Barry Andrew Trimmer - US grants

The long-term objectives of this proposal are to determine how transmitters transfer information between neurons, how individual neurons integrate and respond to this information, and how this processing contributes to behaviors and behavioral changes. Specifically, these studies concentrate on two types of cholinergic receptors that are expressed by an identified motoneuron (PPR) in the tobacco hornworm, Manduca sexta. One of these receptors, the nicotinic acetylcholine receptor (nAChR), is a member of the family of acetylcholine-gated channels found both peripherally and centrally in most mammals. In humans these receptors are targets for the actions of nicotine (usually absorbed from smoking tobacco) and a variety of clinically important drugs. Based on the pharmacological responses of PPR, nAChRs in Manduca appear to have several extraordinary properties. First, perhaps as an adaptation to Manduca's diet of tobacco, PPR has a low sensitivity to nicotine while retaining a normal responsiveness to other agonists. Second, since PPR remains depolarized during exposure to nicotinic agonists, there appears to be no desensitization of these NACHRS. This proposal will investigate whether these unusual properties are intrinsic to Manduca NACHRS by using patch clamp electrodes to isolate single receptors in membrane patches from PPR. Agonist sensitivity of the NACHRS will be measured in outside-out patches and their desensitization kinetics monitored in cell-attached patches. In view of the considerable health risks associated with nicotine addiction in humans, understanding the nicotine resistance and other special adaptations of Manduca NACHRS is of obvious medical importance. The other cholinergic receptor expressed by PPR in a muscarinic acetylcholine receptor (mAChR) that appears to modulate PPR's excitability. In mammals, mAChRs are of enormous importance in a wide range of cellular roles, but it is rare to able to study their actions in individual neurons in situ. This is easily achieved in the nervous system of Manduca. Hence, another of my specific aims is to characterize Manduca MACHRS and to determine their effects in identified neurons such as PPR. The pharmacology, distribution and possible heterogeneity of Manduca MACHRS will be established using radioligand binding assays in conjunction with immunological approaches. The cellular function of MACHRS in particular neurons will then be identified using electrophysiological methods. Because MACHRS can control diverse effects in different cells, an important part of this proposal will use intracellular injections to determine which G-proteins and second messenger pathways are employed by MACHRS in individual neurons. These proposed studies will establish how Manduca NACHRS may be specialized to tolerate nicotine, and further, will identify mechanisms of neuronal modulation by MACHRS.

Lay Abstract PI: Trimmer, Barry A. Proposal Number: IBN-9723507 The objective of this research is to understand how reflex movements are modified and adapted for different tasks so that they are appropriate for different behaviors. Detailed analysis of movement is combined with electrical recordings from muscles both before and after different training regimes. Once the changes are described, the central nervous system mechanisms that mediate these changes are investigated by recording the electrical activity of individual nerve cells and by manipulating the chemical receptors on those nerve cells during behavioral training and during sensory stimulation. This research links adaptive changes in behavior with the cellular mechanisms underlying learning and plasticity. Its results contribute to understanding how basic rhythmic movements, such as those involved in walking, are modified as a result of experience so that the behavior can be appropriately expressed in a variety of situations and challenges.

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Trimmer

RANGE AND SPECIFICITY OF NO/CGMP SIGNALING IN THE CNS Pi Barry A. Trimmer, Tufts University

One of the biggest challenges in biology is to understand how the brain produces thought and actions. It is clear that these processes depend on complex communication between nerve cells (neurons) through the secretion of specific chemicals called neurotransmitters. These chemical messages are non-nally detected and interpreted by receptor proteins on nearby cells. However, some neurons signal by the production of an unstable soluble gas, nitric oxide (NO). This unusual messenger was discovered only recently hence comparatively little is known about its role in the brain. This research project seeks to understand the special functions of nitric oxide when it is released from neurons. We have identified individual nitric oxide-producing and responding neurons in the living nervous system and our goal is to establish how they communicate. In particular, how far does nitric oxide spread in the brain and how is it targeted to specific neurons? These experiments use well-established electrophysiological methods together with a novel technology in which nitric oxide is localized using a fluorescent dye.
Nitric oxide has important regulatory functions in the cardiovascular and immune systems but in the brain its ability to simultaneously contact vast numbers of neurons give it the potential to coordinate complex tasks. By exploring the mechanisms, specializations and limitations of nitric oxide signaling this research will help to understand how groups of neurons work together to produce appropriate behaviors.

The majority of our knowledge about how animals move is based on creatures that walk, fly or swim using rigid articulated bones and exoskeletons. However, animals without backbones invertebrates are the most numerous animals on the planet and nearly all are soft-bodied with hydrostatic skeletons for at least part of their life. These crawling creatures do not escape predators by running but instead use camouflage, chemical defenses and cryptic behavior. As a consequence, crawling has evolved into a highly specialized form of locomotion that allows soft bodied animals to move in complex and confined three dimensional structures such as tubes and branches. With a soft body, joints do not restrict movements. Such animals can crumple, compress and rotate body parts with virtually unlimited freedom. Such complex movements are very interesting from a neural control perspective because movement coordination by the nervous system has co evolved with these biomechanical features. The proposed study uses a caterpillar, the tobacco hornworm, Manduca sexta, as a model system to help understand the neural control of hydrostatic movements. Two specific aspects will be examined in detail: first we will determine how crawling is controlled by the central nervous system and how it interacts with peripheral structures such as muscles and cuticle; secondly, we will examine a unique aspect of caterpillar crawling, the ability to climb using curved hooks at the tips of the abdominal prolegs. This gripping is passive but very strong like Velcro hooks and can be actively released. We will determine how this gripping is controlled and how it is integrated into normal crawling.
Although focused on understanding animal locomotion, these studies have potential applications in the design and control of a new type of flexible robot. Such robots could be used to navigate through pipelines or intricate structures such as blood vessels and air tubes. Finally, in examining the mechanism of proleg gripping it is possible that new adhesive materials could be developed that attach passively but can be released actively to avoid the tearing forces in conventional hook and loop fastenings.

Nitric oxide (NO) is a small, highly reactive molecule produced in both plants and animals. It is used to protect tissues from infections and acts as a signaling molecule in the vasculature of vertebrates and the central nervous system (CNS) of most species. Because NO can dissolve in both cell membranes and the fluid around cells it is thought to spread widely from where it is produced. However, the range of NO signaling has not been rigorously defined and may vary between tissues and species. In the CNS an effective range of 200 microns would enable NO to control hundreds of neurons, but in peripheral tissues such as muscles this range could limit NO to a very local or even intracellular role. Hence, the immense differences in size, metabolic function and cellular architecture of NO target tissues raise important questions about the mechanisms of NO signaling at different locations.

The proposed experiments take advantage of a well-characterized NO signaling system in an insect model system (larval Manduca sexta) to establish the functional roles of NO at central and peripheral sites. A key advantage of studies in this insect is that signaling can be manipulated in the intact, freely moving larva, or in tissues dissected from the insect and maintained alive for several hours under defined conditions. Pharmacological tools are available to block or supplement NO production and to block or increase its effectiveness. It is now also possible to alter the synthesis of key enzymes at the molecular level in intact animals using injections of double-stranded RNA. This treatment interferes with the expression of specific genes in the fully developed animal or in selected parts of the CNS.

The results of preliminary physiological experiments, together with the anatomical distribution of enzymes that make or respond to NO (nitric oxide synthase, NOS; and soluble guanylyl cyclase, sGC; respectively), suggest that NO is involved in the control of feeding (chewing movements and foregut activity), the regulation of normal body wall tension and the direct cellular immune response. The neural and muscular activity will be recorded using electrodes and pressure sensors implanted in normal larvae and in larvae that are experimentally deficient in NOS or sGC. These actions of NO on motor patterns and neuromuscular physiology will also be examined pharmacologically in isolated tissues. The role of NO in the insect immune response will be tested by implanting fluorescent micro spheres into the body cavity and assessing the degree of encapsulation in normal and NO-deficient larvae. It is expected that feeding (and growth), gut movements and activity of body wall sensory neurons will all be strongly modulated by NO. The control of body turgor, motor coordination, and abiotic encapsulation are also potentially very important sites of action.

The long-term goal of the proposed activity is to better understand how NO carries out its diverse functions. In particular, the results will help to define the specializations and limitations of NO signaling in very different tissue environments. Because NO is involved in the locomotion and feeding behavior of this herbivorous insect, the results could also impact the development of pest-specific antifeedants for crop protection.

Despite their lack of joints or rigid limbs, soft-bodied animals are capable of complex and precise movements. Such movements often involve a large number of muscles and yet control is accomplished in most cases with relatively few neurons. One ultimate goal of these studies is to understand how flexible movements are controlled by relatively simple nervous systems. This work will use a large caterpillar, the tobacco hornworm (Manduca sexta), because it offers several powerful advantages for analyzing interactions between the nervous system and the mechanical responses of soft tissues. The experiments will use force measurements, motion capture, and recordings of nerve activity, to establish how Manduca controls its movements in different behavioral contexts. In addition, both the encoding of sensory information by stretch receptors, and the responses of muscles to neural stimuli, will be measured during natural cycles of stretching. Together, these experiments will provide a comprehensive description of how soft-bodied legged locomotion is controlled, and provide new insights into the role of soft materials in stable movements. This work will also have broader impacts in the fields of biomechanics, engineering, and robotics. For example, it is important to know how soft tissues contribute to the control and stability of running animals and equally for prosthetic devices. To this end, these studies are being used in collaborative projects to help design and build new types of maneuverable soft-bodied climbing robots. In addition to its intrinsic scientific importance, this research will provide completely new training opportunities at undergraduate, graduate, and advanced levels of education, but most importantly, across biological, mathematical, and engineering disciplines. The work will be carried out in association with a new biology and engineering facility at Tufts which will support cross-disciplinary research and training in all aspects of soft materials and their control in animals and useful devices.

Worm-like body shapes are thought to be some of the earliest evolved body plans for soft moving organisms. This simple cylindrical shape is often modified through evolution with the addition of limbs and other structures to improve how an animal interacts with the environment. This project asks a very fundamental question: how do such soft appendages change the way a soft-bodied worm-like organism moves? The caterpillar, tobacco hornworm (Manduca sexta), offers a great system to answer this question.

Using a custom-built force sensor array, the investigators directly measure forces the caterpillar legs exert during normal crawling. By matching the movements of the body to forces in different directions it is possible to accurately describe how different legs help the caterpillar crawl and climb. This study will also measure changes in body pressure to establish how caterpillars differ from non-legged soft invertebrates such as leeches and earthworms. It is expected that because the caterpillar has legs to anchor the body, it will rely less on stiffening its body with fluid pressure for fine movement control. The experiments will test if legs allow soft animals to exploit stiff structures in their environment as a continually changing skeleton. In effect the caterpillar can conform to the shapes that it crawls upon.

Understanding the functional benefits of appendages on a worm-like morphology not only provides insights to soft material control, but also gives clues to why different caterpillars have different numbers of legs. Practically, this project pushes the technical limits of biomechanical measurement. It will also expand our understanding of animal locomotion and contribute to current theories of legged systems, both animals and machines. Conceivably, this project can benefit engineering and the development of soft robots and devices.

The objective of this proposal is to obtain support for the purchase of a state-of-the-art, ultra-high-resolution, 3-dimensional printing system for rapid prototyping and rapid manufacturing of parts with complex shapes and complicated material properties. The printing system will find its primary use in the activities of the Tufts University biomechanics, biology and biomedical engineering groups. The term "rapid prototyping" refers to the automated construction of physical objects directly from computer aided design or animation software. A variety of competing rapid prototyping technologies are now commercially available. However, for our purposes, the least costly, easiest to use, fastest and most flexible technology in terms of the variety of different materials that can be processed to fabricate parts is 3-dimensional printing. In this approach, photopolymer liquids are forced through inkjet print heads to create the layers of the part, which then are cured and fused together by ultraviolet light. The printing system will be the centerpiece of a biomimetic biomechanics facility, in which it will be used to fabricate essential parts that are the basis of the research activities of the Tufts University biomechanics group, and that cannot be produced by any other method. Use of this printing system in our interdisciplinary approach to biomechanics will lead to (1) major breakthroughs in the understanding of biologic mechanical systems and (2) novel applications of biologically inspired principles to engineer mechanical devices and systems. A particularly novel aspect of this work is that we seek to understand the role of tissue material properties in living movements and to exploit materials to show similar properties to design, build and control devices emulating biologic mechanical systems. Thus acquisition of the printing system will significantly advance the field of biomechanics, by providing a crucial part of the key instrumentation used in a novel and innovative, biologically inspired, closed loop research and design facility.

By allowing the fabrication of complex parts with advanced, non-uniform material properties, acquisition of the 3D printing system will permit us to undertake a large set of projects that are presently not possible due to part fabrication limitations. These projects will be central to the biomimetic biomechanics facility, allowing us to design and fabricate novel biomimetic devices and systems. Development of such devices and systems can in due course significantly impact understanding of biomechanical systems and thereby underlie new biologically inspired research and design approaches. Furthermore, the proposed project will permit the PIs to continue to engage undergraduate as well as graduate students in our ongoing research efforts, expanding the research training, educational experience and opportunities available to the students involved. Moreover, formal courses in which the printing system will be used have been identified from the Tufts Departments of Biology, Biomedical and Mechanical Engineering, and the Tufts Gordon Institute. In addition, as part of the process of recruitment of students into our projects, full attention will be given to the infrastructure in place at Tufts for recruiting and retaining women and underrepresented minorities in research. These include the New England Board of Higher Education Excellence through Diversity Program, a major forum for recruitment of underrepresented minority students, and formal ongoing outreach programs in Computer Science, Biomedical Engineering and robotics, as well as the Tufts School of Medicine NIH-funded minority outreach program, which has been highly successful in attracting underrepresented undergraduate students to summer internships for the last ten years.

IGERT: Soft Material Robotics.

This Integrative Graduate Education and Research Traineeship (IGERT) award creates an interdisciplinary graduate program to develop advances in the field of soft robotics. These machines, inspired by animals, will be capable of complex tasks that are difficult to achieve with conventional robots, suitable for close interactions with humans, and able to work in environmentally sensitive locations. The research will cross traditional disciplinary boundaries, employing novel biomaterials, exploiting cellular processes and tissue engineering methods, and using control strategies derived from evolutionary principles ? approaches that are comparatively rare in conventional robotics.

Broader Impacts: The development of this new technology provides an exciting opportunity to train inventive and entrepreneurial future science and engineering leaders. IGERT trainees will train in multiple disciplines, including materials science, neuromechanics, mechanical control systems, computer science, artificial intelligence and product design. Novel collaborative training approaches include the formation of mentorship teams and an innovative problem solving-based model of education. IGERT trainees will exploit bioengineering approaches to machine design, fabricate and control new robotic devices to address a wide range of current medical, social and environmental challenges. For example, soft robots could be developed for internal medical diagnosis and delivery of therapeutics, or for search and rescue operations and bioremediation. The international partners, leaders in the emerging field of soft robots, will help trainees form collaborations and affiliations across the world. These students will bridge emerging areas of biology and engineering to create revolutionary new technologies including robots for human assistance and environmentally-friendly biodegradable robots.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to establish new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries, and to engage students in understanding the processes by which research is translated to innovations for societal benefit.

The long-term goal of this work is to develop transformative technologies for the actuation and control of mobile, soft robots in challenging field applications, such as disaster relief. This project is an innovative, feedback-based solution for enhancing soft-robot mobility. This solution is inspired by the caterpillar Manduca sexta, a remarkable, multi-segmented invertebrate that crawls, climbs and burrows in many terrains. Our approach is aimed at controlling the caterpillar-like, periodic movements of a fluid-filled soft robot, in which the robot's deformable structure makes it difficult to sense or model body configuration on-the-fly. The research has two specific research aims. Aim 1 will formulate a control design method for generating caterpillar-like gaits by exciting sustained oscillations (e.g., limit cycles) within a deformable, fluid-filled system. Aim 2 will quantify the in vivo kinematics of burrowing caterpillars to identify how they adapt their gait in response to rich patterns of tactile stimuli. New educational opportunities will be developed to foster interdisciplinary controls teaching for engineering and biology students.

Soft robots offer tremendous potential to enhance mobility, for example, by burrowing through debris or maneuvering through complex terrain not accessible to conventional rigid robots. The research will have a broader impact through its integration with the team's teaching activities. Major advances in soft robot technology are likely to come from insights gained at the interface between systems biology and controls engineering. Hence, existing courses will be modified to introduce undergraduate biologists to engineering control, and to introduce graduate engineers to neurobiology.

All human-made devices, from the very first pre-historic tools to present day robots, have been constructed from non-living materials, most of which are very stiff and synthetic. To make modern devices more suitable for use in close proximity to humans and for work in natural environments it is important that we find new ways to build machines that are biologically compatible, biodegradable, environmentally safe and able to interface with tissues. A major challenge for making such biologically compatible machines is that there are no suitable motors (actuators) to make them move. Attempts to use muscle cells derived from animals such as frogs and mice have had limited success because vertebrate tissues require an intricate blood system and they are easily damaged by changing environmental conditions. It is also hard to replicate the conditions found in a vertebrate embryo that make muscles grow appropriately. This research introduces a new biological approach to making such actuators by growing them from insect cells produced during metamorphosis. Adult insect tissues (such as flight muscles) form directly on existing larval tissues and their growth can be controlled using simple manipulations of insect hormones. Preliminary studies show that insect muscles can be grown in culture at room temperature and that they will survive for many months. This research will identify the conditions needed to generate powerful insect muscles and develop methods to grow them for use in living machines. Successful completion of this work will lead to the production of an engineered muscle that can be sustained for several months and that can generate forces ten times greater than current muscle actuators grown in culture. The work will have wider implications in revealing some of the processes (genetic, biochemical and hormonal) that lead to the re-programming of cells that must occur as part of insect metamorphosis. This is expected to stimulate new experimental approaches to studies of tissue specification, growth and repair.

These studies will test the hypothesis that fully formed tissues can be grown ex-vivo from metamorphic cells of the tobacco hawk moth Manduca sexta. Using the dorsal longitudinal (flight) muscles (DFM) as a target tissue the experiments will focus on four main goals: 1) To characterize the physiological and molecular changes that accompany DFM formation during metamorphosis and in culture. Measurements will be made of in vivo changes in electrical characteristics and the contractile properties using isometric/isotonic tests and dynamic work loops. The gene expression profile (transcriptome) of developing muscles and growing explants will be compared at different stages to help identify gene networks associated with Manduca muscle formation. 2) To characterize the roles of local (cell-cell, mechanical) and systemic (circulating) factors in muscle specification, differentiation and growth. This will involve tissue excision and cross stage transplant methods that are well established for insects. 3) To recapitulate the normal formation of adult dorsal flight muscles from larval precursors in culture by engineering the hormonal and substrate conditions. The transcriptomes of the in-vitro muscles will be compared with both native larval and adult DFMs to look for changes in key developmental and physiological gene networks. 4) To grow and maintain muscle constructs in-vitro for practical actuator applications. The goal is to engineer a muscle that can be sustained for several months and that can generate stress an order of magnitude better than current in-vitro muscle actuators by maximizing survival, cell proliferation and eventual differentiation. It is expected that this process will produce densely packed muscle fibers that can be used for high-stress actuators. The unified contraction of these fibers will be controlled by growing them on micro-electrode arrays or though the expression of light-sensitive channels such as ChR2. This research will engage graduate students in cross-disciplinary research in soft robotics.

The main objective of this National Robotics Initiative (NRI) award is to develop collaborative Modular Soft Robots (MSoRos) that can move in complex terrestrial and climbing environments and change size and shape. A swarm of MSoRos could be used in disaster relief (search and rescue operations), space exploration and precision agriculture. For example, search and rescue scenarios require small robots to autonomously navigate holes and to crawl through narrow cracks/spaces. The collaborative MSoRos will be composed of soft individual units that can deform to penetrate these spaces without prior programming. In agriculture, where the environment is complex, unstructured (soil) and adverse (changes include heat-cold and rain), these robotic modular devices will be capable of multiple behaviors to match their tasks. For example, individual modules could crawl around locally to monitor soil-health and then re-configure as a three-dimensional ball to roll to a centralized station after the task is complete. The ability to form different structures in this way can minimize locomotion costs. Furthermore, this research is easy to disseminate among high-school and undergraduate students as soft robots are cheap, safe to operate and intriguing. The MSoRos will excite young minds by connecting popular robot icons such as Transformers or Big Hero 6, with real-life morphing soft robots. Simultaneously, it will introduce them to futuristic robotics and mechatronics technologies with applications to wearable robotics, collaborative robotics and robots-in-homes, and encourage them to pursue career in STEM and robotics.
This combination of morphing and modularity can dramatically increase the adaptability of a robot. The proposed research will: a) learn principles for robot mobility in complex environments. This is analogous to building reduced-order models (ROMs). Environment-specific ROMs for a highly deformable, soft, continuum robot will be reverse-engineered by learning factors that dominate robot-environment interactions; b) design open-source, untethered MSoRos that will increase the versatility, robustness and cost effectiveness of traditional modular robots; c) establish that environment awareness is a powerful strategy for controlling deformable robots. MSoRos will use their interaction with the environment to learn and deploy appropriate behaviors. This will lead to the development of hybrid robots that will combine environment-centric exploratory learning (this research) with existing model-centric strategies, to carryout complex autonomous tasks.

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.