A Comparative Study of Sensory Neuron Synaptic Activity and the Role of Presynaptic Diversity, Specificity, and Regulation in Caenorhabitis Elegans

Chemical synapses are complex structures that have a diversity of specific activities that shape nervous system computation. To understand how this diversity contributes to specific circuit functions, we sought to characterize synaptic release in the stereotyped and defined neural circuitry of C. elegans. Here we use pHluorin imaging (Vglut-­pH) to monitor presynaptic glutamate release from single chemosensory neurons in intact animals and characterize the dynamics of endo-­ and exocytosis from two types of glutamatergic neurons, AWCON and ASH. In Chapter 1, we describe the optimization of Vglut-­pH, we introduce a reagent for measuring synaptic calcium influx by tethering GCaMP to synaptic vesicles, and we provide our initial characterization of glutamate release in AWCON and ASH. Our results indicate that AWCON and ASH have distinct exocytosis dynamics and that AWCON exhibits synaptic release properties similar to vertebrate photoreceptors and retinal bipolar neurons, aligning with previous results from functional calcium imaging, gene expression, and circuitry. In Chapter 2, we characterize the dynamics of endocytosis in AWCON and ASH. We find that synaptic vesicle endocytosis in these neurons have kinetic features and timescales similar to that of mammalian neurons. We show that endocytosis appears to be homeostatically regulated by previous neuronal activity and is composed of at least two kinetically distinct modes, fast and slow. We show that fast retrieval is dependent on the clathrin adaptor protein AP180/CALM, suggesting that clathrin-­mediated endocytosis is important for synaptic vesicle retrieval in these sensory neurons. In Chapter 3, we compare and contrast the dependences of AWCON and ASH Vglut-­pH responses on the core synaptic vesicle release machinery and its regulators: syntaxin, synaptobrevin, SNAP-­25, unc-­13, unc-­18, RIM, complexin, and tomosyn. We find that Vglut-­pH responses are highly dependent on the core components of the SNARE complex and its regulators, but we detected significant differences in the residual responses in these mutants that suggest AWCON and ASH synapses are distinct from each other and from those of the neuromuscular junction. We find that complexin appears to act as an inhibitor of SV release in AWCON and ASH, and we find an unexpected role for tomosyn in regulating calcium influx. In Chapter 4, we describe activity-­dependent cytoplasmic pH changes in AWCON and ASH, and we conduct a series of experiments to show that these pH changes do not interfere with the measurement or interpretation Vglut-­pH signals. Our results indicate that these activity-­dependent pH changes are consistent with a depolarization-­generated acidosis and are correlated with calcium influx. We show that these pH changes in response to stimulation are not dependent on unc-­13 or unc-­18 and are thus are largely independent of synaptic release. Finally, we show that in contrast to intracellular pH, extracellular pH changes are not detected in response to sensory stimulation. In Chapter 5, we investigate the role of synaptotagmins in AWCON and ASH glutamate release using Vglut-­pH imaging. We find that AWCON basal release is highly dependent on snt-­1, whereas ASH exocytosis is intact in snt-­1 null mutants and slightly diminished in snt-­6 mutants. Our results indicate that AWC and ASH synapses have distinct requirements for snt-­1 and may use a combination of calcium sensors to mediate glutamate release. In Chapter 6, we use a combination of genetics, behavior, calcium imaging, and Vglut-­pH imaging to investigate how loss-­of-­function mutations in pkc-­1 (protein kinase C epsilon) modulate AWCON butanone olfactory preference. We find that pkc-­1 functions in AWCON downstream of presynaptic calcium influx to modulate eat-­4 dependent glutamate release and a second form of AWCON output that is important for specifying butanone olfactory preference. We identify the receptor-­type guanylate cyclase gcy-­28 and Gqα as additional important regulators of AWCON synaptic release, and identify unc-­31 (CAPS) as an additional genetic determinant of butanone olfactory preference. Finally, we suggest a model for a dual function Gqα/DAG/pkc-­1 signaling pathway that regulates synaptic vesicle release and butanone preference in AWCON. Our work in this thesis extends the characterization of C. elegans synapses from the neuromuscular junction to the presynaptic terminals of central synapses and supports a role for presynaptic diversity among distinct neuronal cell types in C. elegans. Our work emphasizes that presynaptic diversity and regulation of neurotransmitter release are important components to specifying circuit function and suggest that C. elegans will provide a deeper understanding of how presynaptic diversity, both in terms of molecular components and activity dynamics, contribute to nervous system function

High-throughput microfluidic assay devices for culturing of soybean and microalgae and microfluidic electrophoretic ion nutrient sensor

In the past decade, there are significant challenges in agriculture because of the rapidly growing global population. Meanwhile, microfluidic devices or lab-on-a-chip devices, which are a set of micro-structure etch or molded into glass, silicon wafer, PDMS, or other materials, have been rapidly developed to achieve features, such as mix, separate, sort, sense, and control biochemical environment. The advantages of microfluidic technologies include high-throughput, low cost, precision control, and highly sensitive. In particular, they have offered promising potential for applications in medical diagnosis, drug discovery, and gene sequencing. However, the potential of microfluidic technologies for application in agriculture is far from being developed. This thesis focuses on the application of microfluidic technologies in agriculture. In this thesis, three different types of microfluidic systems were developed to present three approaches in agriculture investigation. Firstly, this report a high throughput approach to build a steady-state discrete relative humidity gradient using a modified multi-well plate. The customized device was applied to generate a set of humidity conditions to study the plant-pathogen interaction for two types of soybean beans, Williams and Williams 82. Next, a microfluidic microalgal bioreactor is presented to culture and screen microalgae strains growth under a set of CO2 concentration conditions. C. reinhardtii strains CC620 were cultured and screened in the customized bioreactor to validate the workability of the system. Growth rates of the cultured strain cells were analyzed under different CO2 concentrations. In addition, a multi-well-plate-based microalgal bioreactor array was also developed to do long-term culturing and screening. This work showed a promising microfluidic bioreactor for in-line screening based on microalgal culture under different CO2 concentrations. Finally, this report presents a microchip sensor system for ions separation and detection basing electrophoresis. It is a system owning high potential in various ions concentration analysis with high specificity and sensitivity. In addition, a solution sampling system was developed to extract solution from the soil. All those presented technologies not only have advantages including high-throughput, low cost, and highly sensitive but also have good extensibility and robustness. With a simple modification, those technologies can be expanded to different application areas due to experimental purposes. Thus, those presented microfluidic technologies provide new approaches and powerful tools in agriculture investigation. Furthermore, they have great potential to accelerate the development of agriculture

Wright State University\u27s Celebration of Research, Scholarship, and Creative Activities Book of Abstracts from Friday, April 11, 2014

The student abstract booklet is a compilation of abstracts from students\u27 oral and poster presentations at Wright State University\u27s second annual Celebration of Research, Scholarship and Creative Activities on April 11, 2014.https://corescholar.libraries.wright.edu/urop_celebration/1007/thumbnail.jp

From Cleanroom to Desktop: Emerging Micro-Nanofabrication Technology for Biomedical Applications

This review is motivated by the growing demand for low-cost, easy-to-use, compact-size yet powerful micro-nanofabrication technology to address emerging challenges of fundamental biology and translational medicine in regular laboratory settings. Recent advancements in the field benefit considerably from rapidly expanding material selections, ranging from inorganics to organics and from nanoparticles to self-assembled molecules. Meanwhile a great number of novel methodologies, employing off-the-shelf consumer electronics, intriguing interfacial phenomena, bottom-up self-assembly principles, etc., have been implemented to transit micro-nanofabrication from a cleanroom environment to a desktop setup. Furthermore, the latest application of micro-nanofabrication to emerging biomedical research will be presented in detail, which includes point-of-care diagnostics, on-chip cell culture as well as bio-manipulation. While significant progresses have been made in the rapidly growing field, both apparent and unrevealed roadblocks will need to be addressed in the future. We conclude this review by offering our perspectives on the current technical challenges and future research opportunities

Role of DPY-30 Proteins in Flagella

Cilia and flagella are similar organelles found in eukaryotic cells. A microtubule-based axonemal cytoskeleton supports these organelles that project into the extracellular environment to detect various stimuli and propel fluid. These functions enable cells to sense and respond to the changing environment. Because of their importance, the fundamental mechanisms have been conserved throughout evolution. However, it remains largely unknown how axonemes are assembled and how the motility is regulated. This thesis investigated two molecules, RSP2 and RSP23, positioned in the axonemes within a T-shaped complex, the Radial Spoke (RS). Among several axonemal complexes, flagella lacking the evolutionarily conserved RS are paralyzed suggesting that this complex is crucial for motility. It was proposed that the RS transduces mechanical and chemical signals to regulate flagellar beating. These two molecules, RSP2 and RSP23, in Chlamydomonas bind the prototype calcium sensor, calmodulin. They also share a highly conserved DPY-30 domain that resembles the RIIa domain known for dimerization and targeting of cAMP-dependent protein kinase A to specific intracellular locations. Moreover, RSP2 links the head and stalk modules in the RS while RSP23 contains a Nucleoside Diphosphate Kinase (NDK) domain presumably for maintaining the equilibrium of nucleotide species. To reveal the mechanisms mediated by these molecules, mutants defective in these domains were generated. The calmodulin-binding region in RSP2, that is absent in mammalian homologs, is not required for the assembly of RS or the oscillatory beating. However, the motile mutants cannot maintain the typical helical trajectory when cells are exposed to bright light and glass barrier simultaneously. In contrast, mutants lacking the DPY-30 domain in RSP2 are paralyzed, despite the presence of all RSPs. Mutants expressing a fraction of RSP23 with inactive NDK activity generate shorter flagella with reduced amounts of RS. Together these results suggest that DPY-30 domains targets to key locations conserved molecular modules critical for flagellar elongation and motility and the diverged calmodulin-binding regions for steering the biflagellate. Models are proposed to illustrate the various roles of these molecular domains. These discoveries provide new insight on the extraordinary mechanisms in cilia and flagella

Neural Orchestration of the C. elegans Escape Response: A Dissertation

How does a nervous system orchestrate compound behaviors? Finding the neural basis of behavior requires knowing which neurons control the behavior and how they are connected. To accomplish this we measured and manipulated neural activity in a live, behaving animal with a completely defined connectome. The C. elegans escape response is a compound behavior consisting of a sequence of behavioral motifs. Gentle touch induces a reversal and suppression of head movements, followed by a deep turn allowing the animal to navigate away from the stimulus. The connectome provides a framework for the neural circuit that controls this behavior. We used optical physiology to determine the activity patterns of individual neurons during the behavior. Calcium imaging of locomotion interneurons and motor neurons reveal unique activity profiles during different motifs of the escape response. Furthermore, we used optogenetics and laser ablations to determine the contribution of individual neurons to each motif. We show these that the suppression of head movements and turning motifs are distinct motor programs and can be uncoupled from the reversal. The molecular mechanisms that regulate these motifs involve from signaling with the neurotransmitter tyramine. Tyramine signaling and gap junctions between locomotion interneurons and motor neurons regulate the temporal orchestration of the turning motif with the reversal. Additionally, tyramine signaling through a GPCR in GABAergic neurons facilitates the asymmetric turning during forward viii locomotion. The combination of optical tools and genetics allows us to dissect a how a neural circuit converts sensory information into a compound behavior

Self-powered mobile sensor for in-pipe potable water quality monitoring

Traditional stationary sensors for potable-water quality monitoring in a wireless sensor network format allow for continuous data collection and transfer. These stationary sensors have played a key role in reporting contamination events in order to secure public health. We are developing a self-powered mobile sensor that can move with the water flow, allowing real-time detection of contamination in water distribution pipes, with a higher temporal resolution. Functionality of the mobile sensor was tested for detecting and monitoring pH, Ca2+, Mg2+, HCO3-/CO32-, NH4+, and Clions. Moreover, energy harvest and wireless data transmission capabilities are being designed for the mobile sensor