title: From intracellular recording to structural analysis – new approaches on the mode of action of anthropogenic chemicals
Creators: name: Schirmer, Elisabeth
Publisher: name: University of Bayreuth
Year: 2022
Subjects: subject: Mauthner neuron
subject: MALDI-MSI
subject: Ectothermic Vertebrates
subject: intracellular recording
subject: Bisphenol, Benzocaine
subject: 570

description: Despite their many benefits, anthropogenic chemicals also pose potential threats to human and animal health. They can potentially act on various levels of organization – from molecular level, affecting the lipid composition to neuronal key functions, affecting synaptic information transmission and circuit function. However, methods providing comprehensive and, in particular, quick information about the mode of action of anthropogenic chemicals are rare. In this thesis I first focus on functional effects of anthropogenic chemicals on neuronal function. I present the goldfish’s Mauthner neuron, a giant central command neuron, which is individually identifiable from one animal to the next and accessible for intracellular in vivo recording, as a new model to obtain quick and comprehensive information on key aspects of neuronal and circuit function. In this context, I initially present how recording in the Mauthner cell can quickly and systematically help filling the gap that currently exists between the legislation of many countries and informed decision-making on anaesthetic use in ectothermic vertebrates. The pair of Mauthner cells has a crucial role in triggering one of the most studied behavioural responses, the vital escape start in teleost fish. To fulfil this role, the Mauthner neurons need to integrate and process information from all sensory systems. This allows to directly and efficiently assay specific effects of anaesthetic agents on central processing and sensory information transmission. Concentration-effect curves further enable the informed decision-making if and at which concentration a given anaesthetic affects neuronal function. I applied the method to four chemicals commonly used as equivalently anaesthetics in fish. I demonstrate that this approach allows to rapidly obtain comprehensive and reliable information with a small number of animals, emphasizing these experiments also from an ethical point of view. These findings provide the basis for an equally informed decision-making on the anaesthetic use in fish, comparable to what is standard since the last 50 years in higher vertebrates. This is the first step in fulfilling the demands by present legislation, which demands the application of anaesthetic agents for any intervention that could be stressful for all vertebrates. I further present the unique opportunity to use the Mauthner neuron as a new model to assay the impact of anthropogenic chemicals on neuronal function, by revealing the alarming and uncompensated impact of one month of exposure to two plasticizers (bisphenol A and its substitute bisphenol S) on basically every neuronal property. Due to the broad range of application of these plasticizers they are annually released at rates of hundreds of tons into the biosphere, which makes them already ubiquitously present in the environment. Beside their proven effect to act as xeno-estrogens, recent studies also demonstrate effects on juvenile developing brains. However, for both bisphenols, the impact on the intact adult brain, whose powerful homeostatic mechanisms could potentially compensate any effects bisphenols might have on isolated neurons, is completely unknown. Using recording in the Mauthner neuron, I could reveal similar effects of both bisphenols, which strongly affect all basic aspects of neuronal function, and additionally show that these effects occur even at low, environmentally relevant concentrations. Comparable effects on neuronal function observed after exposure of EE2 (ethinyl estradiol), indicate that the examined effects after bisphenol exposition could at least partly occur due to the activation of estrogen receptors. The observed dramatic impact on the mature brain is not only worrying for aquatic organisms, but also for human health. It has been shown that offsetting balance in the brain might be the basis of severe neurological disorders. Hence, I call for the rapid replacement of both plasticizers and recommend this assay, due to its speed and resolution, to be part of early testing phases for new developed plasticizers. In the third chapter of this thesis, I address the lipid composition within two aquatic model organisms (zebrafish and water flea). I demonstrate the capacity of MALDI MSI, an analytical imaging technique being able to identify lipids and visualise their spatial distribution within thin tissue sections, by providing highly resolved histology-linked lipid pattern even down to subcellular resolution. This could serve as a basis for future studies assaying molecular changes caused by anthropogenic chemicals. Using this analytical approach, I could not only visualise the anatomical arrangement of different organs due to their lipid content in the analysed tissue section, but, in the case of zebrafish, additionally provide a deeper insight into characteristic features like different eye layers, brain regions and neuronal structures (e.g. axon of the Mauthner neuron). In summary, I present groundbreaking findings by assaying the impact of anthropogenic chemicals on neuronal and molecular level. Those approaches open up new possibilities for the risk assessment of anthropogenic chemicals.