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.

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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.

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.

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 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.