Aravinthan Samuel - US grants

Understanding how neural systems encode animal behavior is a general goal of neurobiology. This CAREER research program, an interdisciplinary effort between neuroscience and physics, will probe the neural representation of the thermotactic behavior of the nematode worm Caenorhabditis elegans by developing techniques at the interface of biology and physics. Examples of technological innovation will include: 1) the development of new behavioral assays that will quantify the worm's responses to arbitrary patterns of thermal stimuli; 2) optical measurements of neural activity applying genetically encoded indicators of cellular processes; and 3) the application of ultrafast lasers to destroy sub-micron sized neural structures (neuronanosurgery). Through partnerships with other labs, these techniques will see broad applications in the biological study of the nematode and other model organisms. An additional component of the program is directed toward promoting scientific education through the awarding of undergraduate research assistantships, development of course curricula, and outreach programs that encourage high school students towards advanced studies in the natural sciences. Special emphasis in placed on education at the interface between biology and physics.

This award is jointly supported by the Division of Physics in the Mathematical and Biological Sciences and the Division of Integrative Organismal Biology in the Directorate for Biological Sciences.

Abstract The ultimate goal of neural science is to understand how interaction between the peripheral and central nervous systems gives rise to human behavior, how sensory information is processed in our brain and memory to stimulate action. But, as human behavior is mediated by a brain with over 100 billion neurons, a comprehensive and integrative approach all the way from sensory input to motor output is currently unimaginable. The fruit fly Drosophila, with sensory modalities, neural circuits, and complex behaviors that are strongly evolutionarily conserved, has emerged as a model system for neural research. Drosophila is simple enough to be tractable, yet complex enough to be scientifically interesting as well as biomedically relevant. This research program will create new paradigms for understanding perception and voluntary action using larval Drosophila, which has unique advantages for this study. In preliminary work, we have used a novel tracking system to quantify the algorithms that underlie larval chemotactic, phototactic, and thermotactic behavior. Using genetic tools provided by our collaborators and new tools that we are developing for optical physiology and behavior quantification in freely moving animals, we will investigate how pathways within the larval brain use information gathered across the larvum?s sensory periphery to make decisions and result in physical behavior. These individual sensory modality studies are the first steps to understanding this model system?s deeper complexities, the behavioral principles and neural encoding behind the brain?s synthesis of the separate environmental representations provided by multiple senses to result in purposeful and coherent behavior.

In this project the PI will develop new techniques to study worm thermotactic behavior in different types of environments with an approach that draws on technological innovation in both physics and biology. At the level of behavior, the PI will develop new assays to track the thermotactic movements of individual animals at high resolution for extended periods of time in two and three dimensions and at the level of neural circuits, the PI will develop an assay for optical recording of the activity of multiple neurons within the intact nervous system. The research program will provide a comprehensive and rigorous understanding of how a worm performs thermotaxis in different environments where it might have to swim, crawl, or burrow towards its preferred temperature. A better understanding of different modes of thermotactic behavior, along with improved methods for manipulating and monitoring neural activity, will provide new insights into how neural circuits give rise to sensory perception, information processing, and motor control during animal navigation. In addition the research program will promote training in the natural sciences through the awarding of undergraduate and graduate research assistantships. It will promote scientific education by integrating aspects of the program into course curricula at Harvard University that is aimed at introducing undergraduate and graduate students to current problems in biophysics and neuroscience. Outreach programs will provide summer internships in neuroscience research to high school students from a small rural community, and to undergraduates at historically black colleges and universities.

instmctions): A complete understanding of the thermosensory mechanisms that regulate nervous system function and be- havior requires their study in an animal with robust temperature-driven behaviors that is amenable to quantitative behavioral, physiological, and genetic analysis. When the Drosophila larva is placed in a temperature gradient, it immediately navigates towards higher or lower temperatures in pursuit of a preferred temperature range. Owing to the relative simplicity of larval motile behavior and the small size of its nervous system, behavioral and physiological analysis can be used to achieve a complete understanding of how thermosensory information is acquired and used by its neural circuits. By analyzing larval movements in response to defined thermosensory inputs, we can uncover the complete set of sensorimotor transformations that underiie thermotaxis, transformations that systematically convert specific patterns of thermosensory inputs into quantifiable patterns of motor output. The transparency of the larva body and its powerfiji genetic toolbox facilitates the use of optical neurophysiology to manipulate and monitor the activity of neural circuits throughout the lan/al nervous system. We propose to combine genetic analysis with new high-throughput behavioral assays to define the locus of themiosensation in the larval nervous system. Furthermore, combining genetic analysis with optical neurophysiology will allow us to understand the molecular pathways that shape the thermosensory properties of the specific neurons that drive cold avoidance and warm avoidance behavior. Given the high conservation of neuronal gene functions between Drosophila and higher vertebrates, we expect that results from this work will lead to major insights into more complex nervous sys-tems. RELEVANCE (See instmctions): This proposal investigates the molecular mechanisms of TRP channel-mediated thermal sensation. In humans, TRP-based thermosensation is critical for pain, inflammation and body temperature regulation. Thus, the mechanisms studied in thie proposal are of biomedical relevance. In addition, thermosensation is important for host-seeking by insect vectors of human diseases like malaria and West Nile. Thus the study of thermosensation is also relevant to the control of insect-borne human disease.

From birth to adulthood, the brain and nervous system continuously expand and develop to keep up with the growing body. Neurons and connections must constantly be added or changed for any animal, including humans, to gain new behaviors or retain old ones. This project is to develop and apply technology to monitor brain and behavior from birth to adulthood of the roundworm C. elegans, an important and widely studied model for neuroscience. A controlled environment will be created where individual animals are born, roam freely, and grow to adulthood while a microscope continuously scans the activity patterns of every neuron in the worm's nervous system. Because of their rapid maturation (<2 days) and small nervous system (<302 neurons), the technology will be used to answer fundamental questions about the developing brain. For example, how do highly conserved behavioral patterns like forward and reverse movement emerge from a nervous system that adapts to a body that grows ten-fold from birth to adult? Coordinating the parallel development of brain, body, and behavior is a problem faced by all animals. The accessibility of C. elegans will yield the first comprehensive measurements of brain and behavior development in any animal. Bringing together developmental biology, neurophysiology, and neurotechnology will train young scientists who will work at the rich interface between the physical and life sciences.

A system will be developed-- a motorized, rotating agar-coated ball-- upon which a nematode can freely move without interruption as it feeds, grows, molts through four larval stages, and becomes an adult. Throughout the nematode's life, the system will record the activity of the entire nervous system visualized throughout its optically transparent body using a confocal microscope that achieves video-rate volumetric recording. These experiments will provide unprecedented datasets that describe the behavioral life history of an individual animal in temporal correlation with whole brain activity patterns. Comparison of these rich datasets between wild-type animals and informative mutants will allow dissection of a wide range of interconnected and shared processes in neurodevelopment, regulation, learning, and memory. For example, understanding the developmental progression of the motor circuit will be achieved by obtaining circuit-wide measurements that describe how the 20-neuron motor circuit that drives the forward and backward movements of the 0.1-mm long juvenile worm is expanded into the 80-neuron motor circuit that drives the same movements of the 1-mm long adult. These studies will illuminate system-wide changes in the nervous system as it dynamically keeps up with animal growth.

This project was developed during a NSF Ideas Lab on "Cracking the Olfactory Code" and is jointly funded by the Chemistry of Life Processes program in the Chemistry Division, the Mathematical Biology program in the Division of Mathematical Sciences, the Physics of Living Systems program in the Physics Division, the Neural Systems Cluster in the Division of Integrative Organismal Systems, the Division of Biological Infrastructure, and the Division of Emerging Frontiers. The sense of smell is essential for maintaining quality of life in humans, and its decline can be an important harbinger of neurodegenerative disease. Moreover, since nearly all animals aside from primates rely on olfaction for most survival functions, understanding chemical sensing has immense practical value, for example, in the control of agricultural pests or in training animals to detect odors relevant for bomb, drug and cancer detection. In spite of its importance, the understanding of olfaction lags far behind the other senses, which is in part due to the lack of understanding of the physical space of odors. The understanding of the neural bases of vision and audition were greatly advanced by investigations of the physical dimensions of visual and auditory stimuli. It is therefore likely that a similar in-depth investigation of odor space - how natural odors occur and the backgrounds against which they must be detected - will reveal a new depth of richness of neural representations of odors in the brain. Insects such as the fruit fly and honey bee are excellent models for this research because of the accessibility of their central nervous systems, because of their ease of use under controlled laboratory conditions, and because of the functional similarity of how odors are processed in insect and mammalian brains. This research will characterize how odor flowers and fruits with respect to behavioral value for honey bees (food) and fruit flies (food and egg laying sites). Further monitoring of neural activity in early and later stage processing in the brain, when combined with computational modeling, will reveal significantly richer neural representations than have heretofore been described. This new understanding stands to have an impact on understanding how healthy brains encode sensations and memories of odors and how brains fail under disease conditions. It will also have an impact on understanding how the sense of smell may be built into engineered devices. Finally, both insects are also of economic importance to agriculture for crop pollination (honey bees) and damage to fruit (fruit flies). The PIs will teach and work with undergraduate, graduate and postdoctoral students and especially recruit students from underrepresented groups in science.

This research will quantitatively characterize the real-world statistics of multi-component natural odor scenes and investigate how they drive behavior and processing in several brain regions. The focus will be on honey bee as well as fruit fly adults and larva as models, where it will be possible to characterize a library of ethologically relevant natural odors associated with a diversity of behavioral outputs. The work will begin by quantitatively characterizing the detailed statistical properties of natural odor scenes in defined ethological contexts. This will build on the rich literature on identified natural odors in insects and mammals. Naturally occurring plant and fruit odor samples from the natural environments of each insect will be collected and chemically analyzed. Nonlinear dimensionality reduction techniques and approaches based on sparse coding will determine the dimensions of odor space that are most salient for behavioral decisions. Such a quantitative deconstruction of the sensory input would be unprecedented in olfactory neuroscience, and should allow the PIs to effectively and comprehensively drive olfactory circuits for the first time. The hypothesis is that the stimulus dimensions that are most behaviorally relevant to the animal will be most efficiently extracted by the olfactory system. Synthetic odor blends will be specially constructed to vary along relevant sensory dimensions, to probe neural codes and adaptive behaviors in the olfactory system. As in research on the visual system, analysis of such evoked neural responses using statistical methods that take into account natural odor statistics will reveal novel olfactory computations and behaviors that have been previously inaccessible. The project will generate datasets of immediate use and importance to scientists in theoretical biology and mathematics, engineering and biology.

? DESCRIPTION (provided by applicant): The goal of this project is to understand how newly born neurons integrate into existing neural circuits and change sensorimotor responses from juvenile to adult. Throughout development, the nervous system undergoes drastic changes in neuron number, neural connectivity, and neurotransmitter properties. From sensory periphery to neuromuscular junctions, circuits expand as new cellular components, differentiated from progenitors, update sensorimotor responses and adapt to changing body plans at each new life stage. A full understanding of the interplay between anatomical, functional, and behavioral changes across development, requires dynamic and structural models of complete neural circuits at different stages. To construct these models, we need to identify and perform physiological analysis of all circuit components. Because circuit function is flexible and heavily modulated by sensory feedback, we need to perform these studies in vivo in behaving animals where key sensorimotor feedback loops are intact. The nematode C. elegans is a particularly suitable model to unravel the interplay between the developing sensorimotor circuits, and the altering behavioral patterns. The genetic accessibility, known adult neural connectivity, and optical transparency of C. elegans provides an exceptional opportunity to fully dissect relationships between overall animal behaviors and the reshaping of neural circuits by the integration and rewiring of new neurons and synapses. Some C. elegans mechanosensory neurons and many motor neurons are born postembryonically, and incorporated the existing circuit during the larval development to the adult sensorimotor circuit. The adult escape response mediated by touch is one of the few behaviors where we know the complete descending pathway, from sensory input to motor output. We found that the C. elegans escape response changes during development. We hypothesize that changes in neural connectivity and integration of sub-motor circuits are required for the compound motor sequence that comprises the adult escape response. To test this hypothesis, we will use: 1) high-throughput serial-section electron microscopy and computer-aided image analysis to precisely map the C. elegans wiring diagram for escape response at each developmental stage, from juvenile larvae to adulthood; 2) quantitative behavioral analysis and optical neurophysiology, to determine the functional contribution of each circuit component to the escape response across development; and 3) optogenetic and genetic perturbation in freely behaving animals, to pinpoint the causative neural connectivities that underlie the execution, transition and developmental changes of the escape motor sequence. Our studies will unravel how neurons integrate into existing circuits with unparalleled resolution, and how new connections shape behavior throughout development. This studies not only are central to our understanding of neural circuit development, and but also has potential biomedical relevance. Cell replacement is viewed as a promising strategy for brain repair, but transplanted neurons often fail to properly integrate into pre-existing circuits. Understanding on how a complete and functioning circuit continuously integrates new components to generate adaptive behavior is critical for advancing an area of basic biology with great translational significance.

Engineering Core-Abstract The goal of the Engineering Core is to enable our Team members to surmount their most challenging technological and engineering obstacles. There are many needs for engineering solutions in our proposal--from behavioral apparatus to optical and electrophysiological rigs, to software for data acquisition and analyses. Even the most technologically sophisticated of the research groups in our Team require special assistance with engineering problems in order to achieve our ambitious aims. In the Center for Brain Science at Harvard there is an existing Engineering Core that consists of (a) an integrated laboratory to design, prototype, iterate, and produce custom devices for neuroscience experiments and (b) an engineer-neuroscientist who solves the most challenging engineering problems in the user laboratories and trains young scientists to do this themselves. By adding a dedicated staff engineer, the Engineering Core will be able to support the team-based neuroscience research proposed here. ?The specific aims are to implement engineering solutions to problems faced by the Projects and other Cores. The Engineering Core will (a) design and build apparatuses to image rapidly swimming larval zebrafish; (b) build other apparatuses to quantitatively assay complex zebrafish behavior; (c) build rigs to record neural activity in larval zebrafish brains via calcium imaging; (d) fabricate rigs to record neural activity in larval Drosophila and rat; and modify software for data acquisition in many existing experimental apparatus, including microscopes.

The NSF Physics of Living Systems (PoLS) Student Research Network (SRN) strives to unite students and faculty working at the interface of physics and biology at different institutions ("nodes") within the US and internationally. A well functioning virtual network could give students at local nodes the ability to take advantage of global educational and research opportunities in PoLS. PoLS is a diverse field, and is composed of researchers and students from varied backgrounds. No single institution can offer (1) the breadth and depth of research and (2) courses that both cover the relevant intellectual landscape and provide in-depth training for students. Such training is critical to create the next generation of researchers who can contribute quantitatively to biophysics, with the ability to move between biology, physics, mathematics, and engineering; PoLS students have important roles to play in this next generation. In addition, no single institution has the range of equipment needed to study PoLS on the enormous range of time and length scales encountered in biological systems. Finally, few single institutions can fruitfully integrate science and engineering to inspire biomedical, robotic and prosthetic devices that will result from basic PoLS research. The HF-SRN will create an environment for students in which they can work among various disciplines while maintaining the physics mindset (simplified systems, few parameter predictive models) and developing new physics. This network will train students (paraphrasing Philip Nelson in his 2008 Biological Physics textbook) "who can switch fluidly between both kinds of brain: the `developmental/historical/complex' sciences and the 'universal/ahistorical/reductionist'." As significant collaborative and educational flux develops within the HF-SRN, successful activities will be broadened to the other US nodes (ultimately with the expectation to engage PoLS SRN international partners). The evaluation plan will help guide aspects of the HF-SRN that could increase flux in other programs in the NSF Science Across Virtual Institutes initiative. More broadly, PoLS SRN students can be leaders in the next generation of researchers who blend biology and physics research seamlessly. Such students will create materials which will seed future K-12 as well as university PoLS curricula. Efforts will be made to extend the educational and research efforts developed within the HF-SRN (and entire SRN) to a broader community including local minority serving institutions. Advances in PoLS can lead to advances in applications such as genome editing, cancer dynamics, robotics and human-assist devices, among others.

During the last period of funding as part of the SRN, the Georgia Tech, Harvard and Maryland nodes have advanced their respective PoLS programs, developing cohesive local communities. The goal of this project is to further develop opportunities for students (and their ideas) to "flow" more easily within the SRN and thereby discover working principles of increased human network flux that can be transferred into the larger SRN. To do so, significant interactions (and evaluations of those interactions) will be developed among three existing SRN nodes (adding Emory as a subcontract to Georgia Tech), forming a "High Flux SRN" (HF-SRN). The HF-SRN will engage in activities such as 1) Collaborative Focused Research Projects, which span nodes and are "built to succeed" by leveraging student and faculty expertise in current projects; 2) Student-Led Dynamic Working Groups (e.g., in biomolecular, microbial, cellular and organismal physics) leveraging faculty research strengths and student interest to develop cross-node communities for these topics. 3) Student-Led Bootcamps: intense 2-3 day tutorials (e.g., microscopy, robophysics, image analysis) with cross-subgroup cutting themes, open to HF-SRN members and held at a particular node; 4) Student-Led Workshops: composed of talks, poster and discussion sessions, inviting the entire PoLS SRN; 5) Curriculum development via open-source course materials, integrating complementary expertise across nodes. All activities will be evaluated and assessed by a Council composed of the lead PIs at each node. This project is being jointly supported by the Physics of Living Systems program in the Division of Physics, the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences, and the Modulation Program in the Division of Integrative Organismal Systems.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Molecular and cellular determinants of Drosophila larva thermotaxis How nervous systems detect and integrate multiple sensory cues to generate robust behaviors is a major question in neuroscience. Such integration is particularly salient in thermosensing, as animals are frequently required to integrate input from multiple thermoreceptor classes. Temperature's ubiquity also means input from other modalities (e.g., olfaction) is commonly received in the context of ongoing thermosensory stimulation. Achieving a comprehensive understanding of the molecular and circuit mechanisms underlying the integration of information from multiple sensors remains a challenge. We will address this challenge in the Drosophila larva. Its ease of genetic manipulation, synaptic-resolution connectome of thermosensory and olfactory processing areas, amenability to neuronal imaging, and stereotyped behaviors, all make it a favorable system for a comprehensive molecular and circuit level investigation of the mechanisms of sensory integration. We propose to achieve these goals in three aims: Aim 1) Establish the molecular and cellular receptors that provide thermosensory input In aims 1.a. and 1.b., we will identify the molecular basis of thermosensing by thermosensory neurons in the larval Dorsal Organ and examine their roles in guiding behavior through cell-specific inhibition and activation combined with high-resolution behavioral analysis. Aim 2) Probe the activities of the interneurons that process thermosensory input In aim 2.a., we will examine how thermosensory inputs act to modulate the neuronal activity of individually identifiable downstream projection neurons revealed from the larval antennal lobe connectome. This will establish the manner in which peripheral sensory input influences these second-order interneurons. In aim 2.b., we will investigate how thermosensory and olfactory systems interact in multi-sensory integration of chemical and thermal cues. Aim 3) Probe the functions of the interneurons that process thermosensory input In aim 3, we will determine the contribution of each projection neuron to thermotactic navigation through cell- specific inhibition and activation of individual PNs combined with high-resolution behavioral analysis. Taken together, these studies combine molecular genetics, physiology, and high resolution behavioral analyses to perform a comprehensive analysis of how this relatively small neural circuit processes multiple, distinct sensory inputs to control robust and flexible behaviors.