A Novel Long-term, Multi-Channel and Non-invasive Electrophysiology Platform for Zebrafish.

Zebrafish are a popular vertebrate model for human neurological disorders and drug discovery. Although fecundity, breeding convenience, genetic homology and optical transparency have been key advantages, laborious and invasive procedures are required for electrophysiological studies. Using an electrode-integrated microfluidic system, here we demonstrate a novel multichannel electrophysiology unit to record multiple zebrafish. This platform allows spontaneous alignment of zebrafish and maintains, over days, close contact between head and multiple surface electrodes, enabling non-invasive long-term electroencephalographic recording. First, we demonstrate that electrographic seizure events, induced by pentylenetetrazole, can be reliably distinguished from eye or tail movement artifacts, and quantifiably identified with our unique algorithm. Second, we show long-term monitoring during epileptogenic progression in a scn1lab mutant recapitulating human Dravet syndrome. Third, we provide an example of cross-over pharmacology antiepileptic drug testing. Such promising features of this integrated microfluidic platform will greatly facilitate high-throughput drug screening and electrophysiological characterization of epileptic zebrafish

Microfluidics for Investigation of Electric-Induced Behaviors of Zebrafish Larvae

Zebrafish has emerged as a model organism for studying the genetic, neuronal and behavioral bases of diseases and for drug screening. Being a vertebrate, they are phylogenetically closer to humans than invertebrates, possess complex organs and the overall organization of their brain shows structural similarities with human. They are small at larval stages, optically transparent and easy to culture. In addition, zebrafish models of human diseases and genetic mutants are widely available. These characteristics make this vertebrate model an ideal organism for neurodegeneration study and drug screening from the molecule to whole organism level. Despite these attractive features, the conventional zebrafish screening methods used for movement-based behavioral tests are mostly time-consuming, uncontrollable, qualitative, low-throughput and inaccurate. Zebrafish larvae behavioral response to various stimulations including optical and chemical stimuli, have been already investigated. However, zebrafish sensory-motor responses to electrical signals, a controllable stimulus which its potential in inducing locomotion response was proven in research done before, have not been broadly studied. Examples of research questions remaining to be answered are if zebrafish electric induced response is sensitive to different electric current intensities, voltage drops, multiple electrical stimulation, and the electric field direction. The involvement of different pathways and genes in this response and its potential for utilization in disease studies and chemical screening, and drug discovery can also be investigated. This research aims to enhance our understanding of zebrafish electric-induced response via presenting novel microfluidic devices that address the challenges associated with monitoring the behavioral activities of zebrafish larvae in response to various electrical signals. In Objective 1 of the thesis, we designed a microfluidic device to deliver electrical stimuli to the awake and partially immobilized zebrafish larvae, screen and study their phenotypic behavioral responses and analyze the outputs. Behavioral response was characterized in terms of response duration and tail beat frequency. A multi-phenotypic microfluidic device was also developed to study the effect of electric stimulation on the heartrate. In Objective 2, attention was given to investigate the effect of electric current, voltage, and field direction on the zebrafish larvae’s response to find an optimized setting which can induce a traceable response in zebrafish. Using different habituation-dishabituation strategies, we also investigated if the zebrafish larvae show adaptation towards repeated exposures to electric stimuli. In Objective 3, we developed a quadruple-fish device to enhance the behavioral throughput of our microfluidic platform and showed the technique's effectiveness for larger sample size and faster behavioral assay. In Objective 4, our quadruple-fish device was employed to investigate the involvement of dopaminergic neurons in electric-induced movement response of zebrafish larvae. Lastly, since we could monitor the electric-induced behavioral responses of zebrafish larvae, in Objective 5, the applicability of our proposed technique in chemical toxicity and gene screening assays was investigated. This study is expected to introduce a microfluidic platform for on-demand and phenotypic behavioral screening of zebrafish larvae with applications in chemical screening and drug discovery

Insights into the opportunistic fungal pathogen Cryptococcus and neutrophilic inflammation using zebrafish models

The innate immunity provides the first line of defence against infection and inflammation. Zebrafish are a proven model for understanding the in vivo biology of infection and immunity. Here I describe how I have developed and used zebrafish models to understand three different aspects of infection and immunity: 1) The development and use of a zebrafish model of the human fungal infection Cryptococcus neoformans 2) Understanding the virulence of the hypervirulent Cryptococcus gattii and 3) The mechanisms of action of the immunosuppressive drug mycophenolate mofetil (MMF). I have established an innate in vivo model for macrophage response to Cryptococcus by injecting cryptococci into zebrafish embryos. I have developed a high-content imaging method in a zebrafish model of cryptococcosis. This approach enabled me the discovery that while macrophages are critical for control of C. neoformans, a failure of macrophage response is not the limiting defect in fatal infections. I found that phagocytosis is inhibited early in infection and that increases in cryptococcal number are driven by intracellular proliferation. Moreover, macrophages favourably phagocytose cryptococci with smaller polysaccharide capsules and that capsule size is greatly increased over twenty-four hours of infection, a change that is sufficient to severely limit further phagocytosis. I then used the zebrafish model of cryptococcosis to determine the virulence of C. gattii. I have identified a mutant in the hyper virulent strain R265 that is attenuated in vivo. The attenuation of the mutant, R265 GFP14 was further confirmed in a mouse model of infection. I analysed the interaction of macrophages and R265 GFP cryptococci in zebrafish and found that the transgenic R265 GFP was rapidly cleared. Whole genome sequences revealed that R265 GFP14 has 32 kb deletion in chromosome 1, resulting in the loss of six genes. R265 wild-type and R265 GFP14 were characterised for carbon sources utilisation. Finally, following up on colleagues’ use of my zebrafish model of cryptococcosis, I investigated the action of MMF on neutrophilic inflammation. I showed that MMF treatment resulted in neutrophil cell death by apoptosis in vivo, thereby reducing neutrophilic inflammation. Thus in this thesis, I demonstrate how I combined the study of infection and immunity to better understand diseases that cause the biggest disease burden in humans. I pioneered novel approaches to studying cryptococcosis using an experimental zebrafish model, which demonstrated for the first time how the early interactions with macrophages determined the outcome of infection. I subsequently used my model to study the virulence of an emerging pathogen, Vancouver strain R265 of C. gattii, identifying a genome region that may be important for virulence. Finally, from my cryptococcosis model a new mechanism for the immunosuppressant mycophenolate mofetil was identified in macrophages. Using my expertise in neutrophilic inflammation I was able to show that there was a second mechanism in neutrophils and this may explain the usefulness of this drug in treating chronic inflammation

Light-Addressable Measurement of in Vivo Tissue Oxygenation in an Unanesthetized Zebrafish Embryo via Phase-Based Phosphorescence Lifetime Detection

We have developed a digital light modulation system that utilizes a modified commercial projector equipped with a laser diode as a light source for quantitative measurements of in vivo tissue oxygenation in an unanesthetized zebrafish embryo via phase-based phosphorescence lifetime detection. The oxygen-sensitive phosphorescent probe (Oxyphor G4) was first inoculated into the bloodstream of 48 h post-fertilization (48 hpf) zebrafish embryos via the circulation valley to rapidly disperse probes throughout the embryo. The unanesthetized zebrafish embryo was introduced into the microfluidic device and immobilized on its lateral side by using a pneumatically actuated membrane. By controlling the illumination pattern on the digital micromirror device in the projector, the modulated excitation light can be spatially projected to illuminate arbitrarily-shaped regions of tissue of interest for in vivo oxygen measurements. We have successfully measured in vivo oxygen changes in the cardiac region and cardinal vein of a 48 hpf zebrafish embryo that experience hypoxia and subsequent normoxic conditions. Our proposed platform provides the potential for the real-time investigation of oxygen distribution in tissue microvasculature that relates to physiological stimulation and diseases in a developing organism

Light-Addressable Measurement of in Vivo Tissue Oxygenation in an Unanesthetized Zebrafish Embryo via Phase-Based Phosphorescence Lifetime Detection

We have developed a digital light modulation system that utilizes a modified commercial projector equipped with a laser diode as a light source for quantitative measurements of in vivo tissue oxygenation in an unanesthetized zebrafish embryo via phase-based phosphorescence lifetime detection. The oxygen-sensitive phosphorescent probe (Oxyphor G4) was first inoculated into the bloodstream of 48 h post-fertilization (48 hpf) zebrafish embryos via the circulation valley to rapidly disperse probes throughout the embryo. The unanesthetized zebrafish embryo was introduced into the microfluidic device and immobilized on its lateral side by using a pneumatically actuated membrane. By controlling the illumination pattern on the digital micromirror device in the projector, the modulated excitation light can be spatially projected to illuminate arbitrarily-shaped regions of tissue of interest for in vivo oxygen measurements. We have successfully measured in vivo oxygen changes in the cardiac region and cardinal vein of a 48 hpf zebrafish embryo that experience hypoxia and subsequent normoxic conditions. Our proposed platform provides the potential for the real-time investigation of oxygen distribution in tissue microvasculature that relates to physiological stimulation and diseases in a developing organism