Shawn R. Lockery - US grants

The nematode Caenorhabditis elegans is a powerful experimental system whose potential for computational neuroscience is largely untapped. The simplicity of its nervous system, with exactly 302 neurons in the adult hermaphrodite, and the existence of a complete anatomical wiring diagram, raise the prospect of a biophysically accurate model of the entire nervous system. Although detailed genetic analyses and laser ablation studies have delineated the circuits necessary for most C. elegans behaviors, it has not been possible to record from the nervous system. Thus, how these circuits function is not known. The main objective of this project, therefore, is to expand upon pilot studies indicating the feasibility of making intracellular recordings from C. elegans muscles and neurons. One of the main obstacles to recording from C. elegans is the thick external cuticle that breaks conventional microelectrodes. Initial experiments show, however, that the cuticle can be penetrated with electrodes pulled from quartz glass and that dye-fills can be made of a variety of muscles. The fills are long-lasting and reflect the morphology of anatomically defined muscles in the body wall and other structures. This sets the stage for attempts to refine this technique for making intracellular recordings from muscles. The small size of C. elegans neurons (2 mm in diameter) make it unlikely that sharp electrodes can be used to make intracellular recordings. A physiological preparation has been developed, therefore, in which neurons are exposed by a microdissection technique in which a small slit is made in the cuticle. Gigaohm seals (5-20 G) can readily be obtained on the cell bodies of exposed neurons and a variety of single channel currents observed in cell-attached patches. This study will attempt to make intracellular recordings from C. elegans neurons in the whole-cell patch configuration. Whole-cell recordings will be established by mechanical disruption of the membrane patch or by the perforated patch technique. Data from the recordings will form the basis of detailed biophysical models of the circuits underlying locomotion and chemotaxis. Measurement of intrinsic properties such as resting potential, input resistance, and active currents will be used to construct biophysically realistic single-neuron models. Single- neuron models will be assembled into circuits as defined by previous studies and the models tested by comparing the effects of "ablating" neurons in the model and actual circuits. These models constitute the initial phase of a long-term effort to understand the neural basis of behavior of a single organism in its entirety.***//

Neural network optimization algorithms greatly enhance our ability to construct large-scale, dynamical models of highly interconnected networks. Until now, optimization has only been applied to networks of simplistic processing units, ignoring the integrative and temporal response properties of single neurons, thus limiting the predictive power of the models. The long-term goal of this project is to develop a hybrid modeling strategy in which optimization methods are applied to networks of realistic,multicompartmental model neurons. To accomplish this goal, we will construct a hybrid model of an actual distributed processing network composed of repeatably identifiable sensory, motor, and interneurons that computes a well-defined behavioral input-output function. Optimization will be used to predict the connectivity of as-yet-unidentified interneurons in the actual network and the predictions will be tested by identifying the interneurons by physiological and morphological means. Performance of the hybrid model will be assessed by comparing it to the performance of an a priori model in which all connection strengths are determined physiologically. The final model will be used to predict the loci of synaptic plasticity underlying nonassociative conditioning of the reflex by incorporating local learning rules and by optimization methods. The predictions will be tested by determining the actual plastic sites physiologically. This project will have the combined effect of enhancing the predictive power of optimized network models and illuminating the relation between computations at the single-neuron and network levels.

9458102 Lockery There is growing evidence that human behavior is strongly influenced by genes. Because many behavioral genes are believed to exert their influence through the nervous system, it is essential to understand how genes affect the structure and function of individual neurons. A promising approach to this problem is to study how genes control the behavior of simple animals. In many ways, the tiny (1 mm long) roundworm Caenorhabditis elegans is ideal for this investigation: the connectivity among its 302 nerve cells is known completely; the neural circuits for its behaviors have been identified; over 150 genes affecting behavior have been found; and cloning a gene to determine its function is easier than in almost any other animal. The main objectives of my research, therefore, are: (1) to make electrophysiological recordings from identified neurons in C. elegans to determine how behavior is produced in normal animals, and (2) to determine how genes affect the structure and function of the nervous system in C. elegans by recording from identified neurons in behavioral mutants. This research will deepen our understanding of the causes of behavior in this increasingly important system and, because C. elegans have homologs in mammals, it will very likely expand our knowledge of the genetic basis of behavior in more complex organisms, including humans.

DESCRIPTION (provided by applicant): The long-term objective of this research is to improve our basic understanding of the structure and function of neural circuits related to action selection. Defined as the task of resolving conflicts between competing behavioral alternatives, action selection has traditionally been carried out in non-human primates which are not amenable to a fleet of powerful experimental techniques including patch clamp recording and optogenetics. A promising approach to this impasse would be to investigate the neuronal basis of action selection in simpler organisms that are genetically tractable. The proposed research investigates the neuronal basis of action selection in the nematode Caeorhabditis elegans, an experimental system with a compact nervous system of only 302 neurons, an essentially complete anatomical wiring diagram, and a wide range of genetic, electrophysiological, and optogenetic techniques for linking molecules, genes, and neurons to behavior. This research focuses on a form of spatial orientation behavior known as klinotaxis in which rhythmic side-to-side movements of the head are biased in the direction of increasing concentration of a chemical attractant. The project is made possible by an innovative microfluidic device that presents the animal with a binary choice between fluid streams carrying different concentrations of chemoattractant, and a novel tracking system that allows one to image neuronal activity in single identified neurons in freely moving animals. The project will proceed in three phases: (1) Development of a quantitative description of klinotaxis behavior by measuring the spatiotemporal propagation of locomotory undulations and their modulation by chemoattractants in microfluidic devices. (2) Identification of the neuronal circuit for klinotaxis by optical recordings of neuronal activity, neuron- al ablations, and electrophysiological measurement of synaptic connectivity. (3) Validation of a mathematical model of action selection by photo-stimulation of identified chemosensory neurons to mimic chemosensory in- puts. The proposed research is likely to identify novel circuit motifs for action selection that can be used to generate hypotheses concerning the function of circuits regulating action selection in higher organisms. More than half of all human disease genes have a matching gene in C. elegans including diseases known to impair motor and cognitive action selection such as Parkinson's disease, Alzheimer's disease, and schizophrenia. The research is therefore likely to help trace causal connections from genetic differences to mental disorders.

This research addresses the question of how the brain uses sensory information to select the most effective behavior in a given situation. This question is addressed by studying the nematode worm C. elegans, an experimental organism whose compact nervous system of only 302 neurons is unusually well suited to investigating relationships between brain activity and behavior. The main focus of the research is to build and test a computer model of the nematode's neural network for chemotaxis, a simple yet widespread form of spatial orientation behavior in which an animal finds food, shelter, or a mating partner by directing its locomotion toward the source of an odor or taste. The model will be used to test the idea that the nematode's chemotaxis network utilizes separate neuronal pathways to signal increases and decreases in sensory input,
much like the visual system in higher organism, including humans, and should provide new insights into how neural networks function to control adaptive behaviors.

Nematode chemotaxis is accessible to young scientists and the lay public alike. The PI hosts visiting undergraduates and high school students from underrepresented groups. University undergraduates have participated in this project from its beginning and will continue to do so. These researchers (currently two Honors College students and two freshman) do real science, including laser ablation of neurons and quantitative assessment of behavior. Simplified versions of the models developed in NSF-sponsored research are a prominent unit of the PI's course in computational neuroscience.

DESCRIPTION (provided by applicant): The long-term goal of the candidate's research program is to understand as completely as possible the neuronal basis of the entire behavioral repertoire of a simple invertebrate--the nematode worm C. elegans. To this end he has developed several important new techniques that have advanced the research goals of his and other labs. However, achieving a virtually complete account of behavior will require another new technique in C. elegans: optical recording of membrane potential from multiple neurons in intact, behaving animals. The candidate seeks release from teaching and reduced administration in order to develop molecular probes for membrane potential based on green fluorescent protein (GFP), and to use these probes to further the goals of his continuing research on the neuronal basis of spatial orientation behavior in C. elegans. The career development plan has four components. Component I is to coordinate and participate in a new consortium of three PI's and six consultants to re-engineer the FlaSh protein, a promising form of voltage-dependent GFP. The consortium brings together world leaders in structural biology, biophysics, fluorescent probes, and optical recording with the aim of enhancing the speed and sensitivity of the original FlaSh molecule. If successful, the re-engineered protein will be used to elucidate the neuronal basis of chemotaxis, a widespread form of spatial orientation, in the nematode C. elegans. The proposed research is significant because it will likely lead to new genetically targetable probes of neuronal activity that can be used in many other important experimental systems. In addition, this work will deepen our understanding of neural computations underlying a common goal-directed behavior. Components II and III are, respectively, technical courses and tutorial visits to laboratories at the leading edge of optical recording technology. These two activities will give the PI new technical background needed to complete the work of Component I. Component IV is a course the PI will teach in research ethics, drawing on his extensive background in analytical philosophy.

Current techniques that measure fluorescence in single cells are usually limited to a very small optical area, typically several tens of microns in diameter, or about the size of the optical field seen through a normal upright or inverted microscope. Wide field measurements have the potential to be used in many types of studies, ranging from examinations of the effects of long-range connections on the dynamics of neuronal activity, the cellular regulation of neuronal outgrowth, and the correlation between neuronal activity and behavior in unrestrained model organisms such as c. elegans. The PI plans to develop a technically accessible and ultimately inexpensive instrument that harnesses the light-gathering power and simplicity of contact fluorescence imaging (CFI) for wide field optical recordings in cultured neural networks, brain slices, and freely moving model organisms.

The goal is to build and test a prototype of a new type of CFI instrument, the first one designed specifically for neuroscientific applications. The instrument will be tested using genetically targeted fluorescent probes expressed in cultured cortical neurons as well as neurons and muscles in the nematode C. elegans. One of the main goals is to enable the first optical recordings of excitable cells during behavior in a freely moving organism.

The proposed instrument has the potential to facilitate research not only in C. elegans but also in cellular, systems, and developmental neuroscience in general. At the cellular level, the instrument could be combined with microfluidic devices and other spatial patterning methods for high-throughput testing of drugs and other biologically active substances in cell cultures. At the systems level, the new device could facilitate studies of the spatiotemporal dynamics of spontaneous and evoked neuronal activity at centimeter scale with single-neuron resolution, in some cases for the first time. A wide range of acute or cultured preparations could benefit from such an approach, including spinal cord, retina, whole ganglia, cultured hippocampal and cortical neurons, and slice or organotypic slice preparations.

Finally, the system should be quite inexpensive to build and maintain. It is envisioned that that the new device could become the imaging equivalent of a table-top centrifuge, vastly increasing the number of labs to which imaging is available.

DESCRIPTION (provided by applicant): The long-term goal of the candidate's research program is to understand as completely as possible the neuronal basis of the entire behavioral repertoire of a simple invertebrate--the nematode worm C. elegans. To this end he has developed several important new techniques that have advanced the research goals of his and other labs. However, achieving a virtually complete account of behavior will require another new technique in C. elegans: optical recording of membrane potential from multiple neurons in intact, behaving animals. The candidate seeks release from teaching and reduced administration in order to develop molecular probes for membrane potential based on green fluorescent protein (GFP), and to use these probes to further the goals of his continuing research on the neuronal basis of spatial orientation behavior in C. elegans. The career development plan has four components. Component I is to coordinate and participate in a new consortium of three PI's and six consultants to re-engineer the FlaSh protein, a promising form of voltage-dependent GFP. The consortium brings together world leaders in structural biology, biophysics, fluorescent probes, and optical recording with the aim of enhancing the speed and sensitivity of the original FlaSh molecule. If successful, the re-engineered protein will be used to elucidate the neuronal basis of chemotaxis, a widespread form of spatial orientation, in the nematode C. elegans. The proposed research is significant because it will likely lead to new genetically targetable probes of neuronal activity that can be used in many other important experimental systems. In addition, this work will deepen our understanding of neural computations underlying a common goal-directed behavior. Components II and III are, respectively, technical courses and tutorial visits to laboratories at the leading edge of optical recording technology. These two activities will give the PI new technical background needed to complete the work of Component I. Component IV is a course the PI will teach in research ethics, drawing on his extensive background in analytical philosophy.

DESCRIPTION (provided by applicant): The Systems Physiology Training Grant (STG) will support a program to prepare the most qualified doctoral students for professional research careers in integrative neuroscience through a combination of original, hands-on research, and formal courses. The trainees and their mentors have appointments in the Department of Biology, Department of Human Physiology (formerly Exercise and Movement Science), and Department of Psychology at the University of Oregon. Their research programs span diverse model systems from humans to nematodes and employ a variety of techniques including electrophysiology, neural imaging, molecular biology, psychophysics, and computational modeling. Nevertheless, the trainees, regardless of their departmental affiliations, experience a common set of requirements and activities designed to establish a cohort of students that interact across departmental and disciplinary lines. Our intention is that these students will continue to interact throughout their graduate career, mutually enriching their research efforts with novel ideas and perspectives. The continued funding requested is for 8 predoctoral trainees within a neuroscience graduate program of approximately 50 students, of whom 40 are eligible for training grant support.

Behavior is known to be influenced by natural genetic variation among individuals, yet the precise physiological mechanisms that alter behavior as a result of this variation are poorly understood. The solution to this problem depends both on identifying genes responsible for natural variation in behavioral responses and on understanding the physiology of how those genes affect neural function. Caenorhabditis elegans, a free-living soil-dwelling nematode, is an ideal organism to address the effects of natural variation on behavior. It has a relatively simple nervous system and a fully annotated sequenced genome, and wild isolates of this species show extremely different temperature preferences. This study will use genetic crosses to map and clone the genes responsible for natural variation in behavior. Microfluidic devices will then be used to create a precise temperature choice environment, allowing the physiological basis of these differences to be analyzed using calcium imaging of individual neurons. This work has broader impacts via interdisciplinary graduate training, involvement of undergraduates in research, and community-oriented scientific education through a Campus Educational Network. This will be among the first studies to identify specific changes in multiple genes that work together to cause variation in the physiological mechanisms of a complex behavior. These connections are necessary to begin to understand the vast diversity of behavioral responses within the natural world, as well as within human populations.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): Translational Research and specific Challenge Topic, 15-AI-103: Develop drugs for neglected tropical diseases, with a special emphasis on malaria. Neglected tropical diseases are a group of 13 major disabling conditions that are among the most common chronic infections in the world's poorest people. Chief among these are the five main diseases caused by nematode worms, accounting for more than 80% of the global prevalence of neglected tropical diseases. Nematode diseases afflict more than a 1/6 of the world's population, yet the anti-nematode drugs - "anthelmintics" - in use today date from the 1960's and 1970's. There is an urgent need for new medications, as research into new anthelmintics has not kept pace with emergence of drug-resistant strains. This problem arises because the resource-limited countries where neglected tropical diseases prevail do not support markets that can offset the enormous cost of drug development. Reducing the cost of drug discovery could do much to increase the flow of better drugs to the people who need them most. The proposed research and development project addresses a critical bottleneck in almost all anthelmintic discovery programs. Developing a new drug starts with a costly screening process that tests hundreds of thousands of compounds. Despite the fact that the majority of anthelmintics act by knocking out essential functions in nervous system, there is as yet no way to screen for compounds that specifically affect the nervous system. The goal of the project is to develop such a screening procedure. The new procedure is based on the observation that the nematode throat, or pharynx, is extremely sensitive to the anthelmintics that also affect the nervous system. The pharynx is a neuromuscular pump involved in feeding. Like the heart, pharynx beats regularly, emitting electrical signals that can monitored on the surface of the body, as in an electrocardiogram or "EKG". In the past year, we have successfully demonstrated a miniaturized recording system that allows researchers to monitor the electrical activity of the pharynx while applying minute quantities of drugs to microscopic nematodes. In the two-year tenure of the proposed research, we will (1) use microfabrication techniques to transform the prototype into a high- throughput screening system and (2) test its ability to identify new anthelmintic agents in partnership with a U.S. pharmaceutical company that is energetically seeking to address the urgent need for new anthelmintics. PUBLIC HEALTH RELEVANCE: Parasitic worms such as nematodes are a major cause of disease in humans and economically important livestock, both at home and abroad. There is an urgent and continuing need for new treatments to combat the rise of drug-resistant nematode strains. This goal of project is to reduce the cost of finding new anti- nematode drugs by developing a new screening process that combines microtechnology and electrophysiology for the first time.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Building on the success of a previousMRI-funded project, an interdisciplinary group of computer scientists, psychologists, biologists, chemists, and physicists at the University of Oregon is acquiring a large-scale computational resource, the Applied Computational Instrument for Scientific Synthesis (ACISS), to support continued cutting-edge scientific research in these areas. The ACISS hardware will consist of general purpose multicore computing nodes, high performance computing nodes augmented with GPGPU acceleration, a 400TB storage system, high-bandwidth networking infrastructure and additional computing resources that will be incorporated into an existing visualization lab in the Department of Computer and Information Science. A key part of the proposed infrastructure is the unique opportunity to manage ACISS as a computational science cloud.

The ACISS infrastructure will allow an expanded the scope for the current projects in the areas of software tools for performance measurement, programming environments and languages for describing and executing complex simulations and scientific work flows, new algorithms for multiple sequence alignment and phylogenetic inference and undertake new projects in support of the domain sciences. Research projects that will benefit include: a) modeling neural networks in C. elegans to better understand the neural mechanisms responsible for chemotaxis and klinotaxis, and investigation of the evolution of genes involved in development and their role in speciation and phenotypic variation; b) development of neuroinformatic techniques used in brain imaging and analysis, integrating structural information from fMRI and other sources with EEG data; c) molecular modeling research, including the definition of new techniques for meso-scale modeling and applying computational methods to understand phase transitions and nitrogen fixation; d) astrophysical simulations of turbulent plasma flows that influence the early stages of planet formation.

The ACISS infrastructure will provide the computational resources necessary for future multidisciplinary research. ACISS will establish a novel paradigm for computational science research and practice. The experience gained in early adoption of the ACISS cloud computing technologies will allow us to more rapidly apply this knowledge to create new scientific work flows, more productive research collaborations, and enhanced multidisciplinary education programs. Farther reaching, ACISS can be seen as a model for translational computational science, in which ACISS-based services function as cyber-incubators where new work flows for scientific research are prototyped.

Project Summary Dysfunctional decision making can have devastating impacts on individuals and on society. Many types of decision making are therefore under vigorous investigation. This proposal emphasizes value-based deci- sions, in which the chooser selects among options based on his subjective assessment of their value. A deeper understanding of this behavior is needed to develop the best possible treatments for decision making disorders, including the many forms of addiction and the cognitive deficits that accompany mental illness, brain injury, and neurodegenerative disease. Consumer choice is one of the best studied forms of value-based decisions. Studies reveal that our economic personalities have a significant genetic basis, but it is difficult to trace causal links between genes and behavior in humans. In response, geneticists often turn to simpler invertebrate organisms like the nema- tode worm C. elegans in which the functions of genes nearly identical to their human equivalents can be inves- tigated more rapidly, completely, and at a fraction of the cost. Until now, evidence that nematodes are truly ca- pable of value-based decision making has merely been suggestive. However, economists have developed mathematically rigorous testing procedures for determining whether decisions are based on subjective value. The PI's laboratory has developed microfluidic devices that enable this test to be done on nematodes deciding between high-quality food that is relatively abundant and low-quality food that is more scarce. The results meet all the criteria of value-based decision making. Previous work has identified a circuit of sensory neurons, interneurons, and motor neurons that controls head movements as the worm makes decisions about which food to eat. Using a combination of functional im- aging (Aim 1), optical manipulation of neuronal activity (Aim 2), and neuronal ablations (Aim 3), the proposed research will identify contribution each neuron makes to value-based decisions. A central question is how food value and abundance are represented in the circuit and how this representation is read-out in behavior. Aim 4 constructs a mathematical model based on data from Aim 1 and tests it using data from Aims 2 and 3. Successful completion of the proposed research yields a biologically realistic computational model of the neuronal mechanism of value-based decision making in a compact circuit than can, in principle, be under- stood completely. This work provides a foundation for understanding value-based decisions in more complex circuits. The work also lays the cornerstone for genetic analyses, at single-neuron resolution, of orthologs of human genes identified in association studies related to decision making. The research is broadly significant because it establishes a new biological system in which to analyze at single-neuron resolution the interaction of genes previously associated with decision making in humans, and to discover novel genetic pathways in- volved in this behavior.

PROJECT SUMMARY/ABSTRACT The endogenous cannabinoid system (ECS) ? comprising the endocannabinoids, their G-protein coupled re- ceptors, and their metabolic enzymes ? is one of the most important physiological systems involved in estab- lishing and maintaining human health. In some illnesses, ECS dysregulation is an element of disease pathol- ogy whereas in others it is believed to be protective. Thus, there is intense interest in developing pharma- cotherapies that target the ECS to blunt its pathologies or harness its protective effects. One of the main challenges in developing new drugs that target the ECS is the problem of side effects, con- sistent with the nearly ubiquitous expression of this signaling system. Current efforts to increase the functional specificity of ECS drugs include preventing them from crossing the blood brain barrier, and targeting ancillary ECS proteins which modulate principal ECS components without being direct effectors themselves. Until now, discovery of ancillary proteins has proceeded mainly by inference from prior knowledge obtained from isolated cells. This approach can be limiting because knowledge of the underlying genetic and biochemical networks is usually incomplete. Here we propose to develop new high-throughput methodologies to enable the first unbi- ased genetic screens to identify new ECS molecules in the nematode Caenorhabditis elegans. C. elegans is an omnivorous bacterivore but it learns to prefer some species of bacteria more than others. We recently discovered that one of the worm's endocannabinoids increases the worm?s appetite for favored foods over less favored foods, a phenomenon called hedonic amplification. We propose this system as a genetically tractable model of cannabinoid effects on appetitive behavior, providing an easily screenable phenotype that directly corresponds to a well-known human behavior. The research develops two high-throughput microfluidic systems for quantifying hedonic amplification in C. ele- gans. The systems are then utilized to perform two small-scale genetic screens as a prelude to future large- scale screens. In the first, hedonic amplification will be quantified in strains in which homologs of known mam- malian ECS components have been knocked out; this screen further validates the C. elegans ECS as a mam- malian model. In the second screen, hedonic amplification will be assessed in a select set of wild isolate strains of C. elegans to estimate the heritability of this phenotype. The research is significant because it could ultimately lead to the discovery of novel drug targets to mitigate disease and promote health.