons in stage 2 develop one or more immature neurites which are not differentiated into dendrites or axons. The transition from stage 2 to stage 3 occurs when one of the immature
7 Introduction neurites undergoes a rapid growth. The transition to stage 4 is followed by the maturation of the other neurites. At stage 5 the synapse formation takes place (Fig. 1.1). Neurite outgrowth inhibition can be investigated at the stages 1 – 3, while neurite degeneration proceeds at a later stage of development (stage 4 - 5).
1.3 Neurotransmitter systems and ion channels
Neurotransmitter-gated ion channels are responsible for synaptic transmission (Smart and Paoletti 2012). The mechanism depends on the release of neurotransmitters from the presynaptic neuron. Neurotransmitters are chemical substances that bind to specific receptors. Neuronal communication depends on the release of a variety of neurotransmitters. They can be classified in four categories: (1) classical neurotransmitters like glutamate, γ-amino butyric acid (GABA), glycine, acetylcholine, adenosine, and adenosine-triphosphate (ATP), (2) monoaminergic neurotransmitters like dopamine, adrenaline, noradrenaline, serotonin, and histamine, (3) neuropeptides as neurotransmitters, and (4) small membrane permeable mediators like nitric oxide, endocannabinoids, and lipid neurotransmitters (Süudhof 2008).
1.3.1 Glutamate receptors
L-glutamate is the major excitatory neurotransmitter in the mammalian CNS (Fonnum 1984, Stephenson 2006). After release from presynaptic nerve terminals, glutamate acts through a variety of glutamatergic receptors (Kew and Kemp 2005). Glutamate receptors can be divided into two broad categories. Metabotropic G-coupled receptors, where second messengers molecules are involved in the signal transduction pathway, and ionotropic receptors (Kew and Kemp 2005, Mayer 2005, Niswender and Conn 2010). The action of glutamate on ionotropic receptors is always excitatory, where the activation of metabotropic receptors can produce, depending on the activated G-protein, either excitation or inhibition.
Eight members of the G-protein-coupled metabotropic glutamate receptor (mGluR) family have been identified (Anwyl 1992, Niswender and Conn 2010). All family members, with the exception of mGluR6, are expressed in the mammalian CNS in both neurons and glia cells with distinct expression profiles (Björklund and Hökfelt 1983, Flor, Van Der Putten et al. 1997). Metabotropic glutamate receptors can be divided into three groups based on sequence homology, signaling pathways, and pharmacology. Group I consists of mGluR1 and mGluR5 and couple via Gq/Gll to phospholipase C, group II with mGluR2 and mGluR3, and group III which consists of mGluR4, 6,
7, and 8 couple both via Gi/G0 to adenylyl cyclase (Conn and Pin 1997, Pin, Galvez et al. 2003). Group I causes postsynaptic depolarization and leads to prolonged excitatory postsynaptic potentials by closing of K+-channels, while group II provokes hyperpolarization and leads to
8 Introduction prolonged inhibitory postsynaptic potentials by opening of K+-channels (Sherman 2014). Group III mGluR inhibit the release of neurotransmitters (Cartmell and Schoepp 2000, Mercier and Lodge 2014). Members of the mGluR family mediate fast synaptic transmission and generally modulate neuronal excitability, synaptic transmission and plasticity, (Anwyl 2009, Niswender and Conn 2010) as well as second messenger cascades (Gereau Iv and Conn 1994).
The three mayor subtypes of ionotropic glutamate receptors are α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA) receptors, kainat receptors and N-methyl-D-aspartate (NMDA) receptors, named according to the types of specific agonists that activate them (Mayer and Westbrook 1987, Kew and Kemp 2005, Mayer 2005). AMPA receptors (AMPARs) are tetrameric cation channels that mediate fast excitatory synaptic transmission in the mammalian CNS (Hollmann and Heinemann 1994, Dingledine, Borges et al. 1999, Heine, Thoumine et al. 2008). NMDA receptors (NMDARs) are also tetrameric cation channels, whereas their activation requires glutamate and membrane depolarization (Stephenson 2006), which determines long- term potentiation (LTP) or long-term depression (LTD) of AMPARs (Bliss and Lomo 1973, Bliss and Collingridge 1993, Bellone and Nicoll 2007). Native receptors of both families are likely tetrameric assemblies compromising more than one type of subunit. Ionotropic glutamate receptor subunits possess an extracellular amino terminal domain (ATD), which exhibits homology to the metabotropic glutamate receptor bi-lobed agonist binding domain and forms the cation-selective pore. After the first transmembrane domain (TMD) a pore forming membrane-resting domain followed that does not cross the membrane but forms a loop entering from and exiting to the cytoplasm. The second and third transmembrane domains are linked by a large extracellular loop and the third transmembrane domain is followed by an intracellular carboxyl-terminus (CTD) which is involved in receptor localization and modulation (Mayer and Armstrong 2004, Oswald, Ahmed et al. 2007, Sobolevsky, Rosconi et al. 2009, Traynelis, Wollmuth et al. 2010). Ionotropic glutamate receptors include two polypeptide segments, called S1 and S2, that after glutamate binding, linked together and formed a two- domain or clamshell-like structure, which called the S1S2 ligand-binding core (Stern-Bach, Bettler et al. 1994, Kuusinen, Arvola et al. 1995, Armstrong, Sun et al. 1998).
Receptors of the NMDAR family are composed of four out of seven existing subunits, NR1, NR2A- D and NR3A and B, which are all coded by different genes (Kutsuwada, Kashiwabuchi et al. 1992, Dingledine, Borges et al. 1999, Kew and Kemp 2005) and differentially expressed in distinct brain regions (Monyer, Sprengel et al. 1992, Monyer, Burnashev et al. 1994, Rigby, Le Bourdellès et al. 1996). Native NMDARs are composed of NR1 in combination with one or more NR2 subunits (e.g. NR1/NR2A, NR1/NR2B, or NR1/NR2A/NR2B) or NR1 in combination with both NR2 and NR3 subunits (NR1/NR2A/NR3A) (Traynelis, Wollmuth et al. 2010). During late embryogenesis and 9 Introduction early postnatal development NMDARs formed exclusively by NR1 and NR2B and NR2 subunit expression is developmentally regulated (Monyer, Burnashev et al. 1994, Sheng, Cummings et al. 1994). The study of (Clements and Westbrook 1991) demonstrate that NMDAR activation requires occupation of two independent glycine sites and two independent glutamate sites. Thus, the minimal requirement for a functional NMDAR is likely to be a tetramer of two NR1 and two NR2 subunits, based on co-agonist binding sites for glycine and glutamate localized on both NR1 and NR2 subunits (Kuryatov, Laube et al. 1994, Wafford, Kathoria et al. 1995, Anson, Chen et al. 1998, Kew, Koester et al. 2000). All four subunits must bind their respective ligands for pore opening (Benveniste and Mayer 1991, Clements and Westbrook 1991, Schorge, Elenes et al. 2005). The NMDARs which contribute to the late component of the excitatory postsynaptic potentials (EPSPs), based on the slow activation of these receptors, had three properties (Stephenson 2006). The NMDAR is a cation channel of high conductivity that is permeable to Ca2+ as well as Na2+ and K+. Glycine is the coagonist for the NMDAR and required for channel opening (Kuryatov, Laube et al. 1994). The opening of NMDAR depends, in addition to the agonist binding, on membrane voltage. The voltage-dependency is related to a special mechanism of conformational change. Within the NMDAR pore an Mg2+-ion is linked and acts as an open channel blocker (Ault, Evans et al. 1980). At the membrane resting potential of -65 mV the magnesium-ion binds tightly. After membrane depolarization, evoked by mGluRs or AMPARs activation, the Mg2+-ion is expelled from the channel and allows Ca2+ to enter (Stephenson 2006). The NMDAR activation leads to the activation of calcium-dependent enzymes and second messenger proteins in the postsynaptic neuron. These biochemical alterations trigger signaling pathways, which are important for modifications at the synapse and are related to learning and memory processes (Baudry, Zhu et al. 2015). There are numerous sites on the NMDAR at which binding of compounds can lead to inhibition, activation or modulation of the receptor function (Kemp and McKernan 2002). NMDARs can be inhibited by ketamine, phencyclidine (PCP) (Anis, Berry et al. 1983), and by dizocilpine (MK801). These antagonists bind to a site within the open channel pore that is distinct from the Mg2+-binding site (Wong, Kemp et al. 1986). These inhibitors are potent, selective and drug-like antagonists. Blockage of NMDAR signals allows an investigation of AMPAR and kainate receptor evoked signals and is used to prevent excitotoxicity.
AMPARs are homo or hetero tetramers assembled from a four subunit containing family (GluA1- 4) that are separate gene products (Wenthold, Petralia et al. 1996, Collingridge, Olsen et al. 2009). The four subunits that make up each receptor combine in various stoichiometry to form receptors with distinct channel properties (Rosenmund, Stern-Bach et al. 1998). Most AMPARs in adult hippocampus and cortex consist of GluR1 and GluR2, or GluR2 and GluR3 subunits (Craig,
10 Introduction
Blackstone et al. 1993). The extracellular and transmembrane regions of all AMPARs are highly homologous and the four GluAs differ only in the term of their intracellular cytoplasmic tails (Hollmann and Heinemann 1994, Dingledine, Borges et al. 1999). Alternative splicing generates the flop (short cytoplasmic tail) and the flip (long cytoplasmic tail) variants encoded by exons 14 and 15 that differ by a 38 amino acid insertion in a region that forms a part of the ligand binding domain (Sommer, Keinanen et al. 1990). The flip variant of all subunits are predominantly expressed before birth, remaining unchanged during postnatal development, and in the adult. The expression of the flop variant increases throughout development and reaches adult level by postnatal day 14 in rats (Monyer, Seeburg et al. 1991). Flop variants desensitize more rapidly in response to glutamate than flip containing receptors (Sommer, Keinanen et al. 1990). There are numerous binding sites for antagonist on AMPARs (Kew and Kemp 2005). At first, competitive AMPAR antagonists like CNQX (6-cyano-7-nitroquinoxaline-2, 3- dione) and DNQX (6, 7-dinitro- quinoxaline-2, 3-dione) were developed, but these antagonists were also active at the glycine binding site of NMDARs. A more selective competitive antagonist was generated, for example ZK200775 ([1, 2, 3, 4-tetrahydr-7-morpholinyl-2, 3-dioxo-6-(trifluoromethyl) quinoxalin-1- yl]methylphophonate) (Turski, Huth et al. 1998).
AMPARs mediate the majority of fast synaptic excitation (Heine, Thoumine et al. 2008). The precise regulation of AMPAR number and subtype at the synapse is crucial to excitatory neurotransmission, synaptic plasticity and formation of neuronal circuits (Hanley 2014). AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis, and endosomal recycling (Henley, Barker et al. 2011, Hanley 2014). The mechanisms and routes by which AMPARs reach the synapse have not been elucidated completely. The three step model of (Opazo and Choquet 2011) indicates that AMPARs of the intracellular pool are first inserted into the perisynaptic surface via exocytosis, and then AMPARs diffuse to the postsynaptic membrane (Fig. 1.2) (Borgdorff and Choquet 2002, Makino and Malinow 2009).
11 Introduction
Figure 1.2 Regulation of AMPAR trafficking during synaptic plasticity. During LTP AMPARs are inserted either at the dendritic shaft or at the spine lateral to the postsynaptic density (PSD) by a myosin-dependent mechanism or via phosphorylation (step 1). Newly inserted AMPARs rapidly diffuse to the synapse (step 2). They are trapped by a phosphorylation step (step 3). The reverse order of events take place during LTD. AMPARs could be destabilized from PSD95 via dephosphorylation (step 1). AMPARs diffuses out of the synapse (step 2) and then undergoes endocytosis (step 3). From Opazo and Choquet, 2011.
Here they are retained by interaction with scaffold proteins and anchored in the postsynaptic density (Lisman and Raghavachari 2006, Newpher and Ehlers 2008). The synaptic AMPARs are recruited either from intracellular vesicular stores via exocytosis or from extrasynaptic surfaces via lateral diffusion. Under basal conditions, AMPAR trafficking is a highly dynamic process where AMPARs are continuously exchanged between synapses and different store pools (Shepherd and Huganir 2007, Newpher and Ehlers 2008, Triller and Choquet 2008). AMPARs are internalized at sites lateral to the postsynaptic density (Carroll, Beattie et al. 1999) with a time constant of about 40 min (Man, Lin et al. 2000). Therefore, AMPARs undergo rapid clathrin- mediated endocytosis (Carroll, Beattie et al. 1999). Additionally, there is a similar rate of constitutive insertion of AMPARs into the cell membrane to counteract endocytosis, thereby maintaining a constant level of cell surface receptors (Man, Ju et al. 2000). Furthermore, F-actin is involved in maintaining AMPARs at synapses and actin depolymerization is also required for the removal of AMPARs from the synaptic plasma membrane (Zhou, Xiao et al. 2001, Fukazawa, Saitoh et al. 2003, Hanley 2014). AMPAR trafficking plays an important role in LTD and LTP which are characterized by internalization and integration of these receptors (Kim and Lisman 1999, Gu, Lee et al. 2010). AMPARs are rapidly transported into and out of synapses to strengthen or weaken their action depending on the appropriate behavioral responses. LTP is characterized by
12 Introduction long-lasting potentiation of AMPAR mediated EPSCs (Kandel 2001). During the initiation of LTP, NMDARs are activated to enable Ca2+ influx which induce the activation of calcium\calmodulin- dependent protein kinase II (CaMKII), protein kinase C (PKC) and protein kinase A (PKA) as well as extracellular signal-regulated protein kinase (ERK-MAPK) (Malinow, Schulman et al. 1989, Silva, Stevens et al. 1992, English and Sweatt 1997, Esteban, Shi et al. 2003, Man, Wang et al. 2003, Byth 2014). These kinases phosphorylate the GluR1 subunit of AMPAR (Lee 2006). This phosphorylation increase AMPAR insertion into the postsynaptic membrane (Oh, Derkach et al. 2006). Extrasynaptic pools of AMPARs probably serve as a source of receptors for delivery to synapses during LTP (Malenka and Bear 2004). LTD is characterized by the loss of synaptic AMPARs triggered by activation of NMDARs or metabotropic glutamate receptors (Malenka and Bear 2004). GluA2 plays a critical role of AMPAR removal during LTD. Several proteins interact with GluA2 to regulate AMPAR trafficking in LTD (Collingridge, Peineau et al. 2010).
1.3.2 GABA receptors
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS and for around one-third of all CNS neurons GABA is their primary neurotransmitter (Bloom and Iversen 1971). GABAergic inhibition is essential for maintaining a balance between neuronal excitation and inhibition. There are two classes of GABA receptors (GABAR). The ionotropic GABAA receptors, which are fast-acting ligand-gated chloride channels, and the metabotropic GABAB receptors, which are coupled to G-proteins (Sieghart 2006, Bowery 2010). The GABAA receptor is a hetero- oligomer composed of five transmembrane subunits of 19 possible subunits (α1-6, β1-3, γ1-3,
δ, ε, π, θ, and ρ1-3). The majority of GABAA receptors in the CNS consist of two α-subunits, two β-subunits, and a γ-subunit. Other receptors can include δ, ε, or π subunits as a substitute for the γ subunit, or the θ-subunit instead of a β-subunit (Sieghart and Sperk 2002). Based on the structure, two molecules of GABA bind to each receptor between the α- and β-subunit. Comparable to the glutamate receptors, the composition of subunits determines channel conductance and kinetics as well as pharmacology and receptor localization (Farrant and Kaila 2007).
In the developing brain, the intracellular chloride-ion concentration [Cl-] is higher than in the extracellular space (Sánchez Fernández and Loddenkemper 2014). Thus, the opening of GABAA receptors leads in the developing brain to different effects than in the adult brain. In developing neurons activation of GABARs lead to membrane depolarization (Ben-Ari, Cherubini et al. 1989, Chen, Trombley et al. 1996, Owens, Boyce et al. 1996). Instead of an inhibition by Cl-- influx upon channel opening, the Cl--efflux leads to a depolarization of the immature neurons (Ben-Ari 2002, Ben-Ari, Woodin et al. 2012). During development, neurons go from a stage of chloride
13 Introduction accumulation to a stage of chloride extrusion. Cation-chloride-co-transporter are differentially expressed at different stages of development. First, in the developing brain the chloride accumulator Na2+-K+-Cl- co-transporter 1 (NKCC1) is expressed (Clayton, Owens et al. 1998). The outwardly directed transporter K+-Cl- co-transporter (KCC2) expression is significantly increased after the first postnatal week (Rivera, Voipio et al. 1999, Hübner, Stein et al. 2001). The gradient of chloride changes and shifts the neuronal response from excitatory to inhibitory during maturation (Lu, Karadsheh et al. 1999). In the immature brain, the fast synaptic transmission is mediated via GABAA receptors (Cherubini, Gaiarsa et al. 1991, Ben-Ari, Khazipov et al. 1997). GABA promote neuronal growth in vivo, proliferation, migration, and synaptogenesis (Cherubini, Gaiarsa et al. 1991, Barbin, Pollard et al. 1993, Behar, Schaffner et al. 2000, Behar, Smith et al. 2001). GABA could act as a general factor for neuronal development (Schousboe and Redburn 1995). GABA-induced excitation arises in the embryonic brain and early postnatal stages of development as well as in some other neuronal cell types. In addition to differentiated glia cells and sympathetic neurons, DRG neurons isolated from cat, rat, frog, and human embryos also possess excitatory GABAA receptors (Deschenes, Feltz et al. 1976, Valeyev, Hackman et al. 1999, Towers, Princivalle et al. 2000).
1.3.3 Voltage-gated calcium channels
The electrical activity of neurons is based on a number of different voltage- and ligand-gated ion channels that are permeable to ions like sodium, potassium, chloride and calcium. Voltage-gated calcium channels (VGCC) are the mediators of depolarization, which is based on calcium entry into the neuron. Channel opening leads to a Ca2+ influx along the electrochemical gradient, and
2+ rise of the intracellular calcium concentration [Ca ]i from resting conditions (100 nM) up to a micromolar range (Wadel, Neher et al. 2007). The increase in intracellular calcium concentrations triggers calcium-dependent processes (Clapham 2007), including gene transcription, neurotransmitter release, neurite outgrowth, and activation of different kinases (Malinow, Schulman et al. 1989, Wheeler, Randall et al. 1994, Esteban, Shi et al. 2003, Wadel, Neher et al. 2007, Wheeler, Barrett et al. 2008, Wheeler, Groth et al. 2012). VGCCs can be subdivided into various families, including the L-type (Cav1 family), the T-type (Cav3 family), and the P/Q-type, the R-type and the N-type channels, which belong to the Cav2 family (Mikami, Imoto et al. 1989, Dubel, Starr et al. 1992, Williams, Brust et al. 1992, Williams, Feldman et al. 1992, Soong, Stea et al. 1993, Randall and Tsien 1995, Bourinet, Soong et al. 1999, Catterall, Perez-Reyes et al. 2005, Richards, Swensen et al. 2007, Berger and Bartsch 2014). VGCCs are assembled of five different channel subunits (α1, α2, β, γ, δ), with α1 as the central-pore forming subunit. The subunit composition of VGCCs, including alternative splice variants, are responsible
14 Introduction for the channel characteristic, the regulation of physiological processes, and the release of neurotransmitters and might be tissue- and age-dependent (Chaudhuri, Chang et al. 2004, Liao, Yong et al. 2005, Lipscombe 2005, Tan, Jiang et al. 2011, Tan, Yu et al. 2012, Lipscombe, Andrade et al. 2013, Simms and Zamponi 2014). VGCCs possess voltage-gated and/or calcium-dependent inactivation mechanisms to prevent calcium overload to prolonged membrane depolarization. This property is common to all VGCC subtypes (Herlitze, Hockerman et al. 1997, Stotz, Jarvis et al. 2004).
1.3.4 TRP Channels
Transient receptor potential channels (TRP channels) were first discovered in the fruit fly Drosophila melanogaster (Montell and Rubin 1989). TRP channels represent a highly conserved family of cation channels, which can be divided into seven groups according to their sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like) (Montell 2005, Wu, Sweet et al. 2010). The TRP superfamily is involved in a variety of sensory processes like mechanosensation, osmosensation, thermosensation as well as olfaction, taste and pain perception (McKemy, Neuhausser et al. 2002, Voets, Talavera et al. 2005, Damann, Voets et al. 2008). TRP channels contribute to regulatory and homeostatic functions (Dimke, Hoenderop et al. 2011). TRP channel function based on the detection of environmental or endogenous stimuli and their processing (Montell 2001). This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues (Nilius and Owsianik 2011). TRP channels have a homo- tetrameric structure. A typical TRP channel protein contains six putative transmembrane segments (S1 to S6) with a pore-forming reentrant loop between S5 and S6. Intracellular amino and carboxyl termini are variable in length and consist of a variety of ankyrin domains (Owsianik, D'Hoedt et al. 2006). TRPs are expressed in a variety of non-neuronal tissues, central nervous system neurons, and are very important in the peripheral neurons (Mezey, Toth et al. 2000). They play an important role in the influx of Ca2+, which is essential for several physiological processes. TRP channels mediate the perception of taste, temperature and pain. Transient receptor potential vanilloid type 1 receptor (TRPV1) is a ligand-gated non-selective cation channel, which can be activated by heat (temperature above 43°C), low pH, and lipid molecules called endovanilloids (Tominaga, Caterina et al. 1998). In addition, the TRPV1 channel can be activated by capsaicin, the main ingredient of hot chili (Jancso, Jancso-Gabor et al. 1967, Szolcsanyi and Jancso-Gabor 1975, Caterina, Schumacher et al. 1997). TRPV1 are located on
15 Introduction small-diameter sensory nerves and play an important role in the nociception (Caterina, Leffler et al. 2000).
1.4 Neurotransmission and activity-dependent plasticity
Neurotransmission, or synaptic transmission, is the process by which neurons transmit information from one cell to another. Synaptic transmission is based on two different principles. The electric transmission occurs within a neuron or at gap junctions, which link the neurons to each other, to transmit signals (Sudhof and Malenka 2008, Süudhof 2008). The chemical transmission is based on different inhibitory and excitatory neurotransmitter systems and enables the transmission of information (Alexander, Mathie et al. 2008). Well-defined connections between neurons enable the integration of sensory inputs and the generation of appropriate responses (Huang and Thathiah 2015). Chemical synaptic transmission involves several steps and is a special process, which enables communication between neurons. Activation of neurons by external stimuli leads to calcium-dependent membrane depolarization via VGCCs (Simms and Zamponi 2014). VGCCs are the key molecules mediating calcium entry into electrically excitable cells in response to membrane depolarization (Berger and Bartsch 2014). These depolarization processes provoke the opening of additional ion channels and an action potential arise. The arriving action potential at the presynaptic synapse causes voltage- gated calcium channels (VGCC) to open. VGCCs open transiently to an incoming action potential to generate a large but temporally precise calcium influx (Weber, Wong et al. 2010). The opening of VGCCs induces an increase in intracellular calcium concentration which is important for depolarization-evoked release of neurotransmitters by exocytosis in the synaptic cleft (Wadel, Neher et al. 2007, Park and Kim 2009). Neurotransmitter molecules diffuse across the cleft and bind to specific receptors on the membrane of a postsynaptic cell. The binding of neurotransmitters leads to the opening of ion channels, which causes a change in the membrane conductance and membrane potential at the postsynaptic neuron. The action potential is transmitted.
The brain has an astonishing capacity to reorganize its structure and function to adjust to changing conditions. This ability of the central nervous system to remodel itself and to adopt a new functional or structural state in response to extrinsic and intrinsic factors is called activity- dependent plasticity (Ganguly and Poo 2013). The developing as well as the adult brain is able to reshape connectivity at both functional and structural levels based on behaviorally relevant experience (Ganguly and Poo 2013). Activity-dependent plasticity provides the basis for the development of neuronal circuits and endows the capacity to perform signaling processing underlying functions. The complex molecular and cellular machinery for the control of
16 Introduction neurotransmitter release and postsynaptic responses makes the synapse the most sensitive site for activity-induced modifications in the nervous system (Ganguly and Poo 2013). VGCCs are important mediators of depolarization-evoked release of neurotransmitters and to ensure the efficient coupling of calcium influx to rapid vesicle release, calcium channels must be located within the active zone of presynaptic nerve terminals (Wheeler, Randall et al. 1994, Simms and Zamponi 2014). During development, synapses are strengthened and weakened and during life changes and occur as a result of incoming information and learning mechanisms (Kandel 2001). AMPARs are rapidly transported into and out of synapses to weaken or strengthen their action. In some cases, trafficking of these receptors can occur in a matter of minutes (Lissin, Carroll et al. 1999). Short-time modifications play a role in adapting and extending the signal processing capability, whereas long-term modifications provides the basis for learning and memory (Malenka and Nicoll 1993, Abbott, Varela et al. 1997, Zucker and Regehr 2002, Malenka and Bear 2004, Kim and Linden 2007, Ganguly and Poo 2013). Long-term synaptic plasticity is defined by a long-lasting, activity dependent change in synaptic efficacy. Long-term plasticity can bidirectionally modify synaptic strength – either enhancing it via long-term potentiation or depressing it via long-term depression (Yang and Calakos 2013, Grasselli and Hansel 2014). These patterns of increase or decrease in synaptic transmission are mediated by glutamate receptors (Anwyl 2009, Shipton and Paulsen 2014). The most common form of LTP is triggered by activation of NMDARs and calcium-dependent protein kinase cascades which leads to an increase in the number of AMPARs at the synapses (Lisman and Raghavachari 2006, Opazo and Choquet 2011). NMDAR activation triggered the immobilization of surface AMPARs (Heine, Groc et al. 2008, Petrini, Lu et al. 2009).
Synaptic strength is based on changes in trafficking, subunit composition, and signaling of AMPAR and NMDAR as fundamental processes (Bliss and Collingridge 1993, Lu and Roche 2012). During synaptogenesis, the subunit composition and relative abundance of AMPAR and NMDAR adjusts as a crucial step in the establishment of a functional mature synapse (Bassani, Folci et al. 2013). In immature neurons, the synapse is characterized by a low AMPA/NMDA receptor ratio (Hall, Ripley et al. 2007). Maturation is marked by incorporation of specific NMDA receptors, which contain a NRA2 subunit, in the synapse and an increase of the AMPA/NMDA ratio (Crair and Malenka 1995, Isaac, Crair et al. 1997, Lu, Gonzalez et al. 2001, Hall, Ripley et al. 2007). The AMPARs density increases dramatically with cell maturation, whereas the NMDARs density is nearly constant (Schmidt-Salzmann, Li et al. 2014). Ionotropic glutamate receptors desensitize after activation. This rapid event works within milliseconds in AMPA/kainate receptors, but requires more time (ten to hundred milliseconds) in NMDA receptors (Armstrong, Jasti et al. 2006, Gereau and Swanson 2008, Schmidt-Salzmann, Li et al. 2014).
17 Introduction
Cell viability, apoptosis, neurite outgrowth inhibition and neurite degeneration as well as testing of functional parameters of neurotransmission are sensitive endpoints of neurotoxicity testing. As an example of the technique development, the in vitro investigation of neurite outgrowth inhibition is something to mention. Neuronal cell lines, human embryonic stem cell derived neuronal cells, and primary neuronal cultures have all been used in quantitative concentration- response assessments of chemical effects on neurite outgrowth and neurite degeneration (Radio and Mundy 2008, Harrill, Freudenrich et al. 2010, Radio, Freudenrich et al. 2010). This is of particular importance because it is known that changes in neurite growth leads to neurobehavioral deficits. These studies have demonstrated the ability of in vitro models to identify chemicals that specifically inhibit the process of neurite outgrowth and often these measurements were more sensitive than changes in cell number or measures of cytotoxicity. By using high throughput automated-microplate reading microscopes and high content image analysis these measurements are quite rapid and cost-effective (Harrill, Freudenrich et al. 2011).
Furthermore, the developmental nervous system is, based on its neurotransmitter-induced signals which regulate basic functions like proliferation, differentiation, and morphogenesis, especially vulnerable to environmental neurotoxins that target neurotransmitter receptors. A well-known examples are organochlorine pesticides, which are antagonists of the GABAA receptor (Eldefrawi and Eldefrawi 1987, Costa 1988). Organophosphates, pesticides and insecticides are known neurotoxins which impair the nervous system via different routes. Organophosphates, including the ortho-isomer of tri-cresyl phosphate, induced organophosphate induced delayed neuropathy (OPIDN) is a well-known and scientifically very well investigated mode of action (Abou-Donia 1981, Abou-Donia and Lapadula 1990, Glynn 2006, Ehrich and Jortner 2010). Since the last century different large-scale human intoxication events have been documented. Following these intoxication accidents and the identification of TCPs as the cause of neuropathy, large scale research was conducted to investigate OP and TCP poisoning and the underlying mechanisms of OPIDN.
1.5 Organophosphate-induced delayed neuropathy
Organophosphate-induced delayed neuropathy (OPIDN) is a neuropathy caused by acute or chronic exposure to organophosphorus compounds, including tri-ortho-cresyl phosphate (ToCP). The neuropathy occurs after 7 to 21 days after exposure (Abou-Donia and Lapadula 1990). OPIDN occurs in humans and sensitive animals like cats, cows (Smith 1930), lambs (Draper, James et al. 1952), sheeps, and water buffalo. Furthermore, adult hens were used as an animal model for OPIDN. Rats, mice (Smith 1930), and rabbits (Smith and Lillie 1931) did not show clinical signs of OPIDN, whereby acute effects of OPs were severe (Abou-Donia 1981).
18 Introduction
1.5.1 Chemistry of OPIDN inducing compounds
A full list of OPs causing OPIDN is provided at “The handbook of pesticide toxicology” or in the reviews of Abou-Donia and Johnson (Johnson 1975, Abou-Donia 1981). Not all OPs are capable of inducing OPIDN. So far, no specific structural features that positively identify a neuropathy- inducing OP have been recognized, but there are some structural-activity relationships which must be complied (Weiner and Jortner 1999, Ehrich and Jortner 2010). First, the phosphorous has to be in a pentavalent state. Second, the atom which is attached with the covalent bond to the phosphorous has to be an oxygen. A protoxicant with sulfur instead of oxygen can be oxidized to an active neurotoxicant. At last the compound needs one oxygen or amino bridge linking an R-group to the phosphorous. The major group producing OPIDN are phosphates (derivate of the phosphoric acid with four oxygen at the phosphorous), phosphoamides and phosphofluorides. Alkyl substitution of an ortho-position of phenyl phosphates increase the likelihood that a compound can be metabolized into a neurotoxicant (Ajwa, Ale et al. 2010). This mechanism works better with a methyl residue at ortho-position than with a long chain substitution (Abou-Donia 1981). In contrast, substitution of other sides decreased the neurotoxic potential. Finally, chirality can contribute to neurotoxicity (Nillos, Gan et al. 2010), where racemic mixtures tend to be less potent to induce OPIDN.
1.5.2 Neuropathology and clinical signs of OPIDN
The main symptoms of OP poisoning can be divided into three groups. The acute cholinergic syndrome with inhibition of the acetylcholine esterase (AChE), the intermediate syndrome, and the OPIDN (Abou-Donia 1993, Johnson, Jacobsen et al. 2000). The neuropathy is described as Wallarian-type degeneration and is associated with a progressive and symmetrical distal axonopathy (Funk, Henderson et al. 1994, Abou-Donia 2003, Wang, Liu et al. 2009). Morphological features of nerve fiber lesions, like the swelling of affected axons, lead to attenuation of their myelin sheaths (Jortner 2000). Primary lesions are a bilateral change in the distal level of axons and their terminals. Primarily, the longer myelinated central and peripheral nerve fibers break down. Distal regions of long peripheral nerve fibers in chickens and cats are similarly affected (Cavanagh 1954, Cavanagh 1964, Jortner and Ehrich 1987). Another morphological sign is axon swelling. Ultrastructural changes in axonal membrane systems are early morphologic events in OPIDN. Swollen axons contain disorganized cytoskeletal components and in vitro cytoskeletal changes occur after OP exposure (Abou-Donia, Lapadula et al. 1988, Abou-Donia 1993)
Clinical signs of OPIDN are based on the distal degeneration of axons in the nervous system. In humans, 8 to 14 days after exposure patients note tingling of the hand and feet, followed by
19 Introduction sensory loss in both, progressive muscle weakness, flaccidity of the distal skeletal muscles of the lower and upper extremities and ataxia (Glynn 2000, Ehrich and Jortner 2010, Emerick, DeOliveira et al. 2012). Hens show clinical signs after a delayed period (several days, similar to humans). Early signs in hens are abnormal foot placement, leg weakness and affected balance. Later incoordination when walking, wing flapping, and the use of wings for balance were observed. At last, the birds lose the ability to walk. Only hens over 55 days of age showed effects, meaning that there is an age-related susceptibility (Abou-Donia 1981, Funk, Henderson et al. 1994).
The underlying mode of action of OPIDN based on the inhibition and aging of neuropathy target esterase (NTE). In principle, the NTE-related mechanism leads to neuromuscular blockage and muscle weakness, resulting in paralysis (Emerick, Peccinini et al. 2010, Read, Li et al. 2010). NTE ha a conserved domain that includes a serine in the active site (Moser, Stempfl et al. 2000). This serine provides the OP binding site and the binding results in the inhibition of NTE (Read, Li et al. 2009). The inhibition of NTE has to be significant, e.g. about 70 %, and the interaction between OP and NTE has to be irreversible to develop OPIDN (Johnson 1990, Lotti 1991, Lotti, Moretto et al. 1993). NTE-binding OPs have a leaving group attached to a labile oxygen bound (Johnson and Glynn 1995), and by elimination of the leaving group the OP compound – the NTE complex becomes charged. This process of intramolecular rearrangement is known as “aging” (Johnson 1975, Johnson 1990, Johnson and Glynn 1995). Protection from OPIDN developed when reversible NTE inhibitors were given to hens before an OP caused the aging of the enzyme- OP complex (Lotti, Moretto et al. 1993).
1.6 Tri-cresyl phosphate
1.6.1 Structure of TCPs
The class of tri-cresyl phosphates belongs chemically to the group of organophosphorus compounds. The chemical substance group of tri-cresyl phosphates (TCPs) encompasses ten different stereo-isomers (Fig. 1.3) and is part of the substance class of organophosphates.
Three of these isomers are symmetric isomers, meaning that three equal cresol rings are esterified at the phosphate main body. These isomers are named tri-m, m, m-cresyl phosphate (TmCP,), tri-p, p, p-cresyl phosphate (TpCP) and tri-o, o, o-cresyl phosphate (ToCP), based on the position of the methyl group (meta-, ortho- or para- position) at the aromatic ring system. In addition to these symmetric isomers, there are further isomers with different cresol rings at the phosphate ester. TCP isomers, including ToCP, are commonly used as plasticizers, flame retardants, and as jet oil additives in aircrafts in various industries.
20 Introduction
Figure 1.3 Different stereo isomers of tri-cresyl phosphate. (A), (B) and (C) symmetric isomers, (D) - (J) isomers with different methyl substituted cresol rings at the phosphate ester. 1.6.2 Metabolism of TCPs
The ToCP metabolism has been well studied in rats (Casida, Eto et al. 1961, Abou-Donia, Nomeir et al. 1990, Somkuti and Abou-Donia 1990), hens (Abou-Donia, Suwita et al. 1990) and cats (Nomeir and Abou-Donia 1986). Uptake and distribution of radioactive labeled ToCP takes place rapidly and the highest radioactivity was found in the bile, gall bladder, kidney and liver. ToCP has a biological half-life time of 12 h (Somkuti and Abou-Donia 1990), thereby different metabolites are generated. After oral application of ToCP the metabolites di-o-cresylphosphate, mono-o-cresylphosphate, o- cresol, o-hydroxybenzylalcohole, o-hydroxybenzylaldehyde and the cyclic salignin-o-cresylphosphate (CBDP) were excreted in the urine of rats and cats. The initial step of the ToCP metabolism is the oxidation of one methyl residue at the cresol ring (Fig. 1.4). This oxidation process, which is catalyzed by different enzymes including P450 in the
21 Introduction liver, creates an o- hydroxybenzylalcohole derivate (Eto, Casida et al. 1962). When ToCP is activated via an intramolecular cyclization, CBDP is generated. The hydroxyl-group at the intermediate reacts with the phosphate ester in a substitution reaction by elimination of a cresol residue. The formation of the intramolecular ring is only possible with a methyl group in ortho- position at the aromatic ring system. The metabolite CBDP is neurotoxic and discussed as being five times more toxic than ToCP (Bleiberg and Johnson 1965). The final product of the metabolism is o-hydroxybenzoic acid.
Figure 1.4 Metabolism of ToCP. As toxic metabolite the cyclic salignin-o-cresylphosphate (CDP) was generated via an intramolecular cyclization. The hydroxyl-group at the intermediate reacts by elimination of a cresol residue with the phosphate ester in a substitution reaction was generated via intracellular cyclization. Scheme of the metabolism modified from Somkuti and Abou-Donia, 1990.
The TpCP metabolism was investigated in rats after oral application of 14C-TpCP (Kurebayashi, Tanaka et al. 1985). As main urinary, biliary, fecal, and expiratory metabolites, p-hydroxybenzoic acid and di-p-cresylphosphate were identified. Additional metabolites were p-carboxyl phenyl- p-cresylphosphate, di- p-carboxyl phenyl phosphate, and di-p-carboxyl phenyl-p- cresylphosphate (Fig. 1.5). The meta- and para-isomers of the TCPs have a steric hindrance, which makes cyclization of the molecule impossible. There is no data in the literature available for the metabolism of TmCP.
22 Introduction
Figure 1.5 Metabolism of TpCP. TpCP is metabolized via P450 enzymes to mono-p-cresyl phosphate as end product of the reaction. Scheme modified from Kurebayashi et al., 1985.
1.6.3 Toxicity of TCPs
The toxicological, physical and physiological properties of TCPs vary depending on the isomer and are reducible to the position of the methyl substituent. ToCP has the highest neurotoxic potential among the TCPs (Casida, Eto et al. 1961). ToCP leads to a decrease in fertility in males and females (Carlton, Basaran et al. 1987, Chapin, George et al. 1988, Morrissey, Schwetz et al. 1988). Cyclic salignin-o-cresylphosphate (CBDP) is one cause of the reduced fertility (Burka and Chapin 1993). Characteristics of TCP mixtures change depending on the composition. The toxicity is directly dependent on the amount of ToCP in the mixture (Henschler 1958). However, even for these well-known neurotoxins no complete risk assessment had been conducted, particularly in terms of the low-level exposure and chronical low-level exposure conditions. TCPs are converted by cytochrome P450 enzymes to more hydrophilic compounds that often indicate a higher toxic potential than the main compound. The different toxic potential of ToCP may arise from the fact that ToCP can cyclize to the toxic, CBDP through the intramolecular ester exchange of the hydroxymethyl-ToCP (Kurebayashi, Tanaka et al. 1985). ToCP and its metabolite CBDP
23 Introduction have a high affinity to the NTE and AChE. ToCP is known to cause OPIDN and in accordance with the OECD guideline 418, ToCP is used as positive control compound for OPIDN testing. The other symmetric isomers TmCP and TpCP are less toxic, which was assumed based on the fact that the affinity of these other isomers for NTE and AChE is lower (Henschler 1958, Henschler and Bayer 1958). TCPs have a high molecular weight and low vapor pressure, wherefore the dermal absorption seems to play a more important role than intake by inhalation (Tabershaw, Kleinfeld et al. 1957). The dermal resorption of ToCP was investigated by using cat as model organisms by using radioactive labeled ToCP (Nomeir and Abou-Donia 1986). Based on varying temperature and pressure conditions in the cabin air of aircraft, inhalation plays an important role and is particularly associated with aerotoxic syndrome. However, detailed data ise not available regarding the inhalation of TCPs.
1.6.4 Human TCP poisoning
Since the end of the 19th century, numerous cases of ToCP poisoning due to accidental contamination of drink and food have been reported (Inoue, Fujishiro et al. 1988). In 1899, initially six cases of polyneuropathy out of 41 cases of pulmonary tuberculosis, treated with phosphor-cresote, were reported and later proved to contain 15 % ToCP. Between 4,000 and 50,000 residents with ToCP intoxication in the USA were reported in the 1930s. During prohibition, a tri-cresyl phosphate containing preparation was used as an alcohol substitute, the so-called “Ginger Jake” (Morgan and Penovich 1978). This was the most dramatic outbreak of paralysis characterized by muscle weakness and ataxia. Since the 1930s, other situations of exposure to TCPs were identified. For example, in 1959 a great outbreak of poisoning occurred in Morocco. About 10,000 Moroccans were intoxicated by ToCP when intake jet gear oil contaminated cooking oil (Smith and Spalding 1959). Another intoxication took place in Italy where shoe-manufacture workers were exposed to TCPs, according to the use of organophosphates as plasticizer (Inoue, Fujishiro et al. 1988).
1.7 The aerotoxic syndrome
TCPs and specially ToCP awakened the public opinion for the second time in relation to the “aerotoxic syndrome” (AS). Temporarily, this topic was part of different independent magazines and newspapers like “Frankfurter Allgemeine Zeitung” and “Die Zeit”, as well as in radio and television. In several cases, emergency landing of aircrafts were reported after so-called “fume events”. The cause of the contamination, every time associated with ToCP intoxication. The described numbers of cases suggest TCP modes of action in addition to OPIDN, which were related to disruption of central nervous system processes.
24 Introduction
The term “aerotoxic syndrome” was coined by Chris Winder and Jean Balouet and describe a diffuse neurobehavioral disorder (Winder and Balouet 2002), which is characterized by symptoms and clinical signs associated with acute impairment of nervous system processes (Liyasova, Li et al. 2011). Cognitive impairment like headache, confusion, disorientation, and tunnel vision were described as symptoms of AS after short time exposure. Long-term exposure caused dizziness, memory impairment and deficits, and lack or limitation of coordination as well as respiratory, gastrointestinal, cardiovascular, skin, and irritation symptoms (C 2005, Winder and Michaelis 2005, Schopfer, Furlong et al. 2010). These symptoms are related to the central nervous system and were caused by mechanisms different from those related to OPIDN. This topic refers to and discusses possible health damage and injury provoked by breathing of contaminated airliner cabin air (Schopfer, Furlong et al. 2010, de Boer, Antelo et al. 2015). The cabin air is supplied by an on board oxygen generating system that uses bleed air from the jet engine. The air passes through the jet engine to be heated, before it is enriched with oxygen. Bleed air, which might be contaminated with jet gear oil, is transferred unfiltered into the cabin and represents a vast health risk (van Netten 1999, van Netten and Leung 2001, Ke, Sun et al. 2014). As one possible toxic compound TCP come into question, but there are several more potential risk compounds (Nagda and Rector 2003, Denola, Hanhela et al. 2011).
TCPs were used as additives and lubricants in jet gear oil and have been proposed as a causative agent of AS based on their known neurotoxicity. Research was conducted to identify safer additives for jet gear oil and the amount of ToCP was reduced (Bakand, Winder et al. 2005). Some studies investigated flight crews concerning the exposure to TCPs and other OPs (Solbu, Daae et al. 2011, Schindler, Weiss et al. 2013) and discussed the health risk of TCPs (de Ree, van den Berg et al. 2014). The results were discussed controversially in scientific literature and more research is required to investigate new modes of action of TCPs in addition to OPIDN, which might be related to the symptoms of AS.
25 Introduction
1.8 Objectives
Organophosphates, including TCPs, are well-known neurotoxins which cause organophosphate- induced delayed neuropathy by the inhibition of the neuropathy target esterase in sensitive animals and humans (Abou-Donia 1981, Johnson and Glynn 1995, Emerick, Peccinini et al. 2010, Emerick, DeOliveira et al. 2012). The underlying mechanism of OPIDN had already been researched in depth (Abou-Donia and Lapadula 1990, Ehrich and Jortner 2010), while additional modes of action of these organophosphorus esters were completely unknown. TCPs were discussed as causative agents in the context of aerotoxic syndrome. The described symptoms indicate an impairment of central nervous system processes (Liyasova, Li et al. 2011) and the related neurobehavioral deficits can hardly be explained by the OPIDN mechanism.
The aim of this thesis is the detection of new adverse effects of TCPs by the disruption of different biological pathways. TCPs, including ToCP, might impair the function of central nervous system neurons. These possible effects of TCPs have not been addressed previously neither in vivo nor in vitro. The present study investigates for the first time TCP-induced alterations of central nervous processes. Three different neurotoxic structural and functional endpoints are (1) cell viability, (2) neurite outgrowth inhibition and alteration of neurite morphology as well as (3) functional processes of neurotransmission and were addressed to recognize all levels of neuronal impairment. The implementation of a multifaceted approach in neurotoxicological studies is one main objective in the context of the paradigm shift in toxicology and the AOP framework. The selection of these different endpoints was motivated by the expectation of an unequivocal concentration-dependency. ToCP is discussed in the literature as the most toxic isomer (Henschler 1958, Casida, Eto et al. 1961), based on the assumption that the NTE affinity of the other isomers is lower and as a consequence that these compounds are less toxic. ToCP might have a higher neurotoxic potential than the others to disrupt the neuronal pathways. Neuronal plasticity and neurotransmission depends on a stable neuronal network and the connectivity between the neurons. TCPs might be capable of the perturbation of (1) the density, integrity and complexity of neurite networks and (2) calcium-dependent membrane depolarization via voltage-gated calcium channels and signaling by changing the permeability of ionotropic neurotransmitter receptors, especially glutamatergic receptors. In this thesis, TCP effects on signals evoked by the excitatory neurotransmitter glutamate were studied as well as effects on the calcium-dependent depolarization processes of the neurons. TCPs can either have a direct receptor-mediated action or cause a reduction of glutamate sensitivity by secondary processes. The mechanisms underlying the altered glutamate sensitivity were studied by different biochemical and functional approaches.
26 Material and methods
2 Material and methods
2.1 Material
2.1.1 Chemical Reagents and Kits
Table 2.1 Chemical Reagents and Kits
Chemical Reagent Company α-amino-3-hydroxy-5-methyl-4-isoxazole Tocris, Bristol, United Kingdom propionic acid hydrobromide (AMPA) 4’, 6-diamidino-2-phenylindole (DAPI) Gibco, Darmstadt, Germany B 27 serum free supplement Gibco, Darmstadt, Germany
Calcium chloride (CaCl2) Carl Roth, Karlsruhe, Germany Capsaicin Tocris, Bristol, United Kingdom Cell Titer Blue® Assay Reagent Promega, Mannheim, Germany Collagenase Sigma Aldrich, Hamburg, Germany Cresyl salignin phosphate Gift from O. Lockridge Deoxyribonuclease I (DNAse) Sigma Aldrich, Hamburg, Germany DEPC treated water Invitrogen, Darmstadt, Germany D-Glucose Fluka, Buchs, Germany Dimethyl sulfoxide (DMSO) Carl Roth, Karlsruhe, Germany
Disodium hydrogen phosphate (Na2HPO4) AppliChem, Darmstadt, Germany DMEM F-12 glutamax Gibco, Darmstadt, Germany Ethanol, 70 % denatured Merck, Darmstadt, Germany Ethanol, absolute Merck, Darmstadt, Germany Fluor Preserve™ Reagent Calbiochem, Darmstadt, Germany Fetal bovine serum (FBS) Gibco, Darmstadt, Germany Fura-2/AM Tocris, Bristol, United Kingdom Gentamycin 50 µg/ml Gibco, Darmstadt, Germany
27 Material and methods
Glycine Merck, Darmstadt, Germany Hanks Balanced Salt Solution (HBSS) Gibco, Darmstadt, Germany HCl (37 %) Carl Roth, Karlsruhe, Germany HEPES Carl Roth, Karlsruhe, Germany innuPREP RNA mini Kit analytik jena, Jena, Germany iSkript cDNA Synthesis Kit Bio-Rad, Munich, Germany L-glutamic acid-monosodium salt monohydrate Sigma Aldrich, Hamburg, Germany
Magnesium chloride (MgCl2) Carl Roth, Karlsruhe, Germany MK801 malate Tocris, Bristol, United Kingdom Neurobasal medium Gibco, Darmstadt, Germany Normal donkey serum Dianova, Hamburg, Germany Paraformaldehyde (4 %) Carl Roth, Karlsruhe, Germany Penicillin/Streptomycin PAN Biotech, Aidenbach, Germany Phosphate buffered Saline (PBS) Gibco, Darmstadt, Germany Poly-D-lysine Sigma Aldrich, Hamburg, Germany Poly-L-lysine (PLL) Sigma Aldrich, Hamburg, Germany Potassium chloride (KCl) Carl Roth, Karlsruhe, Germany
Potassium hydrogen phosphate (KH2PO4) Merck, Darmstadt, Germany RNA protect Quiagen, Hilden, Germany Sodium chloride (NaCl) Carl Roth, Karlsruhe, Germany Sodium hydroxide (NaOH) Carl Roth, Karlsruhe, Germany Soybean trypsin inhibitor Gibco, Darmstadt, Germany Stable glutamine PAN Biotech, Aidenbach, Germany TaqMan® Assay Mix Applied Biosystems, Karlsruhe, Germany TaqMan® Universal Master Mix II Applied Biosystems, Karlsruhe, Germany Tri-cresylphosphate mixture (TCP mix) TCl Chemicals, Eschborn, Germany
28 Material and methods
Tri-meta-cresylphosphate (TmCP) TCl Chemicals, Eschborn, Germany Tri-ortho-cresylphosphate (ToCP) TCl Chemicals, Eschborn, Germany Tri-para-cresylphosphate (TpCP) TCl Chemicals, Eschborn, Germany Triton X-100 Carl Roth, Karlsruhe, Germany Trypane blue Sigma Aldrich, Hamburg, Germany Trypsin PAN Biotech, Aidenbach, Germany ZK 200775 Tocris, Bristol, United Kingdom
2.1.2 Primary Antibodies
Table 2.2 Primary antibodies
Antibody Origin Company Dilution Polyclonal anti-β-III-tubulin Mouse HISS, Freiburg, Germany 1:500 (IF) (MMS-435P) Polyclonal anti-β-III-tubulin Rabbit HISS, Freiburg, Germany 1:2000 (IF) (PRB-435P)
2.1.3 Secondary Antibodies
Table 2.3 Secondary antibodies
Antibody Origin Company Dilution Anti-mouse DylightTM649 Donkey Dianova, Hamburg, Germany 1:500 (IF) Anti-rabbit DylightTM488 Donkey Dianova, Hamburg, Germany 1:500 (IF)
29 Material and methods
2.1.4 Consumables
Table 2.4 Consumables
Consumable Company Biosphere Filter Tip, 1000 µl, 200 µl, 100 µl, 10 µl Sarstedt, Nümbrecht, Germany Black 96 well plate Greiner Bio, Frickenhausen, Germany Cell Scraper, 25 cm Sarstedt, Nümbrecht, Germany Cell strainer, 70 µm Falcon, Durham, USA Costar® 96 well plate Sigma Aldrich, Hamburg, Germany Cover glass (Ø = 14 mm) Menzel, Braunschweig, Germany Tube, 15 ml Sarstedt, Nümbrecht, Germany Tube, 50 ml Sarstedt, Nümbrecht, Germany Gentle Skin Classic gloves Meditrade, Kiefersfelden, Germany MicroAmp Optical 96 well Reaction Plate Applied Biosystems, California, USA Multiwell plate Sarstedt, Nümbrecht, Germany Parafilm Cole-Parmer, Kehl, Germany Petri dishes, 3.5 cm, 10 cm Falcon, Durham, USA Pipette tips, 1000 µl, 200 µl, 20 µl Sarstedt, Nümbrecht, Germany RNaseZap® RNase Decontamination Solution Ambion, Thermo Fischer Scientific, Waltham, USA SafeSeal 0.5 ml microtube Sarstedt, Nümbrecht, Germany SafeSeal 1.5 ml microtube Sarstedt, Nümbrecht, Germany SafeSeal 2.0 ml microtube Sarstedt, Nümbrecht, Germany Serological pipette, 25 ml, 10 ml, 5 ml Sarstedt, Nümbrecht, Germany Sterile filter, 0.20 µm Falcon, Durham, USA SuperFrost Plus® microscopic slides Thermo Scientific, Braunschweig, Germany Vacuum filtrations flask, 0.22 µm, 250 ml Sarstedt, Nümbrecht, Germany
30 Material and methods
2.1.5 Technical Equipment
Table 2.5 Technical equipment
Equipment Company Application system Harvard Apparatus, Warner Instruments Autoclav, 5075 ELV Tuttenauer Balance Mettler Toledo Camera Primo Vert AxioCam ICm 1 CCD Camera Array-Scan VI hCS Reader Hamamatsu OCRA-ER CCD Camera ImageXpress sCMOS ZYLA 4.2 CCD Camera Leica DMI 6000 DFC 360 FX Centrifuge Biofuge 15 Heraeus Instruments Centrifuge Megafuge 1.0 R Heraeus Instruments Centrifuge mini spin plus Eppendorf
CO2 Incubator, APT Line C150 Binder Freezer, - 20 °C Bosch Freezer, - 80 °C Hera Freeze Freezer, + 4 °C Liebherr Lamina flow hood, hera Safe hS 18/85 Heraeus Instruments Magnetic stirrer, IKAMAG RCT IKA Microscope, Array-Scan VI hCS Reader Thermo Scientific Microscope, Eclipse TS 100 Nicon Microscope, ImageXpress Molecular Devises Microscope, Leica DMI 6000 Leica (Software LASAF, Leica) Microscope, Primo Vert Zeiss (Software ZEN, Zeiss) pH meter, CG 842 Schott Pipette, 8-Kanal Integra Pipettes, Research and Reference Eppendorf 5000 µl, 1000 µl, 200 µl, 100 µl, 20 µl, 10 µl polytetrafluoroethylene measuring chambers Self-build Precision balance Sartorius Real Time PCR System 7500 Applied Biosystems Spectrometer, Infinite M200 PRO Tecan
31 Material and methods
Spectrometer, NanoDrop 2000 Thermo Scientific Thermo shaker peqlab Thermo shaker Grant-Bio Vacuum pump Diaphragm Vacuum Pump, Vacuumbrand Vortex, Vortex-Genie 2 Bender&Hobein Water bath, GFL 1083 Gesellschaft für Labortechnik Water purification system Elga
2.2 Methods
2.2.1 Animals
All experiments involving animals were performed in accordance with the European Union Community Council guidelines. All measures were taken to reduce animal suffering to a minimum. CD1 mice from Charles River Laboratories (Sulzfeld, Germany) or Janvier Labs (France) were housed in standard cages with standard laboratory chow and drinking water ad libitum. In the animal facility a programmed day and night rhythm of 12 hours was present. The animals were handled and cared by a certified animal keeper.
2.2.2 Preparation and cell culture of primary mouse cortical neurons
Pregnant CD1 mice ordered from Charles River/Janvier Labs or from timed mating (from own breeding) were used for cell isolation. Primary mouse cortical neurons were isolated from mouse embryos at embryonic day E 16.5. After anaesthetization with CO2, dams were sacrificed by cervical dislocation. The abdomen was opened and embryos were dissected from uterine tissue. A quick decapitation of each embryo was followed by the opening of the embryo’s skull. The brain was removed and transferred into a petri dish filled with ice-cold Hanks Balanced Salt Solution (HBSS-/-) (Tab. 2.6). On ice, the hemispheres were separated, and the meninges removed. The segregated cortices were transferred to fresh ice-cold HBSS-/-. After washing with HBSS-/-, cortices were trypsinized for 10 min at 37 °C. The trypsin reaction was stopped by the addition of soybean trypsin inhibitor (0.25 mg/ml) and 0.01 % (v/v) DNase. Cortices were then gently triturated with fire polished glass pipettes with different diameter. The cell suspension was centrifuged for 5 min at 1000 rpm. The cell pellet was resuspended in neurobasal media supplemented with 2 % (v/v) B27, 0.5 mM stable glutamine, and 0.1 % (v/v) gentamycin. Cells were seeded on poly-L-lysine (PLL) coated glass cover slides (Ø = 14 mm,
32 Material and methods
Menzel, Germany) or plastic surfaces at a density of 40.000 cells/cm2 and cultured at 37 °C in a humidified atmosphere containing 5 % CO2. The half of media was changed every four days.
Table 2.6 Hanks Balanced Salt Solution
Component Concentration KCl 5.3 mM
KH2PO4 0.44 mM
NaHCO3 4.16 mM NaCl 137.93 mM
Na2HPO4 0.33 mM D-glucose 5.55 mM Phenol red 0.026 mM
2.2.3 Preparation and cell culture of primary rat cortical neurons
Pregnant Wistar rats were ordered from Charles River/Janvier Labs and used for cell isolation. The rat cortical neurons were isolated from rat embryos at embryonic day E 18.5. The isolation protocol followed the instruction of the isolation protocol for mice (see 1.2.2) with minor changes. The rat cortices were trypsinized for 20 min at 37°C, before trituration and centrifugation processes. Cells were seeded on poly-D-lysine (PDL) coated glass cover slides (Ø = 14 mm, Menzel, Germany) or plastic surfaces at a density of 40.000 cells/cm2 and cultured at 37 °C in a humidified atmosphere containing 5 % CO2. For rat pCNs the media with the same composition as for the mouse pCNs were used for culture, with half of media exchanged every four days.
2.2.4 Preparation and cell culture of mouse dorsal root ganglia neurons
Dorsal root ganglia neurons (DRG) were prepared from CD1 mice at postnatal day 5. Mice were sacrificed by decapitation. The spinal column was opened, the spinal cord removed and the dorsal root ganglia dissected. The ganglia were washed in PBS-/- and collected in DMEM media supplemented with 1 % (v/v) penicillin and streptomycin. After collection the DRG were disrupted, incubated with 0.025 % collagenase for 45 min, dissociated by using fire-polished glass pipettes, and centrifuges at 200 g for 4 minutes. The cell pellet was resuspended in fresh F-12 media supplemented with 10 % (v/v) fetal bovine serum, and 1 % penicillin/streptomycin. DRG were seeded in 24 well plates on poly-L-lysine (PLL) coated glass cover slides (Ø = 14 mm, Menzel, Germany) in 50 µl media. After 1 h adhesion time 450 µl media were added to each well and neurons cultured at 37 °C in a humidified atmosphere containing 5 % CO2.
33 Material and methods
2.2.5 Treatment conditions
PCNs were treated with TCP isomers, the TCP mixture or the metabolite CBDP in different experimental designs for the investigation of the cytotoxicity via the resazurin-based CellTiter- Blue® assay, the investigation of neurite outgrowth inhibition or neurite degeneration as well as the investigation of alterations in the neurochemical processes by using fluorescence-based live- cell calcium imaging. TCP isomers were diluted in DMSO and used from stock solutions 1:1000 diluted in medium. The maximum DMSO concentration was 0.1 % in media. CBDP was diluted in acetonitrile and used from stock solutions 1:1000 diluted in media. The maximum acetonitrile concentration was 0.1 % in media. As solvent control for the TCP isomers as well as the TCP mixture 0.1 % (v/v) DMSO was used, while for the metabolite CBDP 0.1 % acetonitrile as solvent control was used. The compounds were tested in concentrations between 1 nM and 10 µM for incubation times between 1 h and 24 h. Long-term ToCP treatment was conducted over seven days in the concentration range between 10 pM and 100 nM. For recovery experiments the neurons were cultured under standardized cell culture conditions for 6 days in vitro, followed by a treatment with different ToCP concentrations or 0.1 % (v/v) DMSO as control for 24 h. After these treatments, neurons were either directly measured in calcium imaging experiments or the ToCP containing media were exchanged. The pCNs were measured after a recovery time of 24 or 48 h.
To investigate acute effects of TCPs on glutamate receptors two different types of measurements were conducted. As control untreated neurons were stimulated three times in succession with 30 µM glutamate to analyze if and how the glutamate-induced response amplitudes were changed by repeated stimulation. During a blocker experiment the neurons were first stimulated with glutamate. Before the second stimulus TCP in either concentration was applied constantly in the measuring chamber to perfuse the cells for one minute, followed by co-application of TCP and glutamate. After wash out of the TCP, a third application of glutamate was performed to investigate if the block of the glutamate-induced response was a reversible process. The selected micromolar concentration range used for these initial studies may appear high, but in this approach, the neurons were only exposed to the respective solution for a very limited time. Therefore, an immediate dilution of the TCP-containing solution occurred.
2.2.6 CellTiter-Blue® cell viability assay
Cell viability of mouse pCNs after neurotoxin treatment was determined by the fluorometric, resazurin-based CellTiter-Blue® (CTB) assay in black 96 well plates according to the manufacturer’s instructions. In brief, cells were seeded at a density of 40.000 cells/cm2 and
34 Material and methods grown for 24 h or six days in vitro (DIV). Cells were then treated with different concentrations of ToCP, CBDP or TCP isomers for 20 h, followed by 4 h incubation with CellTiter-Blue® reagent. The sub chronical ToCP treatment began after 24 h with different ToCP concentrations for seven days, with media exchange every two days. At the last day a 4 h incubation with CellTiter-Blue® reagent followed. The fluorescence for both treatment paradigms was measured at 540/595 nm with the Tecan Infinite M200 PRO plate-reader and the i-control software (version 1.7.1.12). Cell viability was calculated after background subtraction and expressed as percentage of control.
2.2.7 Immunocytochemistry
Mouse cortical neurons were treated with different concentrations of ToCP, CBDP or TCP isomers for 24 h at DIV 2 or DIV 7, washed twice with phosphate buffered saline PBS-/- (Tab. 2.7), and fixed with 4 % (v/v) paraformaldehyde (PFA) for 15 min. After two washing steps with PBS- /-, the cell membranes were permeabilized with 0.1 % (v/v) Triton X-100 in PBS-/- for 10 min. After two washing steps with PBS-/-, the nonspecific binding sites were blocked by using 5 % (v/v) normal donkey serum in PBS-/- for 1 h. Cells were incubated with the primary antibody (e.g. polyclonal mouse anti-β-III-tubulin diluted in 1 % normal donkey serum 1:500) for 2 h at room temperature (RT). After washing with PBS-/- followed the incubation with the secondary antibody (e.g. donkey anti mouse DylightTM649 (1:500 in 1 % normal donkey serum)) for 30 min in the dark at RT. As a last step in the staining protocol, nuclei were stained with DAPI (4’, 6-diamidino- 2-phenylindole) diluted 1:10.000 in PBS-/- for 30 min, followed by washing steps with PBS-/- and distilled water. Coverslips were mounted on microscopic glass slides with Fluor Preserve™ Reagent. Images were analyzed with the Leica DMI 6000 B microscope, the monochrome CCD camera DFC 360 FX, and the Leica LAS AF 600 Software (version 2.6.0.7266).
Table 2.7 Phosphate buffered saline (PBS-/-)
Component Concentration
KH2PO4 1.5 mM KCl 2.7 mM
Na2HPO4 8.1 mM NaCl 137 mM pH 7.4 (NaOH/HCl)
35 Material and methods
2.2.8 Quantitative analysis of neurite outgrowth and neurite degeneration
Cells were treated with different ToCP concentrations and incubated for 24 h under standard cell culture conditions (37 °C and 5 % CO2), either after 24 h adhesion time for neurite outgrowth inhibition or after 6 days in vitro for neurite degeneration. Cells were stained as described above with polyclonal rabbit anti-tubulin, donkey anti rabbit DyLightTM488, and DAPI. After the staining, fixed cells were send to the group of Prof. Dr. Marcel Leist at the University Konstanz to analyze the neurite area. To measure the neurite area, the cell culture plates were loaded into an automated microplate-reading microscope (Array-Scan HCS Reader, Cellomics) with a Hamamatsu OCRA-ER camera. For each well, 10 randomly selected fields of view were scanned in two channels (10-fold objective, 2x2 pixel binning). In channel 1, DAPI was detected with an excitation/emission wavelength of 365 ± 50 nm/535 ± 54 nm to identify nuclei based on size, area, shape, and intensity, whereas β-III-tubulin was detected with an excitation/emission wavelength of 474 ± 40 nm/535 ± 45 nM in channel 2. The nuclear outlines were expanded in each direction to define a virtual cell soma area (VCSA). All β-III-tubulin-positive pixels of the field were defined as neuronal cellular structures (NCSs). In an automatic calculation, the VCSAs were used as a filter in the β-III-tubulin channel and subtracted from the NCS. The remaining pixels (NCS - VCSA) in the β-III-tubulin channel were defined as neurite area.
Cells were treated with different TCP isomers or the metabolite CBDP at various concentrations and incubated for 24 h under standard cell culture conditions (37 °C and 5 % CO2), either after 24 h adhesion time for neurite outgrowth inhibition (DIV 2) or after 6 days in vitro for neurite degeneration (DIV 7). Cells were stained as described above with polyclonal rabbit anti-tubulin, donkey anti rabbit DyLightTM488, and DAPI. To measure the neurite area, the cell culture plates were loaded into an automated microplate-reading microscope (Molecular Devices) with a sCMOS ZYLA 4.2 camera. For each well, one field of view was scanned in two channels (10-fold objective). In channel 1, DAPI was detected to identify nuclei based on size, area, shape, and intensity, whereas β-III-tubulin was detected in channel 2. Different neurite outgrowth and neurite degeneration parameters were calculated (Software: MetaXpress, version 5.3.01).
2.2.9 Quantitative analysis of neurite morphology
IMARIS Software (Bitplane, Zurich, Switzerland) was used to analyze details of neurite morphology. Based on fluorescence images of ToCP-treated and control neurons, a reconstruction of neuronal structures was conducted by using software parameters. After reconstruction the IMARIS software enables the quantification of neurite length and neurite
36 Material and methods diameter. In brief, for ToCP-treated and control cells the neuronal cytoskeleton was immunostained by using polyclonal mouse anti-β-III-tubulin antibody and donkey anti mouse DylightTM649 as secondary antibody (Fig. 2.1). The nuclei were counterstained with DAPI.
Figure 2.1 Quantitative analysis of neurite morphology with the IMARIS software tool. β-III-tubulin stained mouse pCNs (ToCP treated and control neurons) (A) and fluorescence based reconstruction of neurite structure (B). Scale bar: 50 µM
For the analysis, a total of 1227 neurons (control: n = 240, 1 nM: n = 215, 10 nM: n = 212, 100 nM: n = 206, 1 µM: n = 216 and 10 µM: n = 141 neurons) were reconstructed with the IMARIS software tool (Fig. 6B). For each exposure condition, five images of stained cells with an average cell number of 13 were analyzed for three independent biological replicates.
2.2.10 Network Formation Assay
The network formation assay (NFA) allowed the observation of neurite outgrowth inhibition or neurite degeneration of established networks by neurotoxins over time (Frimat, Sisnaiske et al. 2010, Hardelauf, Waide et al. 2014). By micro contact printing a hexagonal pattern of adhesion notes (Ø 70 µm) with connecting channels (2 µm width, 100 µm length) were generated. For mouse pCNs a layer of poly-L-lysine was stamped on a cell repellent surface of polyethylenglycole (PEG). A NFA chip consists of four areas with each nine fields of 202 adhesion points. Mouse pCNs were seeded on the NFA chips. After an adhesion time of 24 h neurons on NFA chips were treated with different ToCP concentration (100 µM, 30 µM, 10 µM, 3 µM, 1 µM and 0.3 mM) or 0.1 % (v/v) DMSO. After 24 h and then every 24 h during four days the connecting neurites were counted and the cpn was calculated. Experiments of neurite outgrowth inhibition were conducted.
37 Material and methods
2.2.11 Calcium Imaging
Calcium imaging studies were performed with mouse primary cortical neurons after different time in culture as well as with rat pCNs, and DRG neurons. Neurons were measured either with TCP isomer treatment or direct application of TCP isomers without pretreatment. Neurons were incubated with 3 mM Fura-2/AM at 37 °C and 5 % CO2 for 35 to 45 min. Glass slides were transferred into inherent polytetrafluoroethylene (PTFE) measuring chambers filled with standard extracellular buffer (Tab. 2.8). The measuring chamber was mounted on a Leica DMI 6000 B microscope equipped with a 20-fold fluorescence-optimized objective (Leica). Fura- 2/AM loaded cells were exited intermittently at wavelengths of 340 nm (80 ms) and 380 nm (20 ms). Emitted light of 510 nM wavelength was detected with the monochrome CCD camera DFC 360 FX. Imaging data were analyzed with the Leica LAS AF 600 Software (versions 2.6.0.7266 and 4.0.0.11706). Changes in intracellular calcium levels were measured as the ratio of emitted light intensity (510 nM) resulting from illumination at 340 nm and 380 nm (f340/f380). For stimulus application a hydrostatic pressure-driven 8-in-1 application system was used. The application cannula was placed in close proximity of the cells to enable precise substance application (Fig. 2.9). Before the first substance application in an experiment takes place the calcium basal level were determined for 20 seconds. At the end of every experiment, neurons were stimulated with a short (5 s) pulse of depolarizing high-K+ extracellular assay buffer containing 45 mM KCl (Tab. 2.9). Time series of the imaging data were exported as Microsoft™ Excel™ compatible files and responses and their respective amplitudes were Figure 2.2 8-in-1 hydrostatic pressure driven application system. The application cannula enables precise substance application direct to the cells. calculated from baseline using Excel™ macros. A threshold criterion of “amplitude plus 4 times standard deviation (4 sigma)” was used to detect such responses from the unspecific fluctuation of the f340/f380 values (baseline variation). The percentage of glutamate responders and percentage of KCl-responsive neurons were given as responders among all identified cells in the view field. Based on the ratio (f340/f380) at the beginning of the measurement (10 s) the basal calcium level was
38 Material and methods calculated. For each cell baseline calcium levels were calculated as the average of 20 consecutive time points.
Table 2.8 Standard extracellular assay buffer
Component Concentration NaCl 140 mM KCl 5 mM
CaCl2 2 mM
MgCl2 1 mM HEPES 10 mM pH 7.4 (NaOH/HCl), 300 mOsmol (Glucose)
Table 2.9 High K+ assay buffer
Component Concentration NaCl 100 mM KCl 45 mM
CaCl2 2 mM
MgCl2 1 mM HEPES 10 mM pH 7.4 (NaOH/HCl), 300 mOsmol (Glucose)
2.2.12 RNA Isolation
ToCP treated and control cells were collected in 500 µl RNA protect solution. RNA isolation of primary cultures of mouse cortex neurons was performed using innuPREP mini kit following manufacturer´s introductions. After cell lysis, the selective removing of genomic DNA took place. After different washing steps and centrifugation steps the RNA pellet was eluted in 30 µl RNase- free water. The RNA integrity and the RNA concentration were determined spectrophotometrically by using the NanoDrop 2000. RNA samples were stored at -20 °C.
2.2.13 cDNA Synthesis
Total RNA (200 ng) were used for the complementary DNA synthesis. Reverse transcriptase PCR was performed with iSkript cDNA Synthesis Kit following manufacturer´s introductions in the TGradient thermocycler. The components for the reaction mixture are listed in table 2.10. The reaction protocol is listed in table 2.11.
39 Material and methods
Table 2.10 Reaction mixture for cDNA synthesis
Component Volume per Reaction 5x iSkript reaction mix 4 µl iSkript reverse transcriptase 1 µl Nuclease-free water x µl * RNA (200 ng total RNA) x µl * Total volume 20 µl * dependent on the RNA concentration
Table 2.11 Incubation protocol for cDNA synthesis
Time Temperature 5 min 25°C 30 min 42°C 5 min 85°C hold 4°C
2.2.14 Quantitative Real-Time PCR
The quantitative real-time PCR was performed with the ABI 7500 Fast Real-Time PCR system. TaqMan probes were used as fluorescence-monitoring system for DNA amplification (Tab. 2.12 for target genes). The expression of gapdh (glyceraldehyde-3-phosphate dehydrogenase) was used as an endogenous control to standardize the samples. The used TaqMan reagents are listed in table 2.13. Samples were run in triplicates. For all reactions a non-template control was included. The thermocycler protocol for qRT-PCR was listed in table. 2.14. Relative quantification was calculated by using the comparative threshold ΔΔ CT - method described previously (Schmittgen and Livak 2008).
Table 2.12 Target genes
Target gene Name Assay number grin2b N-methyl-D-aspartic acid receptor subunit Mm00433820_m1 gria1 α-amino-3-hydroxy-5-methy-4- Mm22433753_m1 isoxazolepropionic acid receptor subunit gapdh glyceraldehyde-3-phosphate dehydrogenase Mm99999915_g1
40 Material and methods
Table 2.13 Reagents for qRT-PCR for one sample
Component Volume per Reaction TaqMan Master Mix 12.5 µl DEPC water 8.75 µl Assay Mix (TaqMan probe for target gene) 1.25 µl cDNA 2.5 µl Total volume per well 25 µl
Table 2.14 Thermocycler protocol for qRT-PCR
Stage Temperature [°C] Time Repetition 1 50 2 min 1 2 95 10 min 1 94 15 sec 3 60 30 sec 40 72 35 sec 95 15 sec 60 20 sec 4 95 15 sec 1 60 15 sec
2.2.15 Statistical Analysis
All statistical analyses were performed using SPSS Version 21 (IBM Corp.). General (GLM) or generalized linear models (GENLIN) were used to analyze the effect of the applied concentrations and stability of possible effects across the biological replicates. The concentration was treated as fixed effect while the biological replicates (3 to 5 repetitions) were treated as random effects. According to the levels of measurement, F or Wald Chi2 values were used to determine the significance of the treatment effect. To account for different variances in the different treatment groups Dunnett-T3 post-hoc tests were used to compare the respective control condition with the various TOCP concentrations. Pairwise comparisons to the control condition using the Wald-type 95 % confidence interval were used to identify significant differences for the number of responders (binary variable). For the analysis of the IMARIS parameter multivariate tests of both parameters were performed to evaluate the overall effect. Subsequently, univariate tests and post-hoc tests were applied. Statistical significance was set at p≤ 0.05 and multiple comparisons were adjusted by Bonferroni correction.
41 Results
3 Results
3.1 Morphology of mouse primary cortical neurons at different stages of the in vitro culture
The toxic and neurotoxic potential of tri-cresyl phosphate (TCP) isomers was investigated in vitro in a model of mouse primary cortical neurons (pCNs). Owing to the different stages of neuronal outgrowth in vitro (Dotti, Banker et al. 1987), two different time points of in vitro culture were investigated. Different endpoints, among them cytoxicity, neurite outgrowth inhibition or alteration of neuronal complexity as well as neuronal processes like glutamate signaling and general responsiveness were analyzed. At 2 days in vitro (DIV) a very early stage of neurite outgrowth with short neurites and at DIV 7 a more mature stage with established neuronal networks were investigated. The cell cultures contained approximately 95 % neurons since the proliferation of glia cells was suppressed. The neuronal morphology of these two different developmental stages was visibly different (Fig. 3.1).
Figure 3.1 Morphology of mouse primary cortical neurons. Increase of the neuronal network density at DIV 7 compared to DIV 2. Transmitted light images of pCNs at DIV 2 20x (A) and DIV 7 20x (B). Scale bar: 50 µm.
At DIV 2, neurons formed initial neurite structures. At DIV 7, high-density neuronal networks with a multitude of neurite structures and several neurites per neuron were visible. The complexity of the neuronal network increased in the course of in vitro culture. Dendrites and axons, specific structures that enable the interconnection of neurons were extended.
42 Results
3.2 Impairment of cell viability
In the study at hand, the cytotoxic potential of the TCPs to assess the toxic concentration range was investigated. The three symmetric TCP isomers as well as a specific TCP mixture containing 2 % ToCP, 31 % TpCP, 41 % TmCP, and 25 % non-symmetric TCPs and the cyclic ToCP metabolite CBDP were analyzed with respect to their potency to cause cytotoxicity in mouse pCNs. The ortho-isomer ToCP has been discussed as being more toxic than the other symmetric isomers TmCP and TpCP (Casida, Eto et al. 1961). Beyond that, CBDP is the toxic metabolite of ToCP and described as more toxic than ToCP (Bleiberg and Johnson 1965).
3.2.1 TCP isomers and TCP mixture impair cell viability
As a first step towards investigating the neurotoxicity of TCPs, mouse pCNs were treated with various concentrations of the different isomers TmCP and TpCP, or the TCP mixture, at concentrations ranging between 10 pM and 10 mM. As the Figure 3.2 Effects of TCP isomers on cell viability. Cell viability of pCNs at DIV 7 after 24 h treatment with TmCP (A), TpCP (B) or TCP mixture (C) was corresponding solvent control 0.1 % determined by CellTiter-Blue® e Assay. Concentration response curves were fitted and IC50 values were determined. Data are given as (v/v) DMSO was used. The cell viability mean ± SEM. was assessed as percent of control and plotted against the concentration. Concentration response curves were fitted based on the
43 Results percentage of viable cells and the cytotoxic potential was thereby determined. The half-maximal inhibiting concentration was 83 µM (95 % confidence interval: 43 - 159) for TmCP (Fig. 3.2A), 122 µM (95 % confidence interval: 93 - 160) for TpCP (Fig. 3.2B), and 96 µM (95 % confidence interval: 40 - 232) for the TCP mixture (Fig. 3.2C). The results indicate a concentration- dependent decrease of cell viability in samples treated with micromolar and millimolar concentrations of the toxins, whereas lower concentrations (˂ 1 µM) do not affect cell viability.
3.2.2 ToCP impairs cell viability
Mouse pCNs were treated with ToCP (100 pM to 10 mM) as well as with 0.1 % (v/v) DMSO as the corresponding solvent control to investigate the cytotoxic potential in vitro. Neurons were incubated for a fixed time period of 24 h starting at two different time points of in vitro culture (DIV 1 or DIV 6).
Figure 3.3 ToCP affects cell viability of mouse pCNs. Transmitted light images of pCNs at DIV 7 after 24 h treatment with different ToCP concentrations and 0.1 % DMSO as the corresponding solvent control (A) Scale bar: 50 µm. Cell viability of pCNs at DIV 2 and DIV7 after 24 h ToCP treatment were determined by CellTiter-Blue® e Assay (B). Cell viability of pCNs after seven days of long-term ToCP treatment (C). Data are given as means ± SEM.
44 Results
Cell viability decreased in a concentration-dependent manner in samples treated with micromolar and millimolar concentrations. After treatment with 1 mM and 10 mM ToCP, no viable cells were detectable, while after exposure to 100 µM ToCP and 10 µM ToCP, living cells were detectable. Cell viability was unaffected by concentrations lower than 10 µM (Fig. 3.3A).
The IC50 value was 89 µM for both time points (95 % confidence interval DIV 2: 84-96 µM; 95 % confidence interval DIV 7: 86-101 µM) (Fig. 3.3B).
As a next step, long-term effects of ToCP on pCNs were investigated in order to study the temporal course of ToCP-induced cytotoxicity. ToCP might reduce the percentage of viable cells after long-term treatment with lower concentrations. In this experimental setup, pCNs were treated for seven days in vitro (Fig. 3.3C). In comparison to the short-term treatment, long-term
ToCP treatment shifted the concentration response curve to the left and reduced the IC50 to 600 nM (95 % confidence interval: 430 – 770 nM).
3.2.3 CBDP impairs cell viability
Next, the toxic potential of the ToCP metabolite cresyl salignin phosphate (CBDP) was investigated (Fig. 3.4). CBDP reduced the cell viability in a concentration-dependent manner with an IC50 value of 15 µM (95 % confidence interval: 7.9 – 29.6). No viable neurons were detected after Figure 3.4 Effects of CBDP on cell viability of mouse pCNs. Cell viability of pCNs treatment with millimolar and at DIV 7 after 24 h CBDP treatment was determined by CellTiter-Blue® e Assay. micromolar concentrations. The concentration response curve was fitted to determine the IC50. Data are given as mean ± SEM.
All symmetric TCP isomers showed comparable cytotoxic potency. According to previous studies, CBDP has a considerably more toxic effect on mouse pCNs than ToCP (Bleiberg and Johnson 1965). Indeed, in the study at hand, CBDP was more toxic than ToCP. Based on the results of the cell viability assays the concentration range for the investigation of the neurotoxic effects and the underlying mechanisms were determined.
The next parts deal with the TCP-induced effects on neurotoxicity related endpoints like neurite outgrowth and neuronal function. For these experiments non-cytotoxic concentrations between 1 nM and 10 µM of the TCP isomers, the TCP mixture as well as the metabolite CBDP were used.
45 Results
3.3 Impairment of neurite outgrowth and neurite degeneration
Neurite outgrowth and neurite degeneration were investigated as specific parameter of neurotoxicity (Harrill, Freudenrich et al. 2011). Neurite outgrowth is a critical process during central nervous system development, and plasticity. Neurons extend specialized processes for the purpose of establishing contact sites (i.e. synapses) that enable the directional flow of information throughout a neuronal network (Sanes, Reh et al. 2006, Harrill, Freudenrich et al. 2011). Primary neuronal cultures, neuronal cell lines, and embryonic stem cell derived neuronal cells have been used in former studies for the quantitative assessment of the effects of chemical substances on neurite outgrowth (Radio and Mundy 2008, Harrill, Robinette et al. 2013). In the present study, high throughput automated microscope systems and high content image analyses were used to quantify parameters of neuronal integrity and complexity in the presence of potential neurotoxins.
Literature data show that organophosphates and TCPs have beside their cytotoxic effects more subtle effects on neurite morphology and growth. The ortho-isomer of the TCPs is known to cause neurite degeneration and morphology alterations in vivo (Abou-Donia, Lapadula et al. 1988, Song, Yan et al. 2009, Song, Zou et al. 2012). The experiments presented below investigate the influence of TCP compounds on neurite morphology, integrity and complexity at two different time points during the in vitro culture. The experiments consider whether there is a link between the in vivo and in vitro situation and whether the other symmetric isomers and the mixture show similar effects compared to ToCP.
3.3.1 Inhibition and degeneration of neuronal networks by TCP isomers
In order to study TCP-induced effects on neurite outgrowth, pCNs cultured either 2 or 7 DIV were incubated with different TCP isomers for 24 h. Then the neurite areas as well as the branching levels were determined (Fig. 3.5A - D). The mean branching level described the number of branches per neuron. At DIV 2, no alterations of neurite area and mean branching level were observed after treatment with TmCP in comparison to the control. After 24 h treatment with 10 µM TpCP the neurite area was significantly reduced by 23 % in comparison to controls (Control: 52 µm2 vs. 10 µM ToCP: 40 µm2), (F = 4.88, p≤0.05). The mean branching level was unaltered by TpCP treatment in the tested concentration range. Incubation with 10 µM TCP mixture decreased the neurite area by 30 % compared to the control (Control: 70 µm2 vs. 10 µM TCP mixture: 50 µm2), (F = 4.55, p≤0.01). The mean branching level was unaffected by treatment with the TCP mixture. At DIV 7, the neuronal complexity and integrity were unaltered by the tested TCP isomers as well as the TCP mixture.
46 Results
Figure 3.5 Quantitative analysis of neurite outgrowth and neurite degeneration parameters of pCNs after 24 h treatment with TCP isomers TmCP and TpCP as well as the TCP mixture. TmCP-, TpCP- and TCP mixture-induced reduction of neurite area (A) and mean branching level (B) after 24 h treatment at DIV 2. TmCP-, TpCP- and TCP mixture-induced reduction of neurite area (C) and mean branching level (D) after 24 h treatment at DIV 7. (A) – (D) show mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicated as * p≤0.05, ** p≤0.01, *** p≤0.001.
Between DIV 2 and DIV 7 in vitro, an increase in neurite area and the mean branching level was observed. In the control condition, the neurite area increased from DIV 2 to DIV 7 by a factor of 8 (60 µm2 to 500 µm2) and the mean branching level increased by a factor of 20 (DIV 2: 1.0 – 1.5 vs. DIV 7: to 28 – 30). PCNs showed only a few neuritic excesses at DIV 2 and a high density neurite network at DIV 7.
The tested isomers had only weak effects on neurite structures. TpCP and the TCP mixture inhibited neurite outgrowth after 10 µM treatment. The established networks were unaltered in growth and morphology by these treatments.
3.3.2 Inhibition and degeneration of neuronal networks by ToCP
ToCP-induced effects on neurite outgrowth inhibition and established neuronal networks were investigated after 24 h treatment at two time points of in vitro culture and the neurite areas were calculated after treatment with different ToCP concentrations. Neurite complexity increased from DIV 2 to DIV 7 under control conditions. Treatment with 1 µM and 10 µM ToCP inhibited neurite outgrowth of pCNs at DIV 2. After 10 µM ToCP treatment pCNs displayed atypical short and thin neurites. Treatment with 1 µM and 10 µM ToCP reduced the density and complexity of established neuronal networks in cultures of pCNs at DIV 7 (Fig. 3.6A and B).
47 Results
Figure 3.6 Quantitative analysis of neurite density and complexity of pCNs at DIV 2 and DIV 7. Fluorescence images of pCNs after 24 h treatment with various ToCP concentrations as well as 0.1 % DMSO as solvent control at DIV 2 (A) and DIV 7 (B). Neuronal marker protein β-III-tubulin in green and DAPI as a marker of cell nuclei in blue. Scale bar: 20 µm. ToCP-induced alteration of neurite area after 24 h treatment with various concentrations at DIV 2 (C) and DIV 7 (D). (C) and (D) are given as mean ± SEM. Results of Dunnett- T3 post-hoc tests are indicated as * p≤0.05, ** p≤0.01, *** p≤0.001.
Neuronal cultures treated with ToCP on DIV 2 showed a significant reduction in neurite area (F = 12.75, p≤0.001), (Fig. 3.6C). Post hoc comparisons revealed that treatment with 1 µM and 10 µM ToCP reduced the neurite area significantly by 40 % or 60 % compared to the control (Control: 66,000 4000 pixels vs. 1 µM ToCP: 40,000 3000 pixels vs. 10 µM ToCP: 26,000 2000 pixels). On DIV 7, treatment with ToCP significantly reduced the neurite area (F = 22.94, p≤0.001), (Control: 387,000 31,000 pixels vs. 1 µM ToCP: 233,000 28,000 (60 % of control) vs. 10 µM ToCP: 70,000 6,000 pixel (20 % of control)).
ToCP-induced effects on neurite outgrowth at DIV 2 were studied in more detail. At the early phase of network development, neurite outgrowth is an ongoing process and single neurons display few and relatively short neurites that can be quantified by specialized software (IMARIS from Bitplane). Parameters like mean branching level, neurite length, and neurite diameter were calculated after the reconstruction of neuronal structures. The complexity of neurites and their structures were concentration-dependently reduced by ToCP (F = 2.97, p≤0.001), (Fig. 3.7). In more detail, after treatment with 10 µM ToCP, many neurons were devoid of neurites and
48 Results remaining neurites were short and swollen. The mean branching level was significantly decreased by 20 % after 10 µM ToCP treatment compared to the control (Control: 2.06 ± 0.048 vs. 10 µM ToCP: 1.69 ± 0.06). The neurite length was decreased by 62 % from 180 ± 9 µm in the control condition to 68.0 ± 9 µm after 10 µM ToCP treatment. The neurite diameter was calculated as parameter for neurite swelling. The neurite diameter was increased from 1.40 ± 0.05 µm in DMSO treated pCNs to 1.9 ± 0.1 µm (135 % of control) in pCNs treated with 10 µM ToCP.
Next, the effects on neurite outgrowth after long-term ToCP treatment in different concentrations for seven days were investigated. The neurite area and the mean branching level were calculated based on Figure 3.7 Quantitative analysis of complexity of pCNs at DIV 2. fluorescence images at DIV 8 (Fig. 3.8). Primary ToCP-induced reduction of mean branching level (A), neurite length (B) and increase of neurite diameter (C) after 24 h cortical neurons showed a significant treatment at DIV 2. (A) – (C) are given as mean ± SEM. Results of Dunnett-T3 post- hoc tests are indicated as * p≤0.05, ** p≤0.01, reduction in neurite area (F = 4.15, p≤0.01) *** p≤0.001. and mean branching level (F = 4.29, p≤0.01) after long-term ToCP treatment. The neurite area was reduced by 55 % after treatment with 100 nM ToCP compared to the control (Control: 270 µm2 vs. 100 nM ToCP: 121 µm2). The mean branching level decreased by 75 % after treatment with 100 nM ToCP to a number of 5.75 branches per neuron compared to 16.7 branches per neuron in the control condition. After the treatment with lower ToCP concentrations no effects on neurite structures were observed. Long-term ToCP treatment produced stronger effects on the mean branching levels, which described the number of branches per neuron, and neurite areas as short-term ToCP treatment. At DIV 7, the mean branching level was in the 24 h DMSO control 30 branches per neuron and the neurite area was 500 µm2. The mean branching level after long-term DMSO treatment was reduced to 16 branches per neuron and the neurite area to 270 µm2.
49 Results
ToCP inhibited neurite outgrowth and caused neurite degeneration of established neuronal networks in a concentration-dependent manner at non-cytotoxic concentrations. The early stage of network formation was more sensitive for ToCP. Long-term treatment decreased the lowest effective ToCP concentration.
3.3.2.1 Time course of neurite outgrowth inhibition – Network Formation Assay
The network formation assay (NFA) is a system which enables the investigation of neurotoxic effects on growing Figure 3.8 Quantitative analysis of neurite degeneration parameters of neuronal networks in vitro (Frimat, pCNs after long-term ToCP treatment with different concentrations. ToCP- induced reduction of neurite area (A) and the mean branching level (B) Sisnaiske et al. 2010, Hardelauf, Waide after seven days ToCP treatment with different concentration). (A) and (B) show mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicated as et al. 2014). The NFA was used to * p≤0.05, ** p≤0.01, *** p≤0.001. investigate the inhibition of neurite outgrowth caused by ToCP. The method allows the monitoring of individual living neuronal cultures for several days. As a parameter of network complexity, the connections per node (cpn, connections between adhesion notes) after different ToCP treatment conditions were counted (Fig. 3.9A - D). In the NFA, neuronal death was observable after 24 h treatment with 100 µM ToCP and after 48 h of incubation with 30 µM ToCP. The time course of the change of cpn was calculated for 10 µM ToCP treatment and the DMSO control condition (Fig. 3.9E). Treatment with 10 µM ToCP resulted in a significant inhibition of the neurite outgrowth compared to the control. The cpn was reduced from 1.5 in the control condition to 1.0 (70 % of control) after 10 µM ToCP treatment. The neurite outgrowth was not inhibited by treatment with lower ToCP concentrations.
50 Results
Figure 3.9 Network formation assay of pCNs treated with ToCP. Calculation of the connections per node (cpn) after 24 h (A), 48 h (B), 72 h (C) and 96 h (D) after treatment. Time course of cpn (E). (A) – (E) are given as mean ± SEM.
3.3.3 Inhibition and degeneration of neuronal networks CBDP
CBDP is the toxic metabolite of ToCP (Casida, Eto et al. 1961). Thus, pCNs might be sensitive to CBDP-induced effects. The CBDP effects on neurite area (Fig. 3.10A, C) and the mean branching level (Fig. 3.10B, D) were investigated at DIV 2 and DIV 7. In neither of the CBDP conditions, changes of the mean branching level or the neurite area could be seen at both tested time points. The metabolite CBDP had no visible influence on neurite outgrowth and caused no degeneration effects on established neuronal networks. The metabolite CBDP had no impact on the neurite structures.
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Figure 3.10 Quantitative analysis of neurite outgrowth and neurite degeneration parameters of pCNs after CBDP treatment. CBDP- induced reduction of neurite area (A) and the mean branching level (B) after 24 h treatment at DIV 2. CBDP-induced reduction of neurite area (C) and the mean branching level (D) after 24 h treatment at DIV 7. (A) – (D) show mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicated as * p≤0.05, ** p≤0.01, *** p≤0.001.
3.4 Effects of TCPs on neurochemical processes
Neurochemical processes were analyzed as endpoints of functional neurotoxicity, by using fluorescence-based live-cell calcium imaging. The disruption of neurochemical processes like glutamate signaling, depolarization and transmission of electrical signals are possible adverse outcomes of neurotoxins that can trigger behavior and lead to neurobehavioral deficits. Effects of the TCP isomers as well as the TCP mixture and the metabolite CBDP on signals evoked by the main excitatory neurotransmitter glutamate were tested. Furthermore, the possible effects of these compounds on neuronal depolarization through the activation of voltage-gated calcium channels (VGCCs) were studied as a marker for general responsiveness of the cells.
Using fluorescence-based live-cell calcium imaging, the baseline responsiveness of mouse pCNs was tested with regard to different neurotransmitters. Neurons were challenged with short pulses of neurotransmitter during live monitoring of intracellular calcium levels. Upon stimulation with the excitatory neurotransmitter glutamate, primary cortical neurons respond with a transient influx of calcium. At an early stage of in vitro culture of pCNs, calcium responses were detected upon stimulation with the inhibitory neurotransmitter GABA showing cellular activation. This situation of excitatory GABA is typical of embryonic central nervous system
52 Results neurons (Ben-Ari 2002, Ben-Ari, Woodin et al. 2012). The shift from excitatory to inhibitory GABA responses takes place later during embryonic and postnatal development (Lu, Karadsheh et al. 1999). The percentage of glutamate responders increased during in vitro culture, whereas the percentage of GABA responders decreased gradually to zero during the maturation process (Khazipov, Khalilov et al. 2004, Tyzio, Holmes et al. 2007).
As a first step towards elucidating possible effects of TCP treatment on glutamate signaling in pCNs, the half-maximal activating glutamate concentration was determined (Fig. 3.11) by randomized application of different glutamate concentrations (1 µM to 3 mM). A concentration response curve, based on the normalized response amplitudes, was fitted and the EC50 value of 33 ± 1.3 µM (95 % CI: 31.5 – 34.5 µM) was calculated.
Figure 3.11 Half-maximal activating glutamate concentration in mouse pCNs at DIV 7. PCNs were stimulated with randomized glutamate concentrations during live-cell calcium imaging measurements. Response curve (A) was fitted and EC50 value was calculated. Exemplary traces of repetitive glutamate stimulation (B). Data are given as mean ± SEM.
Accordingly to the half-maximal activating glutamate concentration, mouse pCNs were stimulated with 30 µM glutamate in further experiments.
3.4.1 TCP isomers impair glutamate signaling
At first, the possible impairment of glutamate signaling by treatment with TCPs was investigated. The functional impairment of neuronal processes represents a completely new mode of action of this group of chemical. The response frequencies (Wald Chi2 = 321.45, p≤0.001) as well as the corresponding glutamate-induced mean response amplitudes (F = 167, p≤0.001) were significantly decreased after 10 µM TmCP treatment (Fig. 3.12B and D). In the control condition, 99 % (CI: 97 – 100) of the neurons responded to glutamate stimulation (i.e. glutamate responders). Of note, treatment with lower TmCP concentrations caused no significant reduction of glutamate responders, whereas incubation with 10 µM TmCP reduced the number of glutamate responders to 30 % (CI: 24 – 36). Neurons treated with 10 µM TmCP showed lower glutamate-induced mean response amplitudes in comparison to the controls (Control: ratio (f340/f380) = 0.21 ± 0.006 vs. 10 µM ToCP: ratio (f340/f380) = 0.008 ± 0.004 (4 % of the
53 Results control)), whereas lower TmCP concentrations were without effect. A significant decrease in frequencies of responses to depolarizing KCl buffer were seen after incubation with 10 µM TmCP in comparison to the DMSO control (Wald Chi2 = 246.19, p≤0.001) (Control: 94 % (CI: 91 – 96) vs. 10 µM TmCP: 38 % (CI: 32 – 44)). Incubation with 10 µM TmCP caused a decrease in the KCl- induced mean response amplitudes (F = 146, p≤0.001), (Control: ratio (f340/f380) = 0.211 ± 0.0062 vs. 10 µM TmCP: ratio (f340/f380) = 0.009 ± 0.0044 (4 % of the control)). The KCl-induced response amplitudes increased after incubation with 100 nM TmCP to ratio (f340/f380) = 0.255 ± 0.008 (120 % of control).
The percentage of glutamate responders (Fig. 3.12G) was significantly reduced after treatment with all tested TpCP concentrations in comparison to the control (Wald Chi2 = 171.43, p≤0.001), (Control: 99 % (CI: 97 – 100) vs. 100 nM TpCP: 93 % (CI: 89 – 95) vs. 1 µM TpCP: 93 % (CI: 86 – 92) vs. 10 µM TpCP: 45 % (CI: 37 - 53). The percentage of KCl responders was decreased after treatment ≥ 100 nM TpCP (Wald Chi2 = 185.37, p≤0.001), (Control: 94 % (CI: 91 – 96) vs. 100 nM TpCP: 87 % (CI: 83 – 90) vs. 1 µM TpCP: 88 % (CI: 85 – 91) vs. 10 µM TpCP: 35 % (CI: 28 – 44)). The glutamate-induced mean response amplitudes (Fig. 3.12E) were reduced after treatment with 1 µM and 10 µM TpCP (F = 144, p≤0.001), (Control: ratio (f340/f380) = 0.213 ± 0.008 vs. 1 µM TpCP: ratio (f340/f380) = 0.157 ± 0.008 (70 % of control) vs. 10 µM TpCP: ratio (f340/f380) = 0.005 ± 0.008 (2 % of control)). Likewise, the KCl-induced response amplitudes (Fig. 3.12F) were decreased after treatment with 1 µM and 10 µM TpCP (F = 146; p≤0.001), (Control: ratio (f340/f380) = 0.211 ± 0.008 vs. 1 µM TpCP: ratio (f340/f380) = 0.159 ± 0.008 (70 % of control) vs. 10 µM TpCP: ratio (f340/f380) = 0.006 ± 0.008 (3 % of control)).
The percentage of glutamate responders (Fig. 3.12J) was reduced after incubation with all tested TCP mixture concentrations compared to the control (Control: 99 % (CI: 97 – 100), 100 nM TCP mixture: 88 % (CI: 84 – 91), 1 µM TCP mixture: 91 % (CI: 88 – 94), 10 µM TCP mixture: 16 % (CI: 12 - 22), (Wald Chi2 = 349.35, p≤0.001). The corresponding glutamate response amplitudes (Fig. 3.12H) were significantly decreased by treatment with micromolar concentrations of the TCP mixture (F = 150, p≤0.001), (Control: ratio (f340/f380) = 0.21 vs. 1 µM TCP mixture: ratio (f340/f380) = 0.129 ± 0.008 (60 % of control) vs. 10 µM TCP mixture: ratio f340/f380) = 0.007 ± 0.008 (3 % of control)).
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Figure 3.12 Effects of TCP isomers on glutamate- and KCl-induced Ca2+-responses of pCNs at DIV 7 after 24 h treatment with various toxin concentrations and 0.1 % (v/v) DMSO as a solvent control. Exemplary traces of glutamate- and KCl-induced Ca2+-responses for 0.1 % DMSO as solvent control and TmCP, TpCP, or TCP mix treated neurons (A). Glutamate-induced response amplitudes (B), KCl- induced response amplitudes (C), and percentage of glutamate and KCl responders (D) of pCNs after 24 h treatment with various TmCP concentrations and 0.1 % DMSO as a control. Glutamate-induced response amplitudes (E), KCl-induced response amplitudes (F), and percentage of glutamate and KCl responders (G) of pCNs after 24 h treatment with various TpCP concentrations and 0.1 % DMSO as a control. Glutamate-induced response amplitudes (H), KCl-induced response amplitudes (I), and percentage of glutamate and KCl responders (J) of pCNs after 24 h treatment with various TCP mixture concentrations and 0.1 % DMSO as control. Bars in (B) – (J) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
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A significant decrease in the percentage of responders (Wald Chi2 = 262.77, p≤0.001) and the corresponding amplitudes (F = 205, p≤0.001) after KCl stimulation (Fig. 3.12I) was observed after treatment with all tested TCP mixture concentrations (Control: 94 % (CI: 91 – 96) with ratio (f340/f380) = 0.211 ± 0.06 vs. 100 nM TCP mixture: 83 % (CI: 79 – 87) with ratio (f340/f380) = 0.218 ± 0.08 vs. 1 µM TCP mixture: 74 % (CI: 70 – 79) with ratio (f340/f380) = 0.143 ± 0.008 (67 % of control) vs. 10 µM TCP mixture: 8 % (CI: 5 -12) with ratio (f340/f380) = 0.001 ± 0.007 (0.5 % of control)).
Calcium is an outstanding signaling component in cells that can trigger a multitude of effects, among them proliferation and apoptosis. Neurons tightly regulate the intracellular calcium concentration and chronically elevated calcium concentrations may be an effect of intoxication. Increased intracellular calcium concentrations have been discussed as a mode of action of TCP neurotoxicity (El-Fawal and Ehrich 1993, Wu, Chang et al. 2003). In the present study, calcium imaging was used to determine the baseline calcium levels as fluorescence ratios for comparison among controls and different treatment groups. The basal calcium levels of TmCP, of TpCP and of TCP mixture treated neurons were determined at DIV 7 (Fig. 3.13).
Figure 3.13 TCP isomers impaired the basal calcium levels in pCNs at DIV 7 after 24 h treatment with different TCP isomers at various concentrations and with 0.1 % (v/v) DMSO as a solvent control. Results of the determination of the steady state calcium level after treatment with TmCP (A), TpCP (B) and the TCP mix (C). Data are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
Treatment with 10 µM TmCP increased the basal calcium level of mouse pCNs significantly in comparison to controls (F = 87.65, p≤0.001), (Control: ratio (f340/f380) = 0.318 ± 0.004 vs. 10 µM TmCP: ratio (f340/f380) = 0.427 ± 0.006). Incubation with 1 µM and 10 µm TpCP increased the basal calcium level significantly in comparison to controls (F = 72.43, p≤0.001), (Control: ratio (f340/f380) = 0.318 ± 0.004) vs. 1 µM TpCP: ratio (f340/f380) = 0.35 ± 0.005 vs. 10 µM TpCP: ratio (f340/f380) = 0.419 ± 0.006). The treatment with 10 µM TCP mixture increased the basal calcium level significantly compared to controls (F = 33.07, p≤0.001), (Control: ratio (f340/f380) = 0.318 ± 0.005 vs. 10 µM TCP mixture: ratio (f340/f380) = 0.398 ± 0.008).
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At micromolar concentrations, the TCP isomers significantly impaired the glutamate sensitivity of pCNs. Glutamate- and KCl-induced response amplitudes were simultaneously reduced after treatment with these concentrations. The baseline intracellular calcium concentration was significantly increased after treatment with 10 µM for all tested isomers and the mixture.
3.4.2 ToCP impairs glutamate signaling
In the following part, the possible effects of ToCP with regard to the impairment of neurochemical processes were investigated. The cytotoxic potential of the TCPs, investigated in the first part of this study, was comparable for the symmetric isomers as well as the mixture. ToCP was described in the literature as the most potent isomer, attributed to the higher affinity to the NTE and other esterases (Henschler 1958, Casida, Eto et al. 1961). The ToCP-induced effects were investigated in more detail in the context of the hypothesis that ToCP impair the glutamate signaling in a time- and concentration-dependent manner. An increase in the induced effects is conceivable. Initially, possible effects on glutamate signaling and depolarization processes were investigated after the short incubation time of one hour. The glutamate sensitivity (F = 23.83, p≤0.001) as well as the general responsiveness of pCNs (F = 40.23, p≤0.001) were reduced after 1 h incubation with ToCP concentrations of 1 µM or higher (Fig. 3.14A – D). In particular, the glutamate-induced response amplitudes were significantly decreased in neurons treated with 1 µM and 10 µM ToCP (Control: ratio (f340/f380) = 0.231 ± 0.008 vs. 1 µM ToCP: ratio (f340/f380) = 0.192 ± 0.009 (83 % of control) vs. 10 µM ToCP: ratio (f340/f380) = 0.125 ± 0.009 (55 % of control)). The KCl-induced response amplitudes decreased after treatment with 10 µM TOCP (Control: ratio (f340/f380) = 0.171 ± 0.009 vs. 10 µM ToCP: ratio (f340/f380) = 0.309 ± 0.008 (55 % of control)). Lower ToCP concentrations had no effect on the general responsiveness of the neurons. The percentage of glutamate responders was reduced after treatment with ToCP concentrations > 100 nM (Wald Chi2 = 75.59, p≤0.001). The percentage of glutamate responders decreased from 100 % in the control to 96 % (CI: 92 - 98) at 100 nM, to 94 % (CI: 89 – 97) at 1 µM, and to 87 % (CI: 81 – 91) at 10 µM ToCP. The frequency of KCl responses was significantly reduced only after treatment with 10 µM ToCP compared to controls (Wald Chi2 = 15.88, p≤0.001) (control: 100 % vs. 10 µM ToCP: 95 % (CI: 90 – 97)).
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Figure 3.14 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 1 h treatment. Exemplary trace of glutamate- and KCl-induced Ca2+-responses for 0.1 % DMSO as a solvent control and ToCP treated cells (A). Glutamate-induced response amplitudes (B), KCl-induced response amplitudes (C) and percentage of glutamate and KCl responders (D) of pCNs after 1 h treatment with various ToCP concentrations and 0.1 % DMSO as a control. Bars in (B) – (D) are given mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
The results indicated an impairment of the glutamate sensitivity of the neurons after treatment with micromolar ToCP concentrations.
Furthermore, the ToCP-induced effects on glutamate signaling were investigated after 4 h incubation. In order to consider the ToCP-induced impact of the glutamate signaling in the temporal evolvement. The percentage of glutamate responders was reduced in all conditions with ToCP (Wald Chi2 = 14.247, p≤0.001). In the control condition 100 % of all living neurons identified by their responses to KCl responded to glutamate, whereas the number of responders decreased concentration-dependently with increasing concentrations of ToCP (100 nM ToCP: 97 % (CI: 94 – 98) vs. 1 µM ToCP: 94 % (CI: 90 – 96) vs. 10 µM ToCP: 87 % (CI: 83 – 91)).
The percentage of KCl responders was reduced after treatment with 10 µM ToCP to 94 % (CI: 90 – 96) (control: 100 %) (Wald Chi2 = 13.14, p≤0.001). No alterations of the percentage of KCl responders was observed after treatment with lower ToCP concentrations. The glutamate
58 Results sensitivity (F = 51.66, p≤0.001) was reduced after 4 h incubation with ToCP concentrations of 100 nM and higher (Fig. 3.15A - D).
Figure 3.15 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 4 h treatment. Exemplary trace of glutamate- and KCl-induced Ca2+-responses for 0.1 % DMSO as a solvent control and ToCP treated cells (A). Glutamate-induced response amplitudes (B), KCl-induced response amplitudes (C), and percentage of glutamate KCl responders (D) of pCNs after 4 h treatment with various ToCP concentrations and 0.1 % DMSO as control. Bars in (B) – (D) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
The glutamate-induced response amplitudes were significantly decreased in all ToCP conditions compared to the control (Control: (f340/f380) = 0.249 ± 0.008, 100 nM ToCP: ratio (f340/f380) = 0.185 ± 0.008 (75 % of control), 1 µM ToCP: ratio (f340/f380) = 0.181 ± 0.007 (72 % of control), 10 µM ToCP: ratio (f340/f380) = 0.115 ± 0.008 (46 % of control)). In contrast to that, only after treatment with 10 µM ToCP the KCl-induced response amplitudes were significantly decreased (F = 42.20, p≤0.001) (Control: ratio (f340/f380) = 0.307 ± 0.008 vs. 10 µM ToCP: ratio (f340/f380) = 0.203 ± 0.008 (66 % of control)).
An extended incubation time from one to four hours lead to a major influence on glutamate signaling. After 4 h ToCP treatment the glutamate sensitivity was reduced with concentrations as low as 100 nM.
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In a next step the incubation time was extended to 24 h. The question is whether the difference in the concentration-dependency of the ToCP-induced reduction in glutamateresponses increased after the extended incubation time. The incubation with different ToCP concentration was started at two different time points, DIV 2 and DIV 7 respectively in order to investigate effects in a very early stage of in vitro culture with short neurites (Fig. 3.16) and a later stage with established neuronal networks (Fig. 3.17). The investigation of ToCP effects on DIV 7 is more related to the situation of the mature brain. ToCP incubation altered response frequencies and amplitudes to glutamate and KCl stimulation in a concentration-dependent manner. At DIV 2, a significantly smaller fraction of pCNs treated with ToCP concentrations ≥ 1 nM displayed calcium responses upon glutamate stimulation in comparison to controls (Wald Chi2: 26.03, p≤0.001), (Control: 61 ± 4 % vs. 1 nM ToCP: 46 ± 4 % vs. 10 nM ToCP: 45 ± 4 % vs. 100 nM ToCP: 40 ± 4 %, 1 µM ToCP: 36 ± 4 % vs. 10 µM ToCP: 4 ± 2 %). The glutamate-induced mean response amplitudes were also significantly affected by all tested ToCP concentrations (≥ 1 nM) (F = 20.1, p≤0.001), (Control: ratio (f340/f380) = 0.08 ± 0.0085 vs. 1 nM ToCP: ratio (f340/f380) = 0.056 ± 0.0069 (70 % of control) vs. 10 nM ToCP: ratio (f340/f380) = 0.038 ± 0.0045 (47 % of control) vs. 100 nM ToCP: ratio (f340/f380) = 0.038 ± 0.0049 (47 % of control) vs. 1 µM ToCP: ratio (f340/f380) = 0.032 ± 0.0044 (40 % of control) vs. 10 µM ToCP: ratio (f340/f380) = 0.0032 ± 0.0012 (4 % of control)). The percentage of KCl responders was significantly decreased (Wald Chi2: 50.18, p≤0.001) after incubation with ToCP concentrations ≥ 10 nM in comparison to controls (10 µM ToCP 0 ± 0 % vs. 1 µM ToCP: 67 ± 4 % vs. 100 nM ToCP: 76 ± 3 % vs. 10 nM ToCP: 84 ± 3 % vs. control: 95 ± 2 %). The corresponding KCl-induced mean response amplitudes (F = 16.29, p≤0.001) were reduced after incubation with 1 µM and 10 µM ToCP, whereas no effects were observed with lower concentrations (Control: ratio (f340/f380) = 0.109 ± 0.0055) vs. 1 µM ToCP: ratio (f340/f380) = 0.06 ± 0.0054 (55 % of control) vs. 10 µM ToCP: ratio (f340/f380) = 0.0 ± 0.0 (0 % of control)). The general responsiveness of mouse pCNs to glutamate increased over the course of culture. In the control conditions, the percentage of glutamate responders increased to 150 % from 61 ± 4 % on DIV 2 to 94 ± 2 % on DIV 7 and the corresponding glutamate-induced mean response amplitudes increased to 170 % from ratio (f340/f380) = 0.08 ± 0.0085 at DIV 2 to ratio (f340/f380) = 0.139 ± 0.0089 at DIV 7. The percentage of KCl responders increased to 130 % from 95 ± 2 % to 99 ± 1 % and the corresponding KCl-induced mean response amplitudes from ratio (f340/f380) = 0.109 ± 0.0055 at DIV 2 to ratio (f340/f380) = 0.146 ± 0.0066 at DIV 7.
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Figure 3.16 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 2 after 24 h treatment. Exemplary trace of glutamate- and KCl-induced Ca2+-responses for 0.1 % DMSO as solvent control and ToCP treated cells (A). Percentage of glutamate responders, (B) glutamate-induced response amplitudes (D), percentage of KCl responders (C), and KCl-induced response amplitudes (E) of pCNs after 24 h treatment with various ToCP concentrations and 0.1 % DMSO as a control. Bars in (B) – (E) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
PCNs treated with ToCP at DIV 7 also showed reduced glutamate sensitivity (Fig. 3.17). Again, the percentage of glutamate responders (Wald Chi2: 25.75; p≤0.001) as well as the corresponding glutamate-induced mean response amplitudes (F = 39.26; p≤0.001) were significantly affected upon treatment with ToCP concentrations of 100 nM - 10 µM (Control:
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94 ± 2 %; ratio (f340/f380) = 0.139 ± 0.0089 vs. 100 nM ToCP: 65 ± 3 %; ratio (f340/f380) = 0.086 ± 0.008 (57 % of control) vs. 1 µM ToCP: 65 ± 3 %; ratio (f340/f380) = 0.053 ± 0.005 (35 % of control) vs. 10 µM ToCP: 23 ± 3 %; ratio (f340/f380) = 0.016 ± 0.0026 (11 % of control)).
Figure 3.17 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 24 h treatment. Exemplary traces of glutamate- and KCl-induced Ca2+-responses for 0.1 % DMSO as solvent control and ToCP treated neurons (A). Glutamate responders (B), KCl responders (C), glutamate-induced response amplitudes (D) and KCl-induced response amplitudes (E) of pCNs after 24 h treatment with various ToCP concentrations and 0.1 % DMSO as a control. Bars in (B) – (E) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
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A significant decrease in the percentage of responders (Wald Chi2: 3.74; p≤0.59) and the corresponding amplitudes (F = 25.17; p≤0.001) after KCl stimulation were observed for 10 µM ToCP, while no effects were observed at lower concentrations (Control: 99 ± 1 %; ratio (f340/f380) = 0.146 ± 0.0066 vs. 10 µM ToCP: 33 ± 3 %; ratio (f340/f380) = 0.042 ± 0.0055 (28 % of control)). The basal calcium levels after 24 h ToCP incubation were calculated at DIV 2 and DIV 7 (Fig. 3.18). At both time points the basal calcium levels were significantly increased after 10 µM ToCP treatment compared to the control condition.
ToCP at nanomolar concentrations significantly reduced the glutamate sensitivity at both tested time points after 24 h treatment. At DIV 2, the lowest effective ToCP concentration was 1 nM, whereas 100 nM ToCP was the effective concentration at DIV 7. The ToCP-induced reduction in glutamatesensitivity was time- and concentration-dependent. More severe effects Figure 3.18 ToCP impaired the steady state calcium levels in mouse pCNs after 24 h continued treatment with different ToCP of the same ToCP concentration on glutamate concentrations and 0.1 % DMSO as control at DIV 2 (A) and DIV 7 (B). Data are given as mean ± SEM. Results of Dunnett-T3 post- sensitivity could be seen with prolonged hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001. incubation times.
Here, it was shown for the first time that very low ToCP concentrations affected central nervous system processes. ToCP had, compared to the other tested isomers, the highest potency to reduce the glutamate sensitivity. Treatment with nanomolar ToCP concentrations reduced the glutamate sensitivity significantly, whereas treatment with these concentrations was without influence on the general functionality.
Additionally, a continued ToCP treatment over seven days in vitro was analyzed with respect to the impairment of glutamate sensitivity. This long-term ToCP treatment possibly causes an increase in effect size concerning the reduced glutamate sensitivity. The long-term treatment reflects the low level chronical exposure to ToCP.
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Figure 3.19 Long-term ToCP treatment impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 7 days of continued treatment. Exemplary traces of glutamate- and KCl-induced Ca2+-responses for 0.1 % DMSO as a solvent control and ToCP treated neurons (A). Glutamate responders (B), glutamate-induced response amplitudes (C), percentage of KCl responders (D), and KCl-induced response amplitudes (E) of pCNs after long-term treatment with different ToCP concentrations and 0.1 % DMSO as control. Bars in (B) – (E) are given as mean ± SEM. Results of Dunnett- T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
Long-term ToCP incubation altered response frequencies and amplitudes seen upon glutamate stimulation in a concentration-dependent manner (Fig. 3.19). In more detail, the percentage of glutamate responders (Wald Chi2 = 18.1, p≤0.01) was significantly reduced by treatment with 100 nM compared to the control condition (100 nM: 89 % (CI: 84 – 93) vs. control: 98 % (CI:
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94 - 99)). For all other tested ToCP concentrations the frequencies of glutamate responders were similar to that of the control condition. The corresponding mean response amplitudes to glutamate were concentration-dependently reduced after ToCP preincubation (F = 8.07, p > 0.001).
Treatment with concentrations ≥ 1 nM led to a significant decrease in the mean response amplitudes, whereas lower ToCP concentrations did not affect glutamate-induced responses (Control: ratio (f340/f380) = 0.245 ± 0.011 vs. 1 nM ToCP: ratio (f340/f380) = 0.197 ± 0.01 (80 % of control) vs. 10 nM ToCP: ratio (f340/f380) = 0.178 ± 0.01 (73 % of control) vs. 100 nM ToCP: ratio (f340/f380) = 0.169 ± 0.01 (69 % of control)). The percentage of KCl responders (F = 1.96) as well as the corresponding KCl-induced response amplitudes were not affected by long-term ToCP treatment indicating that the general excitability of the neurons was unaffected by ToCP.
The glutamate sensitivity was impaired after long-term ToCP treatment. Concentrations as low as 1 nM ToCP significantly reduced the glutamate-induced response amplitudes. Long-term ToCP exposure did not interfere with the general responsiveness of the neurons to a depolarizing stimulus.
3.4.2.1 ToCP affects glutamate receptor expression
To gain insights into the underlying mechanisms of the reduced glutamate sensitivity after ToCP treatment, the expression level of two glutamate receptor subunits were tested by qRT-PCR. Possible AMPAR modifications were tested by the investigation of the gria1 subunit (Fig. 3.20A and C), while the possible impairment of NMDARs was tested by the investigation of grin2b subunit (Fig. 3.20B and D) for both time points studied in the live-cell imaging experiments.
The expression of the endogenous control gapdh was not affected by any of the tested ToCP concentrations. The solvent control had no effects on the RNA expression compared to the untreated control. On DIV 2, a significant decrease in the Figure 3.20 Influence of ToCP on AMPAR subunit gria1 (A, C) and NMDAR subunit expression levels of both, gria1 grin2b (B, D) expression at DIV 2 and DIV 7 after 24 h ToCP treatment. Bars in (A) – (D) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are (F = 9.7, p≤0.001) and grin2b indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
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(F = 8.13, p≤0.001), was observed after treatment with 10 µM ToCP. Lower ToCP concentrations had no effect regarding the RNA expression levels of both tested glutamate receptor subunits. On DIV 7, the NMDAR subunit expression and the AMPAR subunit expression were reduced after 10 µM ToCP treatment, but the observed alterations were not significant. No effects on the expression level were observed by treatment with lower ToCP concentrations.
Long-term ToCP treatment significantly reduced glutamate sensitivity at concentrations as low as 1 nM. The underlying mechanisms of the reduced sensitivity might be connected to the RNA expression of glutamate receptors. With qRT-PCR different subunits of AMPAR and NMDAR (gria1 (AMPAR subunit) and grin2b (NMDAR subunit), Fig. 3.21) were analyzed after seven days of long-term ToCP treatment.
The expression of the endogenous control gapdh was not affected by any of the tested ToCP concentrations after long- term treatment, nor had the solvent control effects on RNA Figure 3.21 Influence of long-term ToCP treatment on AMPAR subunit gria1 (A) expression. After seven days of and NMDAR subunit grin2b (B) expression after ToCP long-term treatment at DIV 8. Bars in (A) and (B) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests treatment, a significant decrease are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001. in the expression levels of both gria1 (F = 27.83, p≤0.001) and grin2b (F = 38.39, p≤0.001) was observed after treatment with 100 nM ToCP. No effects on RNA expression levels were observed at lower ToCP concentrations.
After 24 h treatment, only the highest tested concentration of 10 µM ToCP inhibited glutamate receptor expression. Lower concentrations had no effect on expression levels. Only long-term treatment with 100 nM ToCP significantly reduced the expression levels of the glutamate receptor subunits.
3.4.3 Recovery of the glutamate sensitivity after ToCP exposure
ToCP incubation significantly reduced the glutamate sensitivity of mouse pCNs after 24 h treatment at concentrations ≥ 100 nM. A possible mechanism of reduced glutamate sensitivity might be the internalization of AMPARs. The AMPAR internalization might act as a protective mechanism against ToCP. AMPARs may internalize during ToCP exposure, stay functional and intact after endocytosis and recycle back into the membrane during recovery. The capacity of pCNs to recover from ToCP exposure and possibly AMPAR internalization was investigated. The possibility of regeneration of the glutamate-induced responses is biologically contemplate of
66 Results crucial importance. Herby, it is tested if the ToCP-induced effects on the glutamate signaling are reversible and if there is a concentration-dependent ability to recovery.
The glutamate sensitivity of mouse pCNs was significantly reduced in all conditions with ToCP after 24 h treatment (F = 52.84, p≤0.001) (Fig. 3.22A). These results confirmed the previous results of the time-dependency experiments (Fig. 3.17) and served as further validation.
The possibility of the PCNs to recover was investigated after 24 h and 48 h. After 24 h recovery time, most of the glutamate-induced response amplitudes recovered to control level (F = 27.97, p≤0.001). Only in the 10 µM ToCP treatment condition, amplitudes were still reduced after recovery time (Control: ratio (f340/f380) = 0.231 ± 0.016 vs. 10 µM ToCP: ratio (f340/f380) = 0.038 ± 0.017 (16 % of control)). After 48 h recovery time the glutamate- induced response amplitudes in the 10 µM ToCP condition were still reduced (F = 7.02, p≤0.001), (Control: ratio (f340/f380) = 0.228 ± 0.022 vs. 10 µM ToCP: ratio (f340/f380) = 0.095 ± 0.027 (40 % of control)) (Fig. 3.22B – C).
The mean amplitudes of glutamate as percent of the DMSO control were calculated (Fig. 3.22D) to show the recovery of the glutamate-induced amplitudes. In the 10 nM ToCP condition the neurons recover from 73 ± 3 % without recovery, to 123 ± 6 % after 24 h and to 111 ± 8 % after 48 h recovery time in comparison to controls. After treatment with 100 nM ToCP the glutamate- induced response amplitudes were able to recover (amplitude recovery: 0 h: 80 ± 8 %, 24 h: 89 ± 3 %, 48 h: 108 ± 6 %). After the treatment with 1 µM ToCP the amplitudes were 49 ± 3 %. After 24 h recovery time the amplitudes recovered to 97 ± 8 % of the control level. After 48 h recovery time the glutamate-induced response amplitudes were renewed decreased (70 ± 7 %). After treatment with 10 µM ToCP the glutamate-induced response amplitudes partially recovered compared to the control (amplitude recovery: 0 h: 5 ± 0.9 %, 24 h: 23 ± 4 %, 48 h: 63 ± 17 %).
The results of the 24 h ToCP incubation experiment (Fig. 3.17) were confirmed by the 24 h treatment of the neurons in this experiment. The general responsiveness of pCNs was affected after treatment with 1 µM and 10 µM ToCP (F = 83.84, p≤0.001) (Fig. 3.22E).
The possible regeneration of the pCNs were investigated after 24 h and 48 h recovery time. After 24 h recovery time the KCl-induced response amplitudes in the 1 µM ToCP condition recovered. The KCl-induced response amplitudes were decreased in the 10 µM ToCP condition (F = 41.29, p≤0.001) compared to the control (Control: ratio (f340/f380) = 0.246 ± 0.014 vs. 10 µM ToCP: ratio (f340/f380) = 0.036 ± 0.015 (14 % of control)). After 48 h recovery time the KCl-induced amplitudes were still reduced compared to the control (F = 25.96, p≤0.001),
67 Results
(Control: ratio (f340/f380) = 0.239 ± 0.011 vs. 10 µM ToCP: ratio (f340/f380) = 0.081 ± 0.014 (34 % of control)) (Fig. 3.22F – G).
The mean amplitudes of KCl as percent of the DMSO control show the recovery of KCl-induced responses (Fig. 3.22H). Both lowest tested ToCP concentrations (10 nM and 100 nM) caused no alteration of the general responses to KCl stimulation. After treatment with 1 µM ToCP the KCl- induced responses were significantly reduced without recovery time 88 ± 9 % and after 24 h recovery time the amplitudes recovered to control level (118 ± 14 %). After 48 h the amplitudes were decreased again (74 ± 3 %). Treatment with 10 µM ToCP lead to significantly reduced amplitudes without and with recovery time compared to the control condition (amplitude recovery: 0 h: 11 ± 2 %, 24 h: 19 ± 5 %, 48 h: 23 ± 5).
Treatment with ToCP at nanomolar concentrations significantly reduced the glutamate sensitivity. After recovery time in ToCP-free media, the glutamate-induced responses recovered completely after treatment with nanomolar concentrations and partially after incubation with micromolar concentrations. The recovery of KCl-induced responses was less pronounced than that of glutamate responses.
68 Results
Figure 3.22 Recovery of glutamate-induced responses of pCNs after 24 h ToCP treatment with different concentrations and 0.1 % DMSO as a control. Mean amplitudes of glutamate-induced responses after 0 h (A), after 24 h (B) and after 48 h (C) recovery time. The mean amplitudes of glutamate-induced responses as percentage of control is shown in (D). Mean amplitudes of KCl-induced responses after 0 h (E), after 24 h (F) and after 48 h (G) recovery time. The mean amplitudes of KCl-induced response as percentage of control is shown in (H). Bars in (B) – (H) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
69 Results
3.4.4 Effects of the ToCP metabolite CBDP on glutamate signaling
Cresyl salignin phosphate is the toxic metabolite of ToCP (Eto, Casida et al. 1962). Organophosphates and TCPs are metabolized by cytochrome P450 enzymes and are often more toxic after hepatic conversion (Ma and Chambers 1994, Sams, Cocker et al. 2004, Foxenberg, McGarrigle et al. 2007, Ellison, Tian et al. 2012). In the first part of the study at hand, it was shown that CBDP had a higher cytotoxic potential compared to ToCP. CBDP was six times more toxic as ToCP. These findings are comparable to the other literature (Bleiberg and Johnson 1965). Following that logic of increased toxicity after hepatic conversion, CBDP may have stronger effects on glutamate signaling than ToCP. The effects of CBDP on glutamate- and KCl-induced responses were therefore investigated (Fig. 3.23).
Figure 3.23 CBDP effects on glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7. Exemplary traces of glutamate- and KCl-induced Ca2+-responses to 0.1 % acetonitrile as a solvent control and CBDP treated cells (A). Glutamate-induced response amplitudes (B,) KCl-induced response amplitudes (C) and percentage of glutamate and KCl responders (D) among pCNs after 24 h treatment with various CBDP concentrations and 0.1 % acetonitrile as a control. Bars in (B) – (D) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001. The mean amplitudes of glutamate-induced responses were only weakly affected by CBDP treatment. Only the incubation with 10 µM CBDP led to a reduction of the mean amplitudes of glutamate-induced responses, whereas lower concentrations had no effect (Control:
70 Results ratio (f340/f380) = 0.185 ± 0.01 vs. 10 µM CBDP: ratio (f340/f380) = 0.144 ± 0.008 (77 % of control). The percentage of glutamate responders was unchanged throughout the entire concentrations tested. The KCl-induced responses as well as the number of KCl responders were unaffected after CBDP treatment.
In contrast to the reduced glutamate sensitivity after treatment with concentrations ≥ 100 nM ToCP, the response amplitudes as well as the percentage of responders were much less affected after CBDP treatment.
3.4.5 ToCP induced effects on glutamate signaling in rat cortical neurons
Furthermore, the ToCP-induced effects on glutamate signaling were investigated species overlapping. Rat primary cortical neurons (rat pCNs) from Wistar rats at embryonic day 18 were used at DIV 21 to investigate if the same reduction of glutamate sensitivity appears after ToCP
Figure 3.24 ToCP impaired glutamate- and KCl-induced Ca2+-responses of rat pCNs at DIV 21 after 24 h treatment. Glutamate- induced response amplitudes (A), KCl-induced response amplitudes (B) and percentage of glutamate responders and KCl responders (C) of rat pCNs after 24 h treatment with various ToCP concentrations and 0.1 % DMSO as control at DIV 21. Bars in (A) – (C) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001. treatment (Fig. 3.24). Rat pCNs were treated with various ToCP concentrations as well as 0.1 % (v/v) DMSO as a solvent control. ToCP treatment of rat pCNs had very similar effects on glutamate responses as on mouse pCNs. The glutamate sensitivity was significantly reduced at all tested ToCP concentrations (F = 67.59, p≤0.001), (Control: ratio (f340/f380) = 0.637 ± 0.02 vs. 100 nM ToCP: ratio (f340/f380) = 0.455 ± 0.029 (70 % of control) vs. 1 µM ToCP: ratio (f340/f380) = 0.473 ± 0.02 (74 % of control) vs. 10 µM ToCP: ratio (f340/f380) = 0.2 ± 0.02 (30 % of control)). After treatment with 10 µM ToCP, the percentage of glutamate responders was reduced to 41 ± 2 % (control: 100 ± 2 %), lower ToCP concentrations had no influence (Wald Chi2 = 153.08, p≤0.001).
71 Results
Only the highest ToCP concentration of 10 µM significantly reduced the KCl responders (control: 100 ± 2 % vs. 10 µM ToCP: 38 ± 2 %) (Wald Chi2 = 157.11, p≤0.001) and the corresponding KCl-induced response amplitudes were significantly reduced compared to the control (F = 43.35, p≤0.001), (Control: ratio (f340/f380) = 0.407 ± 0.016, 1 µM ToCP: (f340/f380) = 0.309 ± 0.016 (75 % of control) vs. 10 µM ToCP: ratio (f340/f380) = 0.113 ± 0.016 (27 % of control). Figure 3.25 ToCP impaired the steady state calcium levels in rat pCNs after 24 h treatment with different ToCP concentrations and 0.1 % The basal calcium levels after 24 h ToCP incubation with DMSO as control at DIV 21 (A). Data are given as 10 µM ToCP were significantly increased (F = 6.3, p≤0.001) mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, compared to the control condition (Control: *** p≤0.001. ratio (f340/f380) = 0.432 ± 0.013 vs. 10 µM ToCP: ratio (f340/f380) = 0.507 ± 0.013) (Fig. 3.25).
The concentration-dependent impairment of glutamate signaling by ToCP occurred in mouse and rat cortical neurons in vitro.
3.4.6 ToCP effects on non-glutamatergic signaling
The results of the study at hand give strong evidence for compromised glutamatergic signaling in pCNs after ToCP treatment, which is not accompanied by a general reduction in neural excitability. Further experiments aimed at unraveling whether ToCP might affect other neurotransmitter systems and receptor systems as well. For this, mouse dorsal root ganglion (DRG) neurons were chosen. In contrast to pCNs, DRGs display marked responsiveness to multiple stimuli in vitro and a variety of stimuli generate calcium responses. In DRG neurons the vanilloid capsaicin generates calcium responses via the TRPV1 receptor (Caterina, Schumacher et al. 1997). Beyond that, DRG neurons retain a considerable proportion of neurons that display calcium transients upon GABA stimulation. In DRG neurons GABA acts as excitatory neurotransmitter with the generation of calcium responses based on the developmental stage and the intracellular [Cl]i.
ToCP-induced effects on GABA signaling were investigated in DRG neurons. In calcium imaging measurements the DRG neurons were stimulated with 30 µM GABA and depolarizing buffer containing 45 mM KCl to identify viable neurons. ToCP showed no influence on GABA- or KCl- induced response amplitudes in DRG neurons. The neurotransmitter-evoked responses were
72 Results unaltered after 24 h ToCP treatment. Likewise, the responses to depolarizing buffer containing 45 mM KCl were unaffected by ToCP (Fig. 3.26).
Figure 3.26 ToCP-induced effects on GABA-induced response amplitudes (A) and KCl-induced response amplitudes (B) after 24 h treatment in DRG neurons. Bars in (A) and (B) are given as mean ± SEM.
The next experiment was performed to investigate ToCP-induced effects on TRPV1 evoked responses in DRG neurons (Fig. 3.27). The treatment with ToCP caused no effects on capsaicin- induced response amplitudes and the percentage of capsaicin responders remain unchanged.
Figure 3.27 ToCP-induced effects on capsaicin-evoked response amplitudes (A) and capsaicin responders (B) after 24 h treatment in DRG neurons. Bars in (A) and (B) are given as mean ± SEM.
GABA-evoked responses as example for neurotransmitter-evoked responses were not altered by ToCP treatment. The capsaicin-induced responses and percentage of capsaicin responders were unchanged after ToCP treatment.
73 Results
3.4.7 Direct receptor-mediated action of tri-cresyl phosphates and the metabolite CBDP
Furthermore, the acute effects of the TCP isomers and the metabolite CBDP on the glutamate receptor were investigated. In the context of the aerotoxic syndrome it might be possible that ToCP as well as the other TCP isomers and the metabolite can cause reversible short time effects on the central nervous system function. The three symmetric isomers, the TCP mixture, and the metabolite CBDP were investigated concerning their capacity to alter glutamate receptor- mediated responses. Therefore, three different concentrations (1 µM, 10 µM and 100 µM) of each TCP isomer were coapplied with glutamate direct to the neurons.
Untreated neurons were stimulated three times in succession with 30 µM glutamate to analyze if and how the glutamate-induced response amplitudes were changed by repeated stimulation. Mouse pCNs reacted with constant mean response amplitude to repetitive stimulation with glutamate. After co-application of different TmCP concentrations together with 30 µM glutamate the neurons showed no differences in the response ability and the response amplitudes were unchanged (Fig. 3.28B and E). The co-application of different concentrations of the para-isomer in addition to glutamate also revealed no alterations of the glutamate evoked response amplitudes (Fig. 3.28C and F). After co-application of 100 µM TCP mixture together with glutamate the glutamate-induced response amplitudes were significantly reduced (Fig. 3.28D and G). The response amplitudes were decreased by around 25 % compared to the control. The co-application of lower concentrations of the TCP mixture together with glutamate had no effect on the induced response amplitudes.
74 Results
Figure 3.28 Effects of TCP isomers on glutamate-induced responses of pCNs in an acute stimulation experiment. Exemplary traces showing responses to repetitive stimulation with glutamate (A), effects of co-application of glutamate with TmCP at different concentrations (B), effects of co-application of glutamate with TpCP at different concentrations (C) effects of co-application of glutamate with the TCP mixture at different concentrations (D). Effects of TmCP on glutamate-induced responses (E), effects of TpCP on glutamate-induced responses (F), effects of the TCP mixture on glutamate-induced responses (G). Bars in (E) – (G) are given as mean ± SEM.
75 Results
The effects of acute application of ToCP on glutamate-induced responses were investigated (Fig. 3.29). The effects of the different applied ToCP concentrations were investigated by co- application of ToCP with 30 µM glutamate. The second amplitudes evoked by co-application of ToCP and glutamate were calculated as percentage of the first response, which is evoked by glutamate stimulation alone. The simultaneous application of different tested ToCP concentrations and 30 µM glutamate together reduced the glutamate-induced response amplitudes concentration-dependently.
Figure 3.29 Effects of ToCP on glutamate-induced responses of pCNs in an acute stimulation experiment. Exemplary traces showing responses to repetitive stimulation with glutamate (A) and effects of co-application of glutamate with ToCP at different concentrations (B). Effects of ToCP on glutamate-induced responses (E) and percentage of responders (D). Bars are given as mean ± SEM.
The co-application of 1 µM ToCP together with glutamate had no influence on the glutamate- induced responses. The co-application of 10 µM ToCP and 30 µM glutamate reduced the glutamate-induced response amplitudes to 75 % of the response amplitudes elicited by glutamate alone. The co-application of 100 µM ToCP and 30 µM glutamate reduced the response amplitudes to 20 % of the response amplitudes of the control. The co-application of the highest ToCP concentration simultaneously with glutamate reduced the percentage of
76 Results glutamate responders by 50 %. However, the co-application of lower ToCP concentrations in addition to glutamate did not alter the percentage of responders.
In the next part it was investigated whether the effect of ToCP was specific for the glutamate receptor or whether the voltage-gated calcium channels were altered (Fig. 3.30). Mouse pCNs were capable of repetitively responding to KCl. The repetitive stimulation with depolarizing KCl buffer induced response amplitudes with constant power. The co-application of depolarizing buffer containing 45 mM KCl and 100 µM ToCP together reduced the response amplitudes slightly in comparison to the response amplitudes elicited by KCl stimulation alone. Lower ToCP concentrations applied together with KCl did not alter the response amplitudes.
Figure 3.30 Acute impairment of KCl induced responses by ToCP. Exemplary traces for repetitive stimulation with KCl (A) and blockage of the second response by different ToCP concentrations (B). Block of KCl-induced responses by ToCP (C). Bars in (C) and (D) are given as mean ± SEM.
The effects of acute application of CBDP on glutamate-induced responses were investigated (Fig. 3.31). The metabolite CBDP displayed interestingly other alterations than the blockage of glutamate receptor-mediated responses. After co-application of different CBDP concentrations and 30 µM glutamate, the glutamate responsiveness increased compared to the control condition. After co-application of 1 µM CBDP with 30 µM glutamate the response amplitudes increased to 125 % compared to the control amplitudes evoked by glutamate alone. The co- application of 10 µM CBDP together with 30 µM glutamate caused as an increase of the
77 Results response amplitudes to 120 % of control amplitudes as well. The co-application of 100 µM CBDP and glutamate lead to response amplitudes comparable to the control.
Figure 3.31 Acute impairment of glutamate induced responses by CBDP. Exemplary traces for repetitive stimulation with glutamate (A) and blockage of the second response by different CBDP concentrations (B). Block of glutamate-induced responses by CBDP (C) and percentage of responders (D). Bars in (C) are given as mean ± SEM.
Co-application of glutamate with the ortho-isomer of ToCP led to a reduction of the mean amplitudes of glutamate-induced responses of pCNs whereas the other isomers had no effect. There might be a direct receptor-mediated action of ToCP with a very specific mechanism. The TCP mixture, containing 2 % ToCP, was able to partially block the glutamate receptor-mediated responses.
Interestingly, acute application of low CBDP concentrations simultaneously with glutamate increased the response amplitudes.
3.4.8 Mechanism and target of ToCP-induced block of glutamate responses
In the co-application experiments described above, glutamate-induced responses were reduced in the presence of micromolar concentrations of ToCP. As a next step, the type of glutamate receptors involved in the ToCP-induced reduction of glutamate-induced response amplitudes
78 Results was investigated. Glutamate receptors are sub classified into three groups, namely AMPA, kainate, and NMDA type receptors (Kew and Kemp 2005, Collingridge, Olsen et al. 2009). These receptors can be identified by their specific pharmacological properties. Specific antagonists for NMDARs and AMPARs can be used to identify these receptor subtypes (Fig. 3.32). The specific antagonist for the NMDAR subtype was MK801, a non-competitive open channel blocker. AMPAR responses could be blocked by the competitive antagonist ZK200775 in a concentration of 100 nM. Application of MK801 blocks the NMDA part of the glutamate-induced Figure 3.32 Effects of NMDAR blocker MK801 and AMPAR blocker ZK200775 on glutamate and AMPA induced responses of mouse pCNs in vitro. Exemplary traces: responses and the response Block of glutamate- and AMPA-induced responses by MK801 (A), and block of amplitudes of AMPA-induced glutamate- and AMPA-induced responses by ZK200775 (B). and glutamate-induced response amplitudes were similar under blocker conditions. The specific AMPAR antagonist ZK200775 was investigated. ZK200775 (100 nM) completely abolished responses induced by 30 µM AMPA. The glutamate-induced response amplitudes after simultaneous application of ZK200775 and 30 µM glutamate were inhibited. It seems that only the NMDAR part of the response amplitudes remained.
3.4.8.1 ToCP affects NMDA receptor-mediated responses
The type of glutamate receptors involved in the reduction of glutamate-induced response amplitudes after co-application of glutamate and ToCP was investigated. By the co-application of 10 µM ToCP or 100 µM ToCP with 30 µM glutamate the glutamate-induced response
79 Results amplitudes were significantly reduced. The use of ZK200775 as the specific AMPAR antagonist allowed the investigation of ToCP-induced effects on NMDARs (Fig. 3.33A). The second amplitudes evoked by co-application of ToCP or ToCP and specific antagonist and glutamate were calculated as percentage of the first response, which was evoked by glutamate stimulation alone.
Figure 3.33 Acute impairment of glutamate-induced responses by ToCP and different glutamate receptor blocker. Block of glutamate-induced responses by two different ToCP concentrations, MK801 and MK801 simultaneous with ToCP (A) and block of glutamate-induced responses by two different ToCP concentrations, ZK200775 and ZK200775 simultaneous with ToCP (B). Bars in (A) and (B) are given as mean ± SEM. Results of Dunnett-T3 post-hoc tests are indicted as * p≤0.05, ** p≤0.01, *** p≤0.001.
The co-application of 10 µM ToCP or 100 µM ToCP with glutamate significantly reduced the response amplitudes. The co-application of ZK200775 in addition to glutamate significantly
80 Results reduced the response amplitudes. The AMPAR mediated part of the glutamate-induced response amplitudes was blocked. The co-application of ZK200775 together with 10 µM ToCP or 100 µM ToCP and glutamate significantly reduced the response amplitudes (F = 166.34, p > 0.001), (10 µM ToCP: 84± 4 % of control, 100 µM ToCP: 52 ± 3 %, ZK200775: 33± 4 %, ZK200775 + 10 µM ToCP: 29 ± 3 %, ZK200775 + 100 µM: 25 ± 2 %). In the both conditions with ZK200775 the influence of ToCP on the NMDAR subtype could be investigated. Here, the difference between ZK200775 as single antagonist and the simultaneous block with ZK200775 and 10 µM ToCP or 100 µM ToCP were not significantly different. The simultaneous block with ZK200775 and ToCP (10 µM and 100 µM) did not significantly alter the responses evoked by glutamate compared to the conditions without ZK200775. The NMDAR-mediated responses were not affected by ToCP. The direct receptor-mediated action of ToCP is not based on the block of NMDAR mediated responses after glutamate stimulation.
3.4.8.2 ToCP affects AMPA receptor-mediated responses
The NMDAR antagonist MK801 was used to identify possible alterations of ToCP on AMPAR mediated responses. The block of the NMDA part of the glutamate-induced response amplitudes allowed the investigation of ToCP-induced effects on the AMPAR mediated responses (Fig. 3.33B). The co-application of 10 µM ToCP or 100 µM ToCP with glutamate significantly reduced the response amplitudes. The co-application of MK801 in addition to glutamate significantly reduced the response amplitudes. The NMDA mediated part of the glutamate-induced response amplitudes were blocked. The co-application of MK801 together with 10 µM ToCP or 100 µM ToCP and glutamate significantly reduced the response amplitudes (10 µM ToCP: 84 ± 4 % of control, 100 µM ToCP: 52 ± 3 % of control, MK801: 52 ± 2 % of control, MK801 + 10 µM ToCP: 35 ± 2 % of control, MK801 + 100 µM ToCP: 30 ± 2 % of control) (F = 166.34, p≤0.001). In both conditions with MK801 the influence of ToCP on the AMPAR subtype could be investigated. In addition, there was a significant difference between the single block with MK801 and the simultaneous block with MK801 and both concentrations of ToCP. The block with MK801 was significantly different from MK801 + 10 µM and MK801 + 100 µM ToCP (Dunnett-T3 post hoc test, MK801 vs. MK801 + 10 µM ToCP: ***p≤0.001, MK801 vs. MK801 + 100 µM ToCP: ***p≤0.001). The receptor-mediated action of ToCP block the AMPAR mediated responses after glutamate stimulation. The AMPAR was identified as main target of the ToCP-receptor-mediated action.
81 Discussion
4 Discussion
The nervous system is characterized by its ability to transmit and process information via interconnected neurons and synapses. The brain has an astonishing capacity to reorganize its structure and function and to adapt to extrinsic and intrinsic stimuli. This process is called activity-dependent neuronal plasticity (Stiess and Bradke 2011, Huang and Thathiah 2015). These key features create the basis for the formation of functional networks, which receive, conduct, and transmit signals. The perturbation of these processes by chemicals leads to neurobehavioral disorders in humans and animals (van Thriel, Westerink et al. 2012). A variety of chemicals is known to alter neurite network structures and cause functional impairment. The current knowledge about alternative OP neurotoxicity with respect to functional endpoints like interaction with different neurotransmitter systems (Gant, Eldefrawi et al. 1987, Pung, Klein et al. 2006, Slotkin, Seidler et al. 2008, Slotkin and Seidler 2012) and new pathways of neurotoxicity in general (Bushnell, Kavlock et al. 2010) suggests that TCPs might have alternative modes of action of neurotoxicity compared to OPIDN (Ray and Richards 2001). Therefore, the overall goal of this thesis was to investigate new modes of actions of TCPs in addition to the well-known concept of organophosphate-induced delayed neuropathy (Abou-Donia 1981, Ehrich and Jortner 2010).
A multifaceted approach was used to characterize all levels of neuronal impairment. This approach incorporated structural and functional in vitro endpoints of neurotoxicology. The functional approach, which includes the investigation of the functionality of glutamate receptors, intracellular calcium levels, and depolarization mechanisms via VGCCs as main influx route of calcium, was studied following the idea that these specific endpoints might be more sensitive to toxins than structural endpoints. In the present study highly specific low concentration effects of ToCP could be observed. ToCP lead to (a) reduction and inhibition of glutamate-induced responses in co-application experiments with AMPARs as the main target structures, (b) a time- and concentration-dependent reduction in glutamate sensitivity, possibly by AMPAR modifications, and (c) reduced neurite outgrowth and alterations of neurite morphology and complexity. According to these findings, ToCP has a central nervous system- related, yet unknown mode of action which is characterized by specific interactions with
82 Discussion
AMPARs. The glutamate-induced responses were specifically altered by ToCP at a concentration of 100 nM which is 1000-fold lower than the cytotoxic concentration.
4.1 Impairment of glutamate signaling
Signaling by ionotropic glutamate receptors and calcium-dependent membrane depolarization by specialized voltage-gated calcium channels are essential characteristics of neuronal function and neurotransmission (Catterall, Perez-Reyes et al. 2005, Kew and Kemp 2005, Wadel, Neher et al. 2007, Simms and Zamponi 2014). Glutamate is involved in almost all brain processes (Danbolt 2001). In addition to its role in learning and memory, glutamate is also involved in signal processing in sensory perception and motion control. Furthermore, pharmacological studies underscore the importance of AMPARs and NMDARs in learning and memory (Morris, Anderson et al. 1986, Rammsayer 2001). TCP-induced impairment of glutamate signaling might lead to cognitive deficits and abnormal behavior, and as a result may have a multifactorial influence on all aspects of life, including development. In this thesis, acute, short- and long-term neurotoxic effects of TCPs were investigated with respect to glutamate signaling and calcium-dependent depolarization. The results of this work show for the first time that TCPs, and especially ToCP, impair glutamate-related signaling in mouse and rat primary cortical neurons in vitro. These findings support a mode of action of ToCP, which occurs at non-cytotoxic concentrations and has not been described before.
4.1.1 Direct receptor-mediated action of ToCP
A previous study reported that several OPs, including ToCP, interact with NMDARs (Johnson and Michaelis 1992). Using binding assays, the authors were able to show that ToCP inhibited [3H]CPP binding. Cholinergic agonists (nicotine, mecamylamine, and lobeline) have also been shown to decrease the response of the NMDAR to the agonist (Aizenman, Tang et al. 1991). The phosphonate structure of these compounds may be relevant for the structure-activity relationship between these compounds and the ionotropic glutamate receptors. Therefore, the possible interactions between TCPs with the ionotropic glutamate receptors were investigated in live cell calcium imaging experiments to explore possible direct receptor-mediated actions. More specifically, the capacity of each TCP isomer and the TCP mixture to inhibit or reduce glutamate-induced responses in co-application experiments were investigated. The results of the present study suggest that ToCP and the TCP mixture interact with glutamate receptors. The glutamate-induced responses were partially or completely blocked in a concentration- dependent manner that was also reversible. The TCP mixture, which contains only a low percentage of ToCP, also reduced the glutamate-induced responses. The low amount of ToCP in
83 Discussion the mixture may influence its basic properties, thus, leading to an enhancement of the neurotoxic potential (Henschler 1958, Henschler and Bayer 1958). These observations may results from two different modes of action. ToCP may directly interact with the glutamate receptors or, alternatively, it may cause a decrease of the glutamate responses visible in calcium imaging experiments by impairing the downstream voltage-gated calcium channels, or a combination of both mechanisms. In order to differentiate between these two modes of action, a co-application experiment with ToCP and KCl was conducted. The results clearly indicate that the KCl-induced response amplitudes remain unaltered by ToCP, thus suggesting that ToCP interacts directly with glutamate receptors. The cyclic metabolite CBDP was also investigated with regard to its possible effect on glutamate receptors. Surprisingly, CBDP increased the sensitivity of the glutamate receptor for its agonist.
The initial findings of the present work support a reduction of the glutamate-induced response amplitude in mouse pCNs in the presence of ToCP. The subsequent experiments were therefore conducted in order to identify the glutamate receptor subtype which is involved in the decreased response amplitude. Previous studies had already shown an interaction of ToCP with the NMDAR without showing functional impairment directly (Johnson and Michaelis 1992). Therefore, in the present study, the possible ToCP-induced effects on NMDARs were examined to determine whether ToCP really impairs the glutamate-induced responses via direct interaction with the NMDARs. The AMPAR-mediated porportion of the glutamate-induced response amplitudes were specifically blocked by ZK200775. This allowed the specific analysis of the NMDAR contribution to the overall glutamate response. However, the percentage of the remaining response with ZK200775 and glutamate, or ZK200775 together with ToCP and glutamate were equal in magnitude, thus indicating that NMDAR are not the target structures for ToCP in mouse pCNs.
The specific AMPAR antagonist, ZK200775 and ToCP show structural similarities in reference to the phosphonate moiety (Turski, Huth et al. 1998). Therefore, I investigated whether the AMPARs could represent a possible target for ToCP. The NMDAR-mediated contribution to the glutamate-induced responses was blocked by MK801 (Wong, Kemp et al. 1986, Woodruff, Foster et al. 1987) to evaluate the possible influence of ToCP on the AMPARs. Here, I was able to show that the responses of glutamate and the specific antagonist MK801 after co-application were significantly reduced compared the responses evoked by the simultaneous application of glutamate with MK801 and ToCP. These results indicated that the reduced response amplitudes after co-application of glutamate and ToCP are based on the reduction of the AMPA receptor- mediated proportion of the overall glutamate response. These results allow the conclusion that ToCP acts as an antagonist at AMPARs. In mouse pCNs 84 Discussion
ToCP may act as a competitive AMPAR antagonist based on its efficiency as a blocker, concentration-dependency effect, and its reversibility. The binding mode of glutamate to the glutamate receptors is highly conserved and takes place in the LBD. In this context, it is possible that ToCP interacts with the clamshell-like LBD (Mayer and Armstrong 2004, Stawski, Janovjak et al. 2010). The most conspicuous interaction is a salt-bridge formed between a positively charged arginine side chain and the negatively charged carboxylate of glutamate (D1 lobe). The mutation of this arginine residue led to a complete loss of the channel function (Kawamoto, Uchino et al. 1997, Stawski, Janovjak et al. 2010). After formation of the salt bridge, the amino group of the glutamate interacts with a conserved glutamate residue at the C-terminal end of the TMD (D2 lobe). These residues bring the two loops of the clamshell together upon binding of the ligand. Agonists that bind deeply into the clamshell structure causes the clamshell to close to a large degree (Stawski, Janovjak et al. 2010). Another study showed that the degree of the clamshell-closure correlates with the efficiency of the agonist (Mayer and Armstrong 2004). Crystal structures of the GluR2 ligand-binding core revealed that glutamate closes the clamshell cleft by around 20° whereas the partial agonist, kainate closes it by approximately 12°, and the competitive agonist DNQX only closes the cleft by around 2.5° (Armstrong and Gouaux 2000). Competitive antagonists often interfere with the closure of the clamshell and minor changes in the closing degree can completely change the receptor permeability (Mayer and Armstrong 2004). Known specific agonists of the AMPARs, such as ZK200775 and CNQX or DNQX - also known as quinoxalines (Honore, Davies et al. 1988, Turski, Huth et al. 1998), interact with the D1-arginine but have no contact with the glutamate residue at the D2 lobe. ZK200775 contains an additional phosphonate moiety and forms hydrogen bonds with proline residues on D1. ToCP may have a similar mode of action as ZK200775. However, the degree of clamshell-closure by ToCP might be lower upon interaction with the arginine and glutamate residues, and the permeability of the AMPA receptor is reduced or inhibited. ToCP prevents the cleft-closure by interacting with the LBD. Furthermore, the AMPARs may be stabilized by ToCP in the non- glutamate binding conformation and the glutamate-evoked responses could thereby be reduced. In a previous study, destabilization of the clamshell-closure conformation increased the rate of channel deactivation and the prevention of closure resulted in a loss of receptor activity (Robert, Armstrong et al. 2005).
The current study permits speculation about the underlying mechanisms of the interaction of ToCP with AMPARs. However, fluorescence-based live-cell calcium imaging is not a suitable technique to completely clarify the mode of action. For deeper studies of the precise mechanism of action of ToCP, methods such as electrophysiology and crystal structure analyses are better suited. In their study Yelshanskaya et al. (2014) could show the structure of the closed-state
85 Discussion
AMPA receptor in complex with the competitive antagonist ZK200775. Such an X-ray structure of the ToCP-bound AMPAR would help elucidate the specific binding site of ToCP. In addition, electrophysiological measurements could clarify whether ToCP is a competitive or non- competitive antagonist.
4.1.2 ToCP impairs glutamate sensitivity in a time- and concentration dependent manner
The potency of ToCP to cause cytotoxicity was investigated as a general parameter of toxicity and to confirm that all additional effects observed at lower concentration can be sufficiently distinguished from overall toxicity. Here, I could show that ToCP induced cytotoxicity in mouse pCNs with an IC50 value of 89 µM. Previous in vitro studies showed that neuroblastoma cells lines are sensitive to ToCP in the millimolar concentration range, but revealed that mouse pCNs have a higher susceptibility (Chang and Wu 2006, Chen, Sun et al. 2013). In the present study, the investigation of specific neurotoxicity related endpoints was conducted using non-cytotoxic concentrations. Different short-term and long-term treatment conditions (e.g. incubation with ToCP for various durations) were used to investigate the time-dependency and the effect size of ToCP that could impair glutamate signaling as well as the general responsiveness of the neurons after the different treatments. The AMPARs were identified as target structure of ToCP. The internalization and trafficking processes might play a role in the time- and concentration dependent impairment in glutamate sensitivity. The AMPAR turnover is quite a rapid process (Shepherd and Huganir 2007), with continuous exchange between different store pools and internalization into the cells with a time constant of about 40 min (Man, Lin et al. 2000, Triller and Choquet 2008). Under normal conditions, a similar number of AMPARs are inserted into the membrane to counteract the endocytosis and to conserve a constant level of AMPARs on the cell surface (Man, Ju et al. 2000). The nervous system is known adopted new functional and structural states in response to extrinsic factors (Bliss and Collingridge 1993). The AMPARs are internalized and can recycled back into the membrane depending up on incoming information (Opazo and Choquet 2011). The observed alterations in the percentage of glutamate responders, as well as in the glutamate- induced response amplitudes can be regarded as a reduction in glutamate sensitivity of pCNs. Incubation of pCNs with ToCP reduced the glutamate sensitivity in a time-and concentration- dependent manner after short-term treatment. After one-hour treatment, the lowest effective ToCP concentration was 1 µM. After four hours of ToCP treatment, a concentration-dependent reduction in glutamate sensitivity occurred. The percentage of glutamate responders and the glutamate-induced response amplitudes were reduced by ToCP concentrations ≥ 100 nM. The
86 Discussion
ToCP-induced reduction in glutamate sensitivity was even more pronounced upon increasing incubation time. The percentage of responders and the glutamate-induced response amplitudes were further diminished by extended incubation time. In addition to the 24 h treatment, a seven-day long-term ToCP treatment in vitro produced cytotoxicity and reduced the IC50 value to 600 nM. Furthermore, low level ToCP exposure for seven days in vitro caused significant effects on glutamate signaling with concentrations ≥ 1 nM. The long-term treatment led to a shift in the effective concentration by a factor of 100. Extended incubation time increased the adverse effects of ToCP and decreased the glutamate sensitivity in a time dependent manner.
The possible different developmental stage dependent sensitivity of pCNs cultures were investigated at two different in vitro time points, DIV 2 and DIV 7. DIV 2 reflected the developmental situation with only initially formed short and thin neurite structures. DIV 7 reflected the more mature stage of the brain with several neurites per neuron and a stable neuronal network. It was noticeable that the responsiveness of mouse pCNs to glutamate and depolarization increased over the course of culture as they matured, most likely due to the increased expression and the multiplied assembly of the appropriate ion receptors on the membrane. The minor responsiveness in the early stage of the in vitro culture might be partly due to the stressful conditions during preparation. At DIV 2, the lowest effective ToCP concentration was 1 nM, while at DIV 7 the lowest effective ToCP concentration was 100 nM. The different sensitivity of pCNs towards ToCP was mostly based on the maturation process of the neurons and the increasing neuronal network density during in vitro culture. Apparently, mature neurons in vitro are more resistant against the adverse influence of chemicals.
One possible reason for the observed time-dependency of reduced glutamate sensitivity could be due to the highly dynamic AMPAR trafficking and internalization processes (Henley, Barker et al. 2011, Hanley 2014). The exposure to ToCP might lead to a remodeling of the neurons, where at the start of exposure the neurons continue to internalize and recycle AMPARs normally. The results of the co-application experiments suggest that ToCP could directly interact with this glutamate receptor subtype. Owing to the finding that ToCP indeed appears to interact with AMPARs, the toxin might specifically modify AMPARs. These modifications in turn might change the rate between AMPAR internalization and insertion in the neuronal membrane. Following that line of thought, the ratio between internalization and recycling should be concentration-dependently altered. During shorter incubation times, only pCNs treated with higher ToCP concentrations would undergo a changed ratio of AMPAR internalization and reinsertion. As the incubation time increases, the internalization/insertion ratio of AMPARs changes even at the lower ToCP concentrations. More AMPARs should then become internalized and stored intracellularly without reinsertion in the membrane. This model could explain the 87 Discussion observed reduction of glutamate sensitivity of the ToCP-treated neurons. The reason for increased AMPAR internalization might be associated with an excitation of NMDARs (Malenka and Bear 2004), and it is known in the context of LTD. NMDAR over excitation can provoke excitotoxicity, and indeed, some OPs are known to cause neuronal cell death by inducing excitotoxicity (Rush, Liu et al. 2010). It is conceivable that ToCP follows the same mode of action.
Impairment of glutamate receptor expression as an underlying mechanism of reduced glutamate sensitivity
ToCP might not only modify the AMPARs by direct interaction. Maybe the expression of glutamate receptors can be affected. Glutamate receptor expression after 24 h and after long- term ToCP treatment over seven days was investigated to identify underlying mechanisms of the reduced glutamate sensitivity. Several studies indicate that toxin exposure leads to the up- or downregulation of several genes (Lattanzi, Corvino et al. 2013, Grinberg, Stöber et al. 2014). Prior studies have also shown that organophosphates can to alter gene expression (Slotkin and Seidler 2011, Li, Zhao et al. 2012, Li, Lein et al. 2015). The RNA expression profiles of two glutamate receptor subunits (gria1 for AMPAR modification and grin2b for NMDAR modification) were investigated with regard to the reduced glutamate sensitivity. On DIV 2, the expression levels of both glutamate receptor subunits, gria1 and grin2b were significantly reduced after treatment with 10 µM ToCP. The same trend was observed on DIV 7 after treatment with 10 µM ToCP. After long-term treatment with 100 nM ToCP, there was significant alteration in the expression of the gria1 gene as a marker of a presumed AMPAR modification, as well as of the grin2b gene as a marker of NMDAR modification. This ToCP-induced reduction in glutamate receptor expression might contribute to the reduced glutamate sensitivity observed after 24 h and after long-term ToCP treatment at higher concentrations. However, the results of the glutamate receptor expression analysis cannot explain the reduced glutamate sensitivity of pCNs that were treated with lower ToCP concentrations. Additional mechanisms, like the increased AMPAR internalization discussed above, are likely more important here.
ToCP impairs the responsiveness of mouse pCNs to a depolarizing stimulus
The influx of calcium after a depolarizing stimulus is important for neurotransmission and the release of neurotransmitters (Wadel, Neher et al. 2007, Simms and Zamponi 2014). In addition, the phasic changes in intracellular calcium concentration through specialized voltage-gated ion channels are essential for neuronal function and activation of a multitude of downstream pathways (Clapham 2007), thus constituting a vulnerable point of neuronal function. Treatment with different neurotoxins leads to perturbations of ion channels and relaying of action potentials (Oortgiesen, Leinders et al. 1993, Bowen, Batis et al. 2006, Cao, Shafer et al. 2011,
88 Discussion
Cao, Shafer et al. 2011). The results of this thesis show that only very high concentrations of ToCP reduced the responsiveness of pCNs to a depolarizing stimulus Thus, it can be assumed that voltage-gated ion channels, as the main route of calcium influx, were only impaired by ToCP at the highest tested concentration. The findings therefore suggest that ToCP does, if at all, only act as a weak inhibitor of VGCCs or the VGCC-mediated calcium influx. This reduction of the general responsiveness to a depolarizing stimulus is more likely related to the additional adverse outcomes of the treatment with 10 µM ToCP, which is slightly cytotoxic and reduces neurite area. This reduction in neurite area is accompanied by a decreased number of neurites equipped with VGCCs and is therefore directly associated with the reduced responsiveness to depolarization. The results of this work indicate that ToCP does not affect general neuronal excitability at very low concentrations, but instead specifically impairs glutamate signaling, thus unraveling a yet unknown mode of action for ToCP.
4.1.3 Regeneration capacity of pCNs after ToCP treatment
The process of internalization and recycling of receptors back into the membrane is known for LTP and LTD processes at the synapse (Park, Penick et al. 2004, Opazo and Choquet 2011). It is possible that the concurrent physiological processes of AMPAR internalization and insertion in pCNs were modified by ToCP treatment (Man, Lin et al. 2000, Hirling 2009, Hanley 2014) and that the ToCP-induced reduction in glutamate sensitivity might be a reversible process. The internalization of AMPARs might act as a protective mechanism against ToCP. In this scenario, increasing ToCP concentrations result in the internalization and intracellular storage of AMPARs that do not re-insert into the membrane, thus leading to the decreased glutamate-induced responses after ToCP treatment. Previous studies have hypothesized that the majority of the internalized AMPARs remain intact and functional even after endocytosis, and could be recycled back into the membrane. To address this issue, a recovery experiment was conducted that shows that the reduction in glutamate sensitivity, induced by ToCP concentrations in the nanomolar range was completely recovered after 24 h. The present work showed that low ToCP concentrations cause a specific impairment of glutamate signaling without additional, maybe more structural neurotoxic outcomes. Here, no additional effects interfere with the regeneration of the pCNs. In contrast, the work also shows the partial recovery of the glutamate- induced responses of pCNs, treated with micromolar ToCP concentrations. One possible explanation is that the regeneration of glutamate sensitivity is limited due to cumulative neurotoxic processes, such as cytotoxicity and neurite degeneration. It is possible that the influence of ToCP on neurite structures reduced the amount of surface receptors, and that neurite structures were not able to recover within this timeframe.
89 Discussion
A possible explanation for the recovery of glutamate-induced responses is that the major part of AMPARs remains functional during low ToCP treatment and only a minor proportion was modified and potentially dysfunctional as a result of ToCP binding. The functional AMPARs were inserted back into the membrane during recovery time. Previous studies have shown that the turnover of AMPARs at synapses depended on the actin cytoskeleton (Park, Penick et al. 2004, Petrini, Lu et al. 2009). AMPARs undergo selective sorting between recycling and degradative pathways following activity-dependent endocytosis with trafficking to early or late endosomes (Ehlers 2000). The sorting is activity-dependent and based on activation via NMDA and AMPA receptors, or is NMDAR independent. In the case of ToCP neurotoxicity, the sorting mechanism might depend on the ToCP concentration. It is possible that treatment with low ToCP concentrations only modified a low number of internalized AMPARs. The modified AMPARs undergo degradation and the unaltered AMPARs are trafficked to the early endosomal compartment. Higher ToCP concentrations probably modified more AMPARs and this resulted in the transport of these AMPARs to the late endosomes (Mukherjee, Ghosh et al. 1997, Buckley, Melikian et al. 2000) where they can undergo lysosomal degradation or autophagy processes, possibly activated by ToCP. Indeed, ToCP-induced autophagy was reported in differentiated SH- SY5Y neuroblastoma cells to degrade cytoskeletal proteins (Chen, Sun et al. 2013) as well as in related processes involved in OPIDN in vivo (Song, Kou et al. 2014). The actin cytoskeleton is important for different recycling pathways (Mayor and Pagano 2007, Grant and Donaldson 2009) and in the present work, micromolar ToCP concentrations degraded neurite structures and impaired the actin cytoskeleton, with potential consequences for actin-dependent recycling routes.
4.1.4 Is the reduced glutamate sensitivity a specific mode of action of ToCP?
Impairment of glutamate sensitivity in rat pCNs
In this study, the question arose whether the reduction of glutamate sensitivity was a special mode of action of ToCP on the glutamate receptor, and if whether the effect was specific for mouse primary cortical neurons. The glutamate-specific ToCP effect was investigated with a second rodent model - in primary rat cortical neurons in vitro. As showed in the mouse pCNs, I was able to observe a similar concentration-dependent reduction in glutamate sensitivity in rat pCNs, indicating that the mode of action of ToCP is independent of species.
90 Discussion
ToCP influence on GABA-evoked responses
From previous studies, it is known that OPs influence different neurotransmitter systems and act on the depolarization mechanisms of neurons (Gant, Eldefrawi et al. 1987, Pung, Klein et al. 2006, Slotkin, Seidler et al. 2008). However, the effect of OPs, including ToCP, on GABA receptors remains a controversial topic. An early study of Ali et al. (1994) showed that hens exposed to a single oral dose of ToCP (750 mg/kg body weight) showed no reduction in GABARs. However, another study by Gant and Eldefrawi et al. (1997) indicated that ToCP and other OPs influence GABA-induced Cl--influx.
In the present study, the reduction in glutamate sensitivity a specific mode of action of ToCP was discovered. To confirm this specificity and to investigate possible effects on GABARs DRG neurons were used in supplementary experiments. Using this additional cell model was also motivated by the fact that ToCP is known to interfere with the peripheral nervous system and causes degeneration of the peripheral neuronal structure at high concentrations (Cavanagh 1964, Jortner 2000). DRG neurons were stimulated with GABA after ToCP treatment to investigate the influence of ToCP on GABA-evoked responses. This work indicated that treatment with ToCP does not alter the GABA-induced response and caused no impairment of the general responsiveness. In excitatory GABA-evoked signals, the inhibition of voltage- dependent chloride channels by ToCP, which inhibit the GABA-induced Cl--influx (Gant, Eldefrawi et al. 1987), is irrelevant. In early postnatal DRG neurons, ToCP had no influence on GABA- evoked responses. Based on the described excitatory GABA-evoked responses in DRG neurons (Ben-Ari, Cherubini et al. 1989, Chen, Trombley et al. 1996, Owens, Boyce et al. 1996) and the differentially expressed cation-chloride-co-transporter. GABAA receptor opening leads to a membrane depolarization based on the Cl--efflux (Ben-Ari 2002) depending on the different homeostasis of chloride (Wang, Shimizu-Okabe et al. 2002, Wang and Kriegstein 2009, Waddell, Kim et al. 2011).
ToCP influence on capsaicin-induced responses
In a further step, possible ToCP effects on other receptors were investigated in order to clarify the specificity of the ToCP effect on glutamate-related responses. The impact of ToCP on a member of the TRP channel superfamily was investigated. DRG neurons are equipped with different TRP channels (Montell 2005, Voets, Talavera et al. 2005, Vandewauw, Owsianik et al. 2013) and TRPV1 is highly expressed in DRG neurons (Cortright, Crandall et al. 2001, Frederick, Buck et al. 2007). The response behavior to the TRPV 1 agonist capsaicin with and without ToCP treatment was compared. Interestingly, the capsaicin-induced responses were similar in the different treatment groups. Apparently, ToCP treatment does not interact with the capacity of
91 Discussion the TRPV1 channel to detect capsaicin. The obtained results of capsaicin- and GABA-evoked signals imply that the ToCP-induced reduction of glutamate sensitivity is a specific mode of action.
4.1.5 Impairment of glutamate signaling by CBDP
In vivo, ToCP undergoes metabolic activation by the microsomal cytochrome P450 system to the potent neurotoxic metabolite, CBDP (Casida, Eto et al. 1961, Eto, Casida et al. 1962, Morifusa, Yasuyoshi et al. 1967). CBDP reacts with AChE and NTE with higher affinity and is described as being six times more toxic than the parent compound (Bleiberg and Johnson 1965, Chapin, Phelps et al. 1991, Burka and Chapin 1993). The current study addressed the cytotoxic potential of CBDP in mouse pCNs. Here, I report that CBDP did indeed exhibit six fold higher cytotoxic potential compared to the parent compound, ToCP, which is in agreement with previous studies (Casida, Eto et al. 1961, Bleiberg and Johnson 1965). Based on the higher potential to cause OPIDN and to react with additional esterases (Carletti, Schopfer et al. 2011), but the results indicate that CBDP is less potent in impairing the glutamate sensitivity than the parent compound. The glutamate-induced response amplitudes decreased with the highest tested CBDP concentration, and CBDP treatment did not alter the percentage of glutamate responders. The modes of actions of CBDP that are characterized in the literature are especially predominant in the peripheral nervous system (Jortner and Ehrich 1987, Kart and Bilgili 2009). In the in vitro experiments performed for this thesis, CBDP had no impact on glutamate receptor-mediated responses.
4.1.6 Impairment of glutamate signaling by TCP isomers
Previous studies proposed that the other symmetric isomers of ToCP, namely TmCP and TpCP, are less toxic than ToCP itself due to their lower affinity for NTE and AChE (Henschler 1958, Henschler and Bayer 1958). The results of the present study indicate that both isomers are equally capable of causing cytotoxicity in mouse pCNs, with IC50 values in the same concentration range as that of ToCP. Nevertheless, distinct differences were identified between the tested isomers with respect to alterations of glutamate signaling. Among the TCPs, the ortho-isomer had the highest potency to affect glutamate signaling. These discrepancies were not correlated to the respective cytotoxic potencies. Contrary to ToCP, the other symmetric isomers, TmCP and TpCP, as well as the TCP mixture reduced the glutamate-induced response amplitudes only in the low micromolar concentration range. Their potential to impair glutamate signaling was clearly less than that observed for ToCP. Here, I also observed that TpCP and the TCP mixture were more effective at altering the glutamate signaling than TmCP, but less efficient than ToCP. The non-ortho TCPs had no specialized effect mechanism compared to ToCP. The
92 Discussion specific ToCP-induced mode of action on glutamate receptors is likely based on the structural differences between ToCP and the other isomers. The higher neurotoxicity of ToCP is supposedly based on the alkyl substitution at the ortho-position of the phenyl phosphates, which usually increases the neurotoxicity of a substance (Weiner and Jortner 1999, Ehrich and Jortner 2010). TpCP and TmCP are, in contrast to ToCP, sterically-hindered phenyl phosphates. The ortho- position of the methyl group at the aromatic ring system seems to be more reactive compared to the isomers with methyl groups at the meta- or para-position. The different positioning of the methyl group determines the activity at receptors or the reactivity with enzymes. This may explain why ToCP, but not the other isomers, causes OPIDN by NTE inhibition (Johnson 1975, Johnson 1990, Johnson and Glynn 1995, Emerick, Peccinini et al. 2010), and why it is reactive enough to build adducts with a variety of enzymes (Schopfer, Furlong et al. 2010, Carletti, Schopfer et al. 2011, Marsillach, Richter et al. 2011, Masson, Lushchekina et al. 2013).
4.1.7 Increased basal calcium levels as an endpoint of TCP-induced neurotoxicity
Functional neurons tightly regulate the intracellular calcium concentration because calcium can trigger a multitude of effects as it is an important signaling molecule (Clapham 2007). Among these effects are all aspects of neuronal development including neuronal growth (Rehder and Kater 1992, Zheng and Poo 2007). An increase in the intracellular calcium concentration leads to excitotoxicity. An increased intracellular calcium concentration observed after TCP incubation could represent an additional mode of action of TCP- and ToCP-induced neurotoxicity. In the present study, the intracellular basal calcium levels were estimated based on the fluorescence ratio (f340/f380) at the beginning of calcium imaging experiments. The three symmetric TCP isomers and the TCP mixture, but not the metabolite CBDP, increased the basal calcium level after 24 h treatment at the highest tested concentration of 10 µM. Similar effects were observed in rat pCNs after treatment with 10 µM ToCP. Changes in the calcium concentration were observed in the context of OPIDN as very early signs (Luttrell, Olajos et al. 1993, Song and Xie 2012). A previous study showed a significant increase in calcium in the sciatic nerve of hens after treatment with the ToCP metabolite, CBDP (El-Fawal and Ehrich 1993). Furthermore, Jiang et al. (2014) described that hens treated with ToCP showed a significantly increased calcium concentration in the brain compared to untreated controls. Further studies hypothesized that the influx of calcium and the activation of calcium-dependent kinases are followed by an increase in the phosphorylation levels of cytoskeletal proteins (Suwita, Lapadula et al. 1986, Suwita, Lapadula et al. 1986, Lapadula, Lapadula et al. 1992, Abou-Donia, Viana et al. 1993). The disturbed intracellular calcium concentration may be linked to other neurotoxic outcomes, like
93 Discussion neurite outgrowth inhibition and neurite degeneration, as well as axon swelling. The phosphorylation of cytoskeletal proteins activates different calcium-related processes (CAMKII- pathways) (Leist and Nicotera 1998), and accumulation of these proteins results in neurite degeneration. This may play a critical role in the context of the reduced overall neuronal responsiveness after depolarization of the neurons, which is most likely also related to the loss of neurites and a reduced number of VGCCs.
4.2 Impairment of neurite outgrowth and neurite degeneration
Inhibition of neurite outgrowth as well as degeneration of established neurite networks are specific readouts for in vitro neurotoxicity testing (Frimat, Sisnaiske et al. 2010, Krug, Balmer et al. 2013). Different studies have demonstrated that in vitro models are adequate systems to identify the chemical-dependent inhibition of neurite outgrowth (Radio and Mundy 2008, Anderl, Redpath et al. 2009, Radio, Freudenrich et al. 2010). OPs alter neurite morphology, cause neurite outgrowth inhibition, and lead to the degeneration of established neuronal networks (Howard, Bucelli et al. 2005).
In the present study, the possible impairment of neurite structure and complexity by TCPs was analyzed. Indeed, TCPs altered neurite growth, morphology, and complexity in pCNs. However, there were significant differences among the isomers in their capacity to inhibit neurite outgrowth. Neither the neurite outgrowth nor the established neurite networks were affected after treatment with different TmCP concentrations compared to the control condition. TpCP and the TCP mixture reduced the neurite area at the early developmental stage of the in vitro culture, but established neurite networks were resistant. The mean branching levels, describing the number of branches per neuron, were unaffected by this treatment. The non-ortho isomers had a lower neurotoxic potential compared to ToCP, based on the described steric hindrance and the alkyl substitution in the meta- or para-position that is known to decrease the neurotoxicity of a compound.
The specific life stage-dependent vulnerability for chemical exposure is described in literature (Rice and Barone 2000, Landrigan, Rauh et al. 2010) and is even known in the field of organophosphates and pesticides (Bruckner 2000, Weiss 2000, Xu, Purcell et al. 2000, Smith, Hinderliter et al. 2014). It was previously described that even subtle perturbations of neuronal growth can lead to adverse outcomes and neurobehavioral deficits (Rice and Barone 2000). In the current study, different methods were used to investigate the ToCP-induced effects on neurite morphology and complexity at two different time points of in vitro culture. The neurite area as a growth parameter was quantified by an automated microplate-reading system. I observed an increase in the network density during in vitro culture time showingthe maturation 94 Discussion of the neuronal cultures, which is also correlated to the increase in the responsiveness of the neurons to different stimuli. The results of this study indicate that ToCP is the more potent isomer and affects the morphology of the neurites in a concentration-dependent manner. The ToCP-induced effect was greater at the early, developmental stage of neurite outgrowth, whereas, in a previous study a different age-dependent vulnerability with regard to neurite outgrowth inhibition is reported (Ng and Lozano 1999). On DIV 2, pCNs displayed atypical short and thin neurites and micromolar concentrations reduced the density and complexity of established neuronal networks in cultures of pCNs at DIV 7. A previous study in Wistar rats showed a Wallerian-type degeneration of neurons occurring after ToCP treatment (Inui, Mitsumori et al. 1993). Together, these results suggest that early stages of network formation with short, newly formed neurite structures are more sensitive to ToCP. In addition, a long-term seven-day ToCP treatment was performed in vitro to investigate the capacity of ToCP to alter neurite outgrowth. Under this treatment condition, nanomolar concentrations reduced neurite area. In general, long-term treatment provoked greater effects on neurite outgrowth and significantly reduced neurite area upon treatment with 100 nM ToCP. Thus, long-term treatment increased the ToCP-induced effects on neurite outgrowth.
ToCP is known to affect neurite structures in differentiated, neuronal N2a and PC12 cells, as well as in retinoic-acid differentiated SH-SY5Y neuroblastoma cells in a concentration range between 200 µM and 1 mM (Flaskos, McLean et al. 1998, Flaskos 2012, Chen, Sun et al. 2013). The results of the present study indicate that a concentration dependent effect of ToCP on the morphology of neurites exists even in the low micromolar concentration range. Neurite outgrowth inhibition and neurite degeneration processes were disturbed by lower ToCP concentrations in mouse primary cortical neurons than in neuronal cell lines. PCNs were more susceptible to ToCP- induced effects with regard to neurite morphology. The use of embryonic CD1 mouse primary cortical neurons was also motivated by prior work showing that primary neurons display more accurate in vitro models of the maturation of neurons that takes place in the brain compared to neuronal cell lines (Harrill, Robinette et al. 2013). Cell lines are tumor-derived and often show different sensitivities against neurotoxin-induced effects than native neurons. Primary rodent cultures of cortical or hippocampal neurons are therefore frequently used to investigate neurite outgrowth inhibition and alterations of neurite morphology (Dotti, Banker et al. 1987, Harrill, Freudenrich et al. 2011, Harrill, Robinette et al. 2013).
The ToCP-induced effects on neurite outgrowth were studied in more detail by analyzing parameters such as neurite length, neurite branching, and neurite diameter, to characterize neurite complexity. This investigation constitutes an opportunity to analyze network integrity. ToCP might cause specific modifications to parameters of neurite complexity at lower 95 Discussion concentrations than the characterized decrease in overall neurite area. In the present study, I was able to show that 10 µM ToCP treatment significantly decreased neurite length and the branching level, whereas neurite diameter increased. The neurite diameter is a parameter which can be used to detect axon swelling in vitro. Previous studies indicate that ToCP induces axon swelling as an early event during OPIDN in vivo (Robertson, Schwab et al. 1987, Abou-Donia, Lapadula et al. 1988, Veronesi, Padilla et al. 1991). Various studies have also supported in vivo and in vitro axon swelling (Chang and Wu 2006, Song, Yan et al. 2009, Song, Zou et al. 2012), with potential underlying mechanisms that include the aggregation and deprivation of neuronal proteins, such as neurofilament proteins, β-III-tubulin, and microtubule-associated proteins (MAPs). In OPIDN the earliest observable ultrastructural alteration was the aggregation and accumulation of cytoskeletal neurofilaments and microtubules (Abou-Donia 1993). Cytoskeletal proteins are targets for ToCP and an increased phosphorylation of these proteins appears (Patton, Lapadula et al. 1986, Suwita, Lapadula et al. 1986). Indeed, in the study at hand, the immunofluorescence staining against β-III-tubulin of ToCP-treated pCNs indicated a clustering of β-III-tubulin in the swollen regions of the neurites compared to the DMSO treated controls.
The results of the present study show that ToCP impairs neurite outgrowth of pCNs. The neurite length decreased in a concentration-dependent manner, and, in this context, it would be interesting to investigate the kinetics of neurite outgrowth inhibition by ToCP. The NFA, a patterning method on microchips, was utilized to analyze the ToCP-induced neurite outgrowth inhibition during continued cultivation of individual pCNs cultures. The pCNs cultures were continuously observed for four days, and the connections per node were calculated as a marker of neurite outgrowth. Treatment with 10 µM ToCP resulted in a significant inhibition of neurite outgrowth compared to the control condition. ToCP suppressed the entire process of neurite outgrowth, and right from the start, the number of connections per node was decreased compared to the control. ToCP also delayed the elongation of the neurites, and is has been suggested to also impair the cytoskeletal proteins, the actin network, as well as the microtubule assembly (Abou-Donia 1993, Abou-Donia 1995, Ono 2007).
As described before in OPIDN, the earliest observable ultrastructural changes are the aggregation and accumulation of cytoskeletal neurofilaments and microtubules (Abou-Donia 1993), and with that the degeneration of neurites. CBDP had a higher potential to cause OPIDN compared to the parent compound ToCP. CBDP thus may have a higher potential to inhibit neurite outgrowth and to cause alterations of established neuronal networks. Interestingly, in this study I could show that the neurite structure in pCNs was unaltered after treatment with the cyclic metabolite CBDP. The more toxic CBDP did not interfere with neurite outgrowth or cause degeneration of neuronal networks. However, a previous study could show a decrease in 96 Discussion cytoskeletal proteins as a result of CBDP treatment in vivo (Jortner and Ehrich 1987). This action could not be detected in vitro in mouse pCNs in the thesis at hand.
Comparison of the three different techniques
Comparing the three techniques of neurite outgrowth measurement indicates sensitivity differences. Each technique has distinct advantages and disadvantages with respect to its application. The automated microplate-reading systems which calculate neurite area as growth parameter, are more sensitive compared to the IMARIS reconstruction or the NFA. This technique is quite rapid and highly sensitive. The NFA was used to investigate neurite outgrowth inhibition. The advantage of this system is that individual neurons can be constantly observed facilitating the investigation of the kinetics of neurite outgrowth. Patterning of neurons in a hexagonal array eliminates the measuring of outgrowth length. Furthermore, the NFA requires no fixation and staining steps because the neurons are still living during measurement. However, the NFA is time-consuming because no automated system is available for counting. The IMARIS software tool enables the reconstruction of neurite structure of stained neurons. More specific outgrowth parameters like neurite branching level, neurite length, and neurite diameter can be determined. However, IMARIS analysis can only be performed at early stages of in vitro culture and is not applicable to high-density cultures. Here, it is impossible to assign individual neurites to neurons because of the dense neuronal network.
4.3 ToCP, TCPs and the aerotoxic syndrome
TCPs are discussed as causative agents of the aerotoxic syndrome because of their known use as additives in jet engine oil (Rubey, Striebich et al. 1996, Liyasova, Li et al. 2011). Aircrew members and passengers reported a variety of symptoms after fume events that were described as the AS (Winder and Balouet 2002, Ross 2008). The cabin air as well as the cockpit is supplied with oxygen by an on board oxygen generating system, which passes the air along the jet engines to be heated, followed by an enrichment with oxygen. Even during normal operation mode, the use of bleed air bears hazards because of potential oil leakage. Particularly dangerous is this cabin air generating system during so-called fume events, where an incorrect combustion of jet engine oil takes place. The bleed air, which is potentially contaminated with jet oil, is transferred unfiltered into the cabin (van Netten 1999, van Netten and Leung 2001, Ke, Sun et al. 2014). TCPs have been detected in air supply system components, where they become airborne by pyrolysis (Van Netten and Leung 2001, Denola, Hanhela et al. 2011). The number of reports of fume events has increased (Rayman and McNaughton 1983), but are not always officially registered. Most pilots have reported some experience with the above described events at least a few times during their work life (de Boer, Antelo et al. 2015). Inhalation as a major route of 97 Discussion exposure is most relevant as TCPs enter the gaseous phase due to their evaporation at the jet engines. In the 1960s, Siegel et al. (1965) conducted an inhalation study of tri-aryl phosphates, showing some neurotoxic effects in rabbits and chickens. The authors investigated signs of OPIDN, but did not investigate neurotoxic effects via different pathways. The tri-cresyl phosphates normally have a high vapor pressure and most data referred to toxicity caused by dermal or oral uptake. The results of this study indicate that possible TCPs are more toxic in the vapor phase.
The perturbed glutamate-induced postsynaptic signaling, indicated in the conducted in vitro experiments, might be associated with the neurobehavioral symptoms of AS. Headache, confusion, disorientation, and tunnel vision were described as symptoms of AS after short time exposure (Winder, 2005). In the present study, ToCP and the TCP mixture reversibly blocked the glutamate-induced response amplitudes in co-application experiments in a concentration- dependent manner. The effect of the TCP mixture is more likely related to the exposure situation in aircrafts. The commercial oils contain only a very low percentage of ToCP. Since the 1950s, it is known that the characteristics of TCP mixtures change depending on the composition, and that low amounts of ToCP increase the neurotoxic potential (Henschler 1958). The symptoms of AS often vanish after a short time of recovery; therefore, the conducted recovery experiment in the present study with completely recovered glutamate sensitivity is highly relevant. Long-term exposure caused neurotoxicity-related AS symptoms like dizziness, memory impairment, and lack of coordination as well as respiratory, gastrointestinal, cardiovascular, skin, and irritation symptoms (Winder, 2005). ToCP significantly reduced the glutamate sensitivity in both a time and concentration dependent manner. Chronic exposure may potentially cause health problems leading to higher disqualification rates of pilots for medical reasons. In vivo, the ToCP metabolite CBDP has been described as more toxic than ToCP itself based on the results of binding studies with different esterases (Casida, Eto et al. 1961, Eto, Casida et al. 1962, Schopfer, Furlong et al. 2010, Marsillach, Costa et al. 2013, Marsillach, Hsieh et al. 2013). CBDP was discussed to be of greater relevance for AS. The results of this thesis suggest a lower potential of CBDP to impair the glutamate signaling and thus question its higher relevance for AS.
The results obtained in this thesis clearly indicate that TCPs are probably not the sole cause of AS. The measured concentrations of TCPs in flight desk air were maximally at 50 – 100 ng m-3, and often the TCPs were under the limit of detection (Denola, Hanhela et al. 2011, Solbu, Daae et al. 2011). TCP concentrations might be much higher during a fume event but together with the studies of Schindler et al. (2013) and de Ree, et al. (2014), it has emerged that the health effects in the AS can hardly be attributed only to TCP exposure (de Boer, Antelo et al. 2015). TCPs are not the only toxic ingredients and additives in jet gear oil. Beside TCPs, the cabin air 98 Discussion may contain a number of other organophosphates (Schindler, Weiss et al. 2013), for example tributyl phosphate and triphenyl phosphate, as well as tris-(2-chloroethyl) phosphate and (tris- (2-chloropropyl) phosphate from other plastics (Solbu, Daae et al. 2011). The formation of trimethylolpropane phosphate (TMPP) from TCP and trimethylolpropane ester at temperatures of 550°C, which do occur in the jet engines, was previously reported (Centers 1992, Wyman, Pitzer et al. 1993). The temperature of several hundred degrees also pyrolizes mineral oil constituents and polycyclic aromatic carbons. Plasticizer and flame retardants might also be problematic in aircrafts (Strid, Smedje et al. 2014). Other important factors include cosmic radiation, the presence of ozone, and different pressure and humidity conditions, which all cause additional health effects (Tveten, Haldorsen et al. 1997, Spengler and Wilson 2003, Hubbell, Hallberg et al. 2005, Morse 2013, Silva, Folgosa et al. 2013, Bekö, Allen et al. 2015).
4.4 Conclusion
The endpoints investigated in this study, cell viability, alteration of neurite growth, and impairment of neurochemical processes, respectively are structural and functional in vitro endpoints in the field of neurotoxicology (Bowen, Batis et al. 2006, Harrill, Freudenrich et al. 2011, van Thriel, Westerink et al. 2012, Krug, Balmer et al. 2013, Sisnaiske, Hausherr et al. 2014). The functional approach with fluorescence-based live-cell calcium imaging as a technique allows the investigation of neurotransmitter receptor function, depolarization mechanisms, and calcium concentration, and reflects the in vivo situation of the functional nervous system. The selection of the different endpoints was motivated by the expectation of a clear concentration- dependency of ToCP effects. In accordance with this hypothesis, the results of the present work show that TCPs affected mouse pCNs in vitro in the nanomolar to micromolar concentration range. Furthermore, the different tested endpoints were affected in a concentration-dependent manner. Reduction of cell viability was observed in the high micromolar and millimolar concentration range for all tested TCP isomers and their metabolites. Neurite outgrowth and neurite degeneration were affected at non-cytotoxic concentrations in the micromolar concentration range. The most sensitive endpoint of TCP-induced neurotoxicity was the perturbation of functional neurochemical processes, which was affected by nanomolar concentrations. Finally, the impairment of glutamate signaling was found to be the most sensitive endpoint of the three selected neurotoxic endpoints.
This thesis was motivated by the challenge to discover and investigate new modes of actions of TCPs, in addition to the well-known concept of OPIDN. In the present thesis, I could for the first time show that low concentrations of TCPs that do not affect cell viability or neurite structure, impair glutamate-related signaling in mouse primary cortical neurons in vitro. Moreover, the
99 Discussion
TCP isomers exhibited different potentials with respect to their ability to impair glutamate signaling, and ToCP was the most potent isomer and that may be based on the structural differences of the compounds. TpCP and TmCP are, in contrast to ToCP, sterically hindered phenyl phosphates with lower neurotoxic potential (Henschler 1958, Henschler and Bayer 1958). The ortho-position of the methyl group at the aromatic ring system seems to be more reactive compared to the other isomers with methyl groups in the meta- or para-position. The difference in the position of the methyl group might determine the activity/reactivity with the glutamate receptors.
The reduction of glutamate sensitivity has been a completely unknown mode of action for ToCP toxicity. ToCP impaired the glutamate signaling in two ways, at concentrations as low as 1 nM. The AMPAR endocytosis and its recycling back in the membrane may act as a protective mechanism of the neurons against excitotoxicity probably induced by ToCP. PCNs were able to recover their glutamate sensitivity after treatment with nanomolar ToCP concentrations. Besides the reduced glutamate sensitivity after short- and long-term treatment, ToCP affected glutamate-induced responses in acute co-application experiments, via direct receptor mediated action. ToCP also specifically reduced the AMPAR-mediated contribution to the overall glutamate response.
The different endpoints to investigate neurotoxicity exhibit distinct differences in their sensitivity to detect neurotoxic effects of ToCP. The different endpoints show a clear hierarchy in their sensitivity. The most sensitive endpoint of ToCP-induced neurotoxicity was the impairment of neurochemical processes (e.g., glutamate signaling). Neurite morphology as another endpoint for ToCP neurotoxicity was less sensitive. In addition, cell viability is more related to general toxicity. The use of fluorescence-based live-cell calcium imaging to investigate functional processes of neurotransmission is the most sensitive technique to explore and elucidate neurotoxicity. The investigation of functional endpoints in the field of neurotoxicology is a relatively new approach. The inclusion of functional parameters is especially important to investigate critical processes for the formation of functional neuronal networks. The present work illustrates the necessity to investigate chemicals in a more multifaceted approach that recognizes all levels of neuronal impairment and include functional endpoints, which reflects the in vivo situation of the functional nervous system. The investigation of only structural endpoints, in developmental toxicology and neurotoxicology can underestimate the hazardousness of chemicals.
The results of this study can be interpreted in the context of the adverse outcome pathway framework. The AOP concept is a new tool in toxicology and provides a mechanistic
100 Discussion representation of a toxicological event. The ToCP interaction with the glutamate receptor is supposed to be the molecular initial event. The results suggest that the AMPA receptor is the main target structure of ToCP and that the mode of action is specific, because ToCP in mouse pCNs caused no alterations of GABA- or capsaicin-induced responses. The reduction in the glutamate sensitivity lead to adverse outcomes, such as neurite degeneration and destabilization of neuronal networks. This structural and functional modifications lead to cognitive deficits, which might be observable in studies with ToCP exposed animals.
101 List of Figures
5 List of Figures
Figure 1.1 Neuronal development and neurite outgrowth...... 7 Figure 1.2 Regulation of AMPAR trafficking during synaptic plasticity...... 12 Figure 1.3 Different stereo isomers of tri-cresyl phosphate...... 21 Figure 1.4 Metabolism of ToCP...... 22 Figure 1.5 Metabolism of TpCP...... 23 Figure 2.1 Quantitative analysis of neurite morphology with the IMARIS software tool ...... 37 Figure 2.2 8-in-1 hydrostatic pressure driven application system...... 38 Figure 3.1 Morphology of mouse primary cortical neurons...... 42 Figure 3.2 Effects of TCP isomers on cell viability...... 43 Figure 3.3 ToCP affects cell viability of mouse pCNs...... 44 Figure 3.4 Effects of CBDP on cell viability of mouse pCNs...... 45 Figure 3.5 Quantitative analysis of neurite outgrowth and neurite degeneration parameters of pCNs after 24 h treatment with TCP isomers TmCP and TpCP as well as the TCP mixture...... 47 Figure 3.6 Quantitative analysis of neurite density and complexity of pCNs at DIV 2 and DIV 7...... 48 Figure 3.7 Quantitative analysis of complexity of pCNs at DIV 2...... 49 Figure 3.8 Quantitative analysis of neurite degeneration parameters of pCNs after long-term ToCP treatment with different concentrations...... 50 Figure 3.9 Network formation assay of pCNs treated with ToCP...... 51 Figure 3.10 Quantitative analysis of neurite outgrowth and neurite degeneration parameters of pCNs after CBDP treatment...... 52 Figure 3.11 Half-maximal activating glutamate concentration in mouse pCNs at DIV 7...... 53 Figure 3.12 Effects of TCP isomers on glutamate- and KCl-induced Ca2+-responses of pCNs at DIV 7 after 24 h treatment with various toxin concentrations and 0.1 % (v/v) DMSO as a solvent control...... 55 Figure 3.13 TCP isomers impaired the basal calcium levels in pCNs at DIV 7 after 24 h treatment with different TCP isomers at various concentrations and with 0.1 % (v/v) DMSO as a solvent control...... 56 Figure 3.14 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 1 h treatment...... 58 Figure 3.15 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 4 h treatment...... 59 Figure 3.16 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 2 after 24 h treatment...... 61
102 List of Figures
Figure 3.17 ToCP impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 24 h treatment...... 62 Figure 3.18 ToCP impaired the steady state calcium levels in mouse pCNs after 24 h continued treatment with different ToCP concentrations and 0.1 % DMSO as control at DIV 2 (A) and DIV 7 (B)...... 63 Figure 3.19 Long-term ToCP treatment impaired glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7 after 7 days of continued treatment...... 64 Figure 3.20 Influence of ToCP on AMPAR subunit gria1 (A, C) and NMDAR subunit grin2b (B, D) expression at DIV 2 and DIV 7 after 24 h ToCP treatment...... 65 Figure 3.21 Influence of long-term ToCP treatment on AMPAR subunit gria1 (A) and NMDAR subunit grin2b (B) expression after ToCP long-term treatment at DIV 8...... 66 Figure 3.22 Recovery of glutamate-induced responses of pCNs after 24 h ToCP treatment with different concentrations and 0.1 % DMSO as a control...... 69 Figure 3.23 CBDP effects on glutamate- and KCl-induced Ca2+-responses of mouse pCNs at DIV 7...... 70 Figure 3.24 ToCP impaired glutamate- and KCl-induced Ca2+-responses of rat pCNs at DIV 21 after 24 h treatment...... 71 Figure 3.25 ToCP impaired the steady state calcium levels in rat pCNs after 24 h treatment with different ToCP concentrations and 0.1 % DMSO as control at DIV 21 (A)...... 72 Figure 3.26 ToCP-induced effects on GABA-induced response amplitudes (A) and KCl-induced response amplitudes (B) after 24 h treatment in DRG neurons...... 73 Figure 3.27 ToCP-induced effects on capsaicin-evoked response amplitudes (A) and capsaicin responders (B) after 24 h treatment in DRG neurons...... 73 Figure 3.28 Effects of TCP isomers on glutamate-induced responses of pCNs in an acute stimulation experiment...... 75 Figure 3.29 Effects of ToCP on glutamate-induced responses of pCNs in an acute stimulation experiment ...... 76 Figure 3.30 Acute impairment of KCl induced responses by ToCP...... 77 Figure 3.31 Acute impairment of glutamate induced responses by CBDP...... 78 Figure 3.32 Effects of NMDAR blocker MK801 and AMPAR blocker ZK200775 on glutamate and AMPA induced responses of mouse pCNs in vitro...... 79 Figure 3.33 Acute impairment of glutamate-induced responses by ToCP and different glutamate receptor blocker...... 80
103 List of Tables
6 List of Tables
Table 2.1 Chemical Reagents and Kits ...... 27 Table 2.2 Primary antibodies ...... 29 Table 2.3 Secondary antibodies ...... 29 Table 2.4 Consumables ...... 30 Table 2.5 Technical equipment ...... 31 Table 2.6 Hanks Balanced Salt Solution ...... 33 Table 2.7 Phosphate buffered saline (PBS-/-) ...... 35 Table 2.8 Standard extracellular assay buffer ...... 39 Table 2.9 High K+ assay buffer ...... 39 Table 2.10 Reaction mixture for cDNA synthesis ...... 40 Table 2.11 Incubation protocol for cDNA synthesis ...... 40 Table 2.12 Target genes ...... 40 Table 2.13 Reagents for qRT-PCR for one sample ...... 41 Table 2.14 Thermocycler protocol for qRT-PCR ...... 41
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8 Publication List
In peer-review journals
Hausherr, V., C. van Thriel, A. Krug, M. Leist and N. Schöbel (2014). "Impairment of Glutamate Signaling in Mouse Central Nervous System Neurons In Vitro by Tri-Ortho-Cresyl Phosphate at Noncytotoxic Concentrations." Toxicological Sciences 142(1):274-84.
Sisnaiske, J., V. Hausherr, A. K. Krug, B. Zimmer, J. G. Hengstler, M. Leist and C. van Thriel (2014). "Acrylamide alters neurotransmitter induced calcium responses in murine ESC-derived and primary neurons." NeuroToxicology 43: 117-126.
Hardelauf, H., S. Waide, J. Sisnaiske, P. Jacob, V. Hausherr, N. Schöbel, D. Janasek, C. van Thriel and J. West (2014). "Micropatterning neuronal networks." Analyst 139(13): 3256-3264.
Pfeiffer-Guglielmi, B., B. Dombert, S. Jablonka, V. Hausherr, C. van Thriel, N. Schöbel and R. P. Jansen (2014). "Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons." BMC Neurosci 15: 70.
Fraatz, M., S. Naeve, V. Hausherr, H. Zorn and L. Blank (2014). "A minimal growth medium for the basidiomycete Pleurotus sapidus for metabolic flux analysis." Fungal Biology and Biotechnology 1(1): 1-8.
In preparation
Hausherr, V., Liebing, J., Schöbel, N., van Thriel, C. „Tri-cresyl phosphates – neurotoxic effects beside OPIDN.“ Toxicological Sciences.
Conferences
Presentations
Hausherr, V., Schöbel, N., van Thriel, C. “ToCP imapirs glutamate signaling of central nervous system neurons.” 14th annual meeting of the International Neurotoxicology Association (INA 2013). Egmond ann Zee, The Nethderlands. 9. – 13. June 2013.
Hausherr, V., Sisnaiske, J., Schöbel, N., van Thriel, C. “Tri-ortho-cresyl phosphate and TCP isomers – neurotoxic effects in addition to OPIDN?” GCAQE Meeting. London, United Kingdom. 24. – 25. Feburary 2015.
Hausherr, V., Sisnaiske, J., Schöbel, N., van Thriel, C. “Tri- ortho cresylphosphate and TCP isomers – neurotoxic effects beside OPIDN.” 15th annual meeting of the International Neurotoxicology Association (INA 2015). Montreal, Canada. 26. June – 1. July 2015.
Pacharra, M., Kleinbeck, S., Hausherr, V., Sisnaiske, J., van Thriel, C. “The aerotoxic syndrome: Is there a new low-level neurotoxic syndrome in the air?” 15th annual meeting of the International Neurotoxicology Association (INA 2015). Montreal, Canada. 26. June – 1. July 2015.
Poster
Sisnaiske, J., Hardelauf, H., Waide, S., Schöbel, N., Hausherr, V., Hengstler, JG., West, J., van Thriel, C. „The Network Formation Assay.” Bio.Dortmund, Dortmund, Germany, 28. October 2011.
Schöbel, N., Hausherr, V., Sisnaiske, J., Henstler, JG., van Thriel. „A live-cell imaging-based screening for toxin effects on neurotransmitter function.“ 78. Jahrestagung der Deutschen Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie e.V., Dresden, Germany, 19. – 22. Juni 2012, Naunyn-Schmiedeberg´s Archives of Pharmacology, 385, Suppl. 1, p. 85.
Sisnaiske, J., Hardelauf, H., Hausherr, V., Waide, S., Schöbel, N., Janasek, D., Henstler, JG., West, J., van Thriel, C. „Patterned growth of primary cortical neurons on microprinted surfaces.” Bio.Dortmund. Dortmund, Germany, 23. October 2012.
Schöbel, N., Sisnaiske, J., Hausherr, V., Waide, S., Jacob, P., Janasec, D., Hardelauf, H., Hengstler, JG., West, J., van Thriel, C. „A network formation assay for the generation of patterned neuronal
networks in vitro.” 41th annual meeting of the Society for Neuroscience (Neuroscience 2012), New Orleans, USA, 13. – 17. October 2012.
Hausherr, V., van Thriel, C., Schöbel, N. „Tri-ortho cresylphophate impairs neurite outgrowth and glutmate sensitivity of central nervous system.“ 42th annual meeting of the Society for Neuroscience (Neuroscience 2013), San Diego, USA, 9. – 13. November 2013.
Sisnaiske, J., Hausherr, V., van Thriel, C., Schöbel, N. „ Single cell imaging identifies toxic effects on functions relevant for neuronal communication.“ 42th annual meeting of the Society for Neuroscience (Neuroscience 2013), San Diego, USA, 9. – 13. November 2013.
Hausherr, V., van Thriel, C., Schöbel, N. „Neurotoxic effects of tri-ortho cresylphophate in vitro.” 53th annual meeting of the Society of Toxicology (SOT 2014), Phoenix, USA, 23. – 27. March 2014.
Hausherr, V., Sisnaiske, J., Schöbel, N., van Thriel, C. “Comparison of neurotoxic effects of tri- ortho cresylphosphate and its metabolite cresyl salignin phosphate in vitro.” Bio.Dortmund. Dortmund, Germany, 28. October 2014. van Thriel, C., Sisnaiske, J., Hausherr, V. „Neurotoxic effects of tri- cresyl phophates (TCPs) and cresyl salignin phaophate (CBDP) in vitro.” 54th annual meeting of the Society of Toxicology (SOT 2015), San Diego, USA, 22. – 26. March 2015.
Sisnaiske, J., Hausherr, V., Krug, AK., Fluri, D., Leist, M., van Thriel, C. „Defeating the animal: Alternative systems for neurotoxicology testing.” 54th annual meeting of the Society of Toxicology (SOT 2015), San Diego, USA, 22. – 26. March 2015.
Sisnaiske, J., Schäfer, D., Hausherr, V., Leist, M., Ramirez-Hernandez, T., Landsiedel, R., van Triel, C. “Neuronal cell models and methods simulating nervous system funbction to screen for neurotoxic compounds.” 15th annual meeting of the International Neurotoxicology Association (INA 2015). Montreal, Canada. 26. June – 1. July 2015.
9 Curriculum Vitae
Personal
Name: Vanessa Hausherr Date of birth: 20.03.1984, Hattingen, Germany Email: [email protected] Nationality: German
Academic Career 11/2011 - Graduate Student, International Graduate School of Bioscience (IGB), Ruhr University Bochum Research associate at the Leibniz Research Centre of Working Environment and Human Factors, Project Group Neurotoxicology and Chemosensation, Supervisor: PD. Dr. C. van Thriel Title of the dissertation: “Neurotoxicity of tri-cresyl phosphate - Impairment of glutamate signaling in mouse central nervous system neurons in vitro” 10/2008 – 12/2010 Master of Science in Chemical Biology at the Technical University Dortmund Title of master thesis: “Characterization of different neuronal cell types” performed at the Leibniz Research Centre of Working Environment and Human Factors, Project group Systems Toxicology, Supervisor: Prof. Dr. J. Hengstler 10/2004 – 09/2008 Bachelor of Science in Chemical Biology at the Technical University Dortmund Title of bachelor thesis: “Pleurotus Sapidus: Minimal growth media development and metabolic flux analysis” performed at the Faculty of bio and chemical engineering, Department of Technical Biochemistry, Technical University Dortmund, Supervisor: Prof. Dr. H. Zorn
School Training 2000 – 2004 Gymnasium Holthausen, Hattingen, Germany, Qualification: Higher education for university entrance qualification 1994 – 2000 Realschule Grünstraße, Hattingen, Germany
Awards
June 2013 Student travel award at the 14th annual meeting of the International Neurotoxicology Association (INA 2013), Egmond ann Zee, The Nethderlands.Title of presentation: “ToCP imapirs glutamate signaling of central nervous system neurons.”
10 Danksagung
Ich möchte mich bei allen Menschen bedanken die mir geholfen haben diese Arbeit zum Erfolg zu führen!
Zuerst möchte ich mich ganz herzlich bei PD. Dr. Christoph van Thriel für die Möglichkeit bedanken die vorliegende Arbeit unter seiner Führung zu verwirklichen. Vielen Dank für die Unterstützung, die vielen Diskussionen und die stetige Förderung und Motivation.
Bei Prof. Dr. Hermann Lübbert möchte ich mich ganz herzlich für die Übernahme des Korreferates bedanken.
Mein Dank gilt auch Prof. Dr. Jan Hengstler für viele Diskussionen und zusätzliche Ideen.
Bei Anne Krug und Prof. Dr. Marcel Leist von der Universität Konstanz möchte ich mich für die Zusammenarbeit und die Hilfe beim Auslesen und Auswerten der Cellomics-Platten bedanken.
Liebe Nicole, dir gilt ganz besonderer Dank. Danke für die Einführung in die Technik des Calcium Imagings, die diese Arbeit erst ermöglicht hat. Vielen Dank für dein konstantes Interesse, die vielen hilfreichen Diskussionen, die immerwährende Unterstützung, den Beistand wenn es nicht rund lief und natürlich für die lustigen Stunden im Büro.
Liebe Dr. Julia Liebing und liebe Ramona Lehmann: Ihr wart immer für mich da. Danke für die vielen lustigen Begebenheiten im Labor und Büro und genauso für die vielen wissenschaftlichen Diskussionen mit der Kaffeetasse in der Hand. Danke für die gemeinsame Zeit.
Vielen Dank an die ganze Arbeitsgruppe NBTox für die tolle Arbeitsatmosphäre, die vielen Gespräche und die Unterstützung. Weiterhin möchte ich mich bei Regina Stöber, Katharina Rochlitz und den vielen anderen aus der Projektgruppe Systemtox für die vielen gemeinsamen Stunden im Labor und das „offene Ohr“ bei Problemen bedanken.
Als nächstes möchte ich mich recht herzlich bei allen bedanken die die Arbeit Korrektur gelesen haben, insbesondere bei Nicole, Julia und Ramona, Dušan Grgas, Michael Schäper, und Dr. Rosemarie Marchan.
Anschließend möchte ich mich bei meinen Freunden bedanken, die mich immer unterstützt haben und die immer die richtigen Worte gefunden haben um mich zu motivieren.
Ich möchte mich bei meinen Eltern und Großeltern für die jahrelange Unterstützung und die Begleitung durch das Studium und die Promotionszeit bedanken – ihr seid die Besten.
Als Letztes gilt mein Dank meinem Mann Benjamin Hausherr der mich wirklich immer bedingungslos unterstützt und mit den passenden Sätzen und Taten motiviert hat. Danke mein Schatz!
Erklärung
Hiermit erkläre ich, dass ich die Arbeit selbstständig verfasst und bei keiner anderen Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Schrift völlig übereinstimmende Exemplare.
Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.
Bochum, den 15.10.2015
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Ort, Datum Unterschrift