Towards high-throughput chemobehavioral phenotypic screening in drug discovery and neurotoxicology

Industrial chemical waste is discharged into freshwater ecosystems across the world at a rate of approximately two million tons per day. Estuarine and coastal environments are exposed to this waste from sources of mining, manufacturing and wastewater treatment plants. Pharmaceuticals, micropollutants, pesticides, personal care products, surfactants, industrial additives and perfluorinated compounds are all released from wastewater treatment facilities as the current technologies underlying the treatment processes are unable to completely remove all these pollutants. This exponentially increasing exposure to anthropogenic pollutants is beginning to pose long-term and understudied risks to ecosystems, with growing evidence to suggest that chemical pollutants, at currently detected environmental concentration levels, can adversely impact the nervous systems of aquatic organisms. Aquatic ecotoxicity studies are conventionally conducted by assessing endpoints such as survival, reproduction, growth rate and estimating a median lethal or effective concentrations of a pollutant on an aquatic species. Typically small model organisms are employed in such biotests, aimed at water quality assessment and quantifying adherence to country-specific regulatory standards. Significant effort is being undertaken to increase understanding of sub-cellular mechanisms of toxicity in addition to developing novel molecular biomarkers. There is also a need for development of new laboratory models using phenotypically anchored, integrative physiological bioassays that can be utilized to extract data that can allude to population and ecosystem level conditions. The acute and chronic effects of many industrial pollutants on nervous systems remain understudied, despite the recent progress and increase of molecular techniques in aquatic ecotoxicology. This is mostly due to the lack of bioassays and analytical technologies amenable for higher throughput screening of chemicals with potential neurotoxic properties. Cell-based assays that can be run at high throughput are not easily translated to organism-level neurotoxicology. This is because cell-based assays are unable to recapitulate the functionalities of an intact central nervous system (CNS) and the associated underlying complex behavioral manifestations. As all neurotoxicants are capable of altering CNS functions and our knowledge of the fundamental mechanistic aspects of neurotoxicity is still relatively undeveloped, such effects can only be discovered, and their effects quantified, using behavioral phenotyping analysis. Neurotoxicity studies must integrate both physiologically and ecologically relevant endpoints on small model organisms that possess intact biological pathways in order to rapidly identify potential neurotoxic risks and understand the potential impacts of aquatic pollution. The aim of my thesis was to develop modular, innovative neuro-behavioral analysis systems, to be used for non-invasive assessment of neuro-behavioral endpoints in small aquatic model organisms. To address this challenge, I performed the following: In Chapter 1, I reviewed the literature with respect to technical measures to acquire basic behavioral data and subsequently the variety of innate, sensory motor and cognitive behavioral endpoints that can be obtained from small model organisms. In Chapter 2, I developed a straightforward workflow to enable high throughput screening with post-processing, and demonstrated techniques that can improve the consistency of animal tracking results. In Chapter 3, I delve into environmental chamber design factors that can influence an organisms innate behavioral preferences in addition to simple sensory motor stimulation techniques that can induce altered behaviors. In Chapter 4, I developed an actuated photic stimulus system designed to stimulate an organism’s phototactic response and deployed the system on larval zebrafish to measure their startle response following exposure to toxicants. In Chapter 5, I developed a system which could create a stable thermal gradient, enabling studies of thermal preferences on a variety of organisms and conducted a subsequent high content behavioral screen on a variety of psychoactive compounds at environmentally relevant concentrations. In Chapter 6, I leverage the techniques developed in video acquisition from prior chapters to conduct a high throughput study on the impact of long term exposure to environmentally relevant concentrations of a psycho-active pollutant (fluoxetine) on behavior, reproduction and morphology. My research targets novel applications and research avenues in neurotoxicology and behavioral ecotoxicology. The project developed fundamental analytical techniques that can enable new discoveries in eco-neurotoxicology and advanced aquatic risk assessments.