Christopher Fang-Yen - US grants

interferometry; biomedical resource; lasers; clinical research;

microscopy; interferometry; biomedical resource; lasers; clinical research;

bioimaging /biomedical imaging; heterodyning; interferometry; biomedical resource; lasers; clinical research;

interferometry; neural transmission; biomedical resource; biological signal transduction; lasers; clinical research;

interferometry; neurons; action potentials; biomedical resource; lasers; clinical research;

DESCRIPTION (provided by applicant): Neural circuits are the fundamental functional units of the nervous system. A basic understanding of circuit function will provide an important basis for understanding how these circuits malfunction in neurological disorders. The study of neural circuits in small and relatively simple model animals such as C. elegans and Drosophila has many advantages, including genetic manipulability and amenability to optical techniques. Circuit analysis in these organisms has been buoyed by the recent development of 'optogenetic' methods for stimulating and inhibiting neural activity using light-sensitive ion channels and pumps [1]. Progress in optogenetics requires not only development and optimization of new opsin molecules but also new strategies and technologies for perturbing specific opsin-expressing neurons. In this project, we will develop optical and genetic methods for manipulating neural circuits with single- neuron resolution in freely moving C. elegans. This project extends previous work by Dr. Fang-Yen, in which machine vision algorithms and lasers patterned by a digital micromirror device (DMD) were used to achieve spatiotemporal control of neural activity in freely behaving worms [2]. This earlier system was limited to a spatial resolution of about 20-30 microns, which is insufficient to selectively illuminate single neurons in the animal's nerve ring (brain). In this project we will develop a next-generation system capable of resolving single neurons and subcellular features. We will approach this goal in three directions. First, we will develop instrumentation and machine vision algorithms to automatically image and track individual neurons and processes using fluorescence imaging. We will use a dual-magnification optical system to simultaneously track behavior of the entire worm and fluorescence in a smaller region. Second, we will design and implement predictive algorithms to illuminate tracked targets with compensation for the latency due to image processing and data transfer. This system will be designed with real-time feedback such that fine-tuning of its parameters can be done in an automated manner. Third, we will use our system, in combination with other methods, to elucidate the mechanisms of modulation of locomotory behaviors by dopaminergic and serotonergic circuits. By enabling, for the first time, the dynamic perturbation of individual or multiple neurons in a behaving animal, the technology we develop will become an important tool for the analysis of neural circuits, with numerous advantages compared with existing methods. In addition to improving our understanding of the circuit basis of behavior, these studies will help provide a circuit-level context for interpreting genetic mutants, for example in C. elegans models of synaptic transmission, neuronal development, and neurodegeneration. While the focus of this project is on C. elegans, we expect that our methods will be readily extensible to other model organisms. This project will be centered in Dr. Fang-Yen's laboratory but will draw on the expertise of several unpaid consultants at the University of Pennsylvania or nearby. These include Dr. David Raizen (Dept. of Neurology), an expert in C. elegans genetics and behavior, Dr. Brian Chow (Dept. of Bioengineering), an expert on optogenetic reagents, and Dr. Niels Ringstad (New York University), an expert in C. elegans genetics and neurotransmitter signaling.

? DESCRIPTION (provided by applicant): Despite the importance of sleep to our well-being and its impact on disease and work productivity, our cellular and molecular understanding of sleep regulation is limited. Sleep/wake regulation arises via the interaction between neurons. We are investigating the regulation of a sleep behavior during larval transitions called lethargus in the roundworm C. elegans. Lethargus shows both behavioral and molecular genetic similarities to sleep in mammals and Drosophila. Since identifying the neuronal circuit regulating sleep is the next step in the analysis of this behavior, we propose to identify sleep-regulating neurons in C. elegans. In Aim 1 we will conduct an optogenetic screen for interneurons that modulate feeding and locomotion rate, as sleep is characterized by cessation of these behaviors. In Aim 2 we will test whether candidate sleep-modulating interneurons identified in Aim 1 affect sleep-like sensory arousal thresholds and behavioral quiescence. We will also establish the order of action of sleep/wake active neurons. Our studies will make use of innovative optogenetic stimulation, optical imaging, and image processing technologies for perturbing neurons, quantifying feeding movements, measuring quiescence behavior, and measuring arousal thresholds. Our overall goal of identifying every interneuron modulating sleep/wake in an animal is a goal never before accomplished in any animal, yet it is feasible in C. elegans due to the simplicity of its nervous system. Our experiments will provide a knowledge base for mechanistic studies of genes affecting C. elegans sleep-like behavior as well as for physiological studies of sleep-regulating neurons.

PROJECT SUMMARY A substantial fraction of the variation in aging can be explained by genetics, but little is known about what these genetic factors are and how they modulate healthy aging. Much of our fundamental understanding of mechanisms that modulate aging, including insulin signaling and proteostasis pathways, come from the study of the model nematode Caenorhabditis elegans. Nearly all C. elegans aging studies use a single laboratory- adapted strain with little connection to natural variation. In this project we aim to establish C. elegans as a model for natural variation in aging. First we will develop a system for multi-modal automated healthspan assays based on a previously developed `WorMotel' microfabricated multi-well imaging platform. Second, we will carry out longitudinal assays on 16 genotypically diverged wild-isolate C. elegans strains to determine traits correlated with healthspan decline. Our results will set the stage for comprehensive mechanistic analysis of genes underlying natural variation in aging. The combination of high-throughput healthspan analysis and quantitative genetics methods will be particularly powerful for delineating genetic causes of a complex healthspan phenotype, providing the first mechanistic understanding of natural variation in metazoan aging.

PROJECT SUMMARY The roundworm C. elegans is one of the most widely studied and powerful genetic model organisms in biology. Many experiments in C. elegans genetics require time-consuming and low throughput manual procedures. For example, the forward genetic screen, through which genes influencing a phenotype are found by identification of mutants, often involves assessment of tens of thousands of individual animals. Full exploitation of C. elegans as a model system will benefit tremendously from automation. We will develop automated methods for performing genetic screens and many other tasks routinely done in C. elegans labs. The key components of the system will be a moving-camera machine vision system for monitoring individual worms on various substrates, and a robotic arm capable of transferring individual worms from one plate to another. To test and refine our methods, we will apply our automation system to perform a saturation genetic screen for modifiers of developmentally-timed sleep, which has molecular and behavioral similarities to sleep in mammals.