Patsy Dickinson, PhD - US grants

9310003 Dickinson Most of the movements that animals make can occur under a variety of internal external conditions, thus placing different demands on the animal. Consequently, the central pattern generators that are responsible for producing the motor outputs to drive rhythmic movements are highly flexible. Under the influence of different neuromodulators, these pattern generators can produce a variety of different outputs. In addition, many movements require the coordination of large numbers of muscle groups, including ones that may also be involved in controlling other behaviors. This is particularly clear in the case of functionally related rhythmic patterns, such as those that drive the four parts of the foregut in crustaceans. Recent research on this system has shown that the four pattern generators, which were once thought to be four discrete entities, interact in a variety of ways. The research to be undertaken will examine a number of aspects of the neuromodulation of the rhythmic motor systems in the stomatogastric nervous system, focusing on the interactions of the pattern generators. In addition, this research will address another issue that has arisen recently, namely the effects of modulation on spatially distributed pattern generating networks.***

Students graduating in biology must be intimately familiar with both the process and content of biological investigations. They should be able to integrate information and use multiple levels of analysis. In addition, they should be comfortable using a variety of data acquisition tools. Scientifically literate non-majors must also understand the process of scientific investigations, as well as fundamental concepts. This project is enhancing the investigative laboratories in physiology at all levels of the biology curriculum. Additionally, because most biology laboratories emphasize data collection while analysis is done individually by the students, modern data acquisition and analysis techniques are being incorporate that promote analysis in class. Students are active participants in their own learning, and are involved in all aspects of the experimental design and execution, including hypothesis generation, experimental design, data collection and analysis using modern equipment, and presentation of results in a variety of formats. In three courses, Introductory Biology, Comparative Physiology, and the Human Physiology course for non-majors, two investigative laboratories are being added or enhanced. In the upper-level neurobiology course, the laboratory is being entirely restructured to include three semi-investigative laboratories ion fundamental concepts in neurobiology, followed by a longer inquiry-based project. By enhancing courses at all levels of the curriculum, the department is helping develop students better critical-thinking skills as well as understanding the process of science and the fundamental concepts of each level. In addition, a large proportion of the students taking biology are being exposed to more realistic science, more discovery-oriented laboratories, and more modern scientific equipment.

Lay Abstract PI: Dickinson, Patsy S. Proposal Number: IBN-9723885 Most behaviors can occur under a variety of internal and external conditions. Because of this, movements may vary from time to time to support the behavior in an appropriate way. It is now clear that the nervous system networks that underlie rhythmic behaviors can each produce a variety of outputs, allowing for this flexibility. These changes in output are largely controlled by chemicals called neuromodulators, which are released from nerve cells. There are many such neuromodulators, each of which can alter behavior in different ways. This project investigates how nervous system output, and hence rhythmic movements, are altered when several neuromodulators are present in the system at the same time, as well as the how such neuromodulators can alter the way nerve cells communicate with each other over a long time period. These studies increase the understanding of the roles of central nervous system chemical substances in generating behavioral flexibility on a number of levels, as well as increasing the understanding of the way the brain controls rhythmic movements so that they are most appropriate for a given situation. Such movements are fundamental to such important behaviors as walking, hand movements, speech, and visceral functions.

Animals must alter their behavior to meet the demands of a changing environment. One mechanism that allows such behavioral flexibility in the pattern generators that drive rhythmical movements, such as locomotion and chewing, is the functional reconfiguration of neural networks by neuromodulators. The stomatogastric system, which contains the pattern generators that drive the pyloric filter and the gastric mill in the crustacean foregut, provides an ideal system in which to study neuromodulation. Both the pattern generators and the modulatory inputs in the stomatogastric system have been extensively characterized. More than 20 modulatory substances have been identified in this ganglion, raising the following question: Why are so many modulators involved in modulating such a relatively simple system? This project asks whether a comparative approach can shed light on this question and thus provide insights into the fundamental mechanisms and effects of neuromodulators.
The pyloric pattern and its responses to modulators have been studied to some extent in a number of different species of decapod crustaceans. The present study will further examine the responses of six species - two closely related species of crab, two related lobster species, the California spiny lobster, and a kelp crab - to a test group of four neuromodulators. Using standard recording techniques, the basic pyloric pattern and its responses to these substances will be recorded and characterized qualitatively and quantitatively. Additionally, some of the mechanisms involved in modulation will be compared, as will the effects of these four modulators on muscle activity and contraction in the six species.

Steroid hormones exert profound effects on social behavior, but those effects are typically slow because steroids activate molecules that turn genes on and off, and changes in gene activity take a long time to produce effects on behavior. However, it has recently been shown that steroids can also affect behavior through more rapid mechanisms that do not depend on interactions with genes. Thompson will test the hypothesis that two sex steroids, testosterone and estradiol, affect reproductive behavior in male goldfish by rapidly changing how goldfish see the world, particularly female sexual stimuli. The proposal outlines a series of behavioral, electrophysiological, and neuroanatomical experiments that will also determine the kinds of molecules in the brain that these steroid hormones act upon to produce behavioral effects. Additionally, the project will identify where within the brain hormones produce those effects. Together, these experiments will increase our understanding of the fundamental cellular mechanisms through which steroid hormones affect behavior, particularly those related to reproduction. The principal investigator will perform many experiments related to this grant in an upper level laboratory course at Bowdoin College, providing meaningful research experiences to undergraduate students. Dr. Thompson will also teach a summer neuroscience outreach program at Bowdoin College for teams of high school teachers and their students. This will be followed by his students setting up presentations in the classes of teachers who have participated.

With this award from the Major Research Instrumentation (MRI) program, Professor Elizabeth Stemmler from Bowdoin College and colleagues Patsy Dickinson, Dharni Vasudevan, Danielle Dube and Benjamin Gorske will acquire a liquid chromatograph-quadrupole, mass spectrometer with tandem capabilities and various accessories. The proposal will enhance research training and education at all levels, especially in areas such as (a) use of transcriptomics and peptidomics strategies to neuropeptide identification and to explore the role of hormonal cycles on differential neuromodulatory responses in crustaceans, (b) characterization of bacterial glycosylation, (c) studies of protein methylation, (d) characterization of transformations of molecules of importance to the environmental degradation of contaminants, and (e) development of methods to synthesize and characterize oligothioamides.

Mass spectrometers (MS) are used to identify the chemical composition of a sample by measuring the mass of the molecular constituents in the sample after they are ionized and detected by the mass spectrometer. This instrument couples a high-resolution liquid chromatography system with the mass analysis ability of mass spectrometry. The liquid chromatograph separates mixtures into their molecular components. These components then flow into a mass spectrometer where their masses, and those of their fragments, are measured. This mass spectrometer has a special design that provides relatively high mass accuracy, sensitivity and resolution that allows detection and determination of the structure of molecules in a complex mixture. The instrumentation will be used not only for research but also in laboratory courses to train significant numbers of students in the use of this important analytical technique and for research and outreach activities with regional institutions such as Bates College, Colby College and Mount Desert Island Biological Laboratory.

Although behavior is controlled by the nervous system, neuronal output is filtered through the neuromuscular system and is altered by feedback pathways. All components of such systems can be altered (modulated) by hormones or neurotransmitters. However, because these components interact in complex ways, the behavioral effects of modulating individual components are difficult to predict. The goal of this project is to extend our understanding of the control and modulation of the multi-component neuromuscular systems that underlie rhythmic behaviors, particularly the ways in which the multiple components that make up complete rhythmic neuromuscular systems are modulated to produce adaptive behaviors. The project will use physiological recordings in a simple model system (the crustacean heart) to examine modulation at all levels, including electrical activity in the central nervous system, movements generated by the muscles, and the effects of the known feedback systems. The approach will be comparative, examining the effects of two structurally related molecules that have similar effects on the whole system, and the effects of a third molecule, also structurally related to the first two, whose effects on the neuromuscular system as a whole differ substantially from the first two. The general principles uncovered should advance our understanding of both the extent to which different components of a system, particularly feedback systems, are modulated, and the ways in which such modulatory effects are able to interact to produce a predicted final output or behavior.

In addition, because this work will be conducted at an undergraduate college, it will contribute to the preparation, retention and interest of undergraduates in science. Students, who will contribute both intellectually and in hands-on work, will include both underrepresented minorities and women, as has been the case in the PI's lab in the past.

The overarching goal of this project is to understand the processes that underlie variability in behavior. Specifically, the project will focus on variability in the responses of neuronal networks generating the activity that controls rhythmic movements to one hormone. This project will utilize the lobster cardiac neuromuscular system, a simple model for understanding this phenomenon. In this system, some hearts respond to the hormone allatostatin-C with increased contraction force, while others respond by decreasing contraction force. The project is multi-disciplinary, using physiological, molecular and biochemical approaches. Experiments will include recording changes in activity of the lobster heart when exposed to the hormone, using molecular biological techniques to determine the genes that contribute to the differential responses to the hormone in different animals, and analyzing proteins within the heart to determine how they differ among animals that respond differently to the hormone. This project is a collaboration between researchers at Bowdoin College (Brunswick, Maine) and the University of Hawaii at Manoa (Honolulu). It will train undergraduates, including underrepresented minority students, at both institutions in a wide variety of chemical, molecular and physiological techniques. It will provide interdisciplinary training and will introduce undergraduates to the importance of examining scientific questions from multiple perspectives. The molecular components of the project will also provide resources that can be used by other scientists to address additional issues in lobster biology, including the origin and physiological consequences of shell disease.

To determine the mechanisms and triggers that underlie the variability in the responses of neuronal networks to modulators, this project will examine a simple pattern generator, the lobster cardiac ganglion. This project will combine transcriptomics and physiology to look for differences in gene expression that correlate with differential responses to the peptide hormone, allatostatin-C. At the same time, the project will ask whether these differences may be triggered by changes related to the molt cycle. The project, which will use Illumina sequencing, will assemble de novo transcriptomes from lobster tissues. These transcriptomes will provide references onto which RNA-Seq data from lobsters that respond differentially to allatostatin-C in physiological experiments will be mapped to determine changes in gene expression. The investigation will target specific genes and will use a genome-wide assessment to look for additional changes. The project will also use mass spectrometry to ask whether specific proteins show different post-translational modifications in lobster hearts that respond differentially to the neuropeptide.

Part 1: This research will examine how steroid hormones, which are produced by all vertebrates, directly activate receptors on the surfaces of neurons in the brain to influence behavior. Once activated, these receptors rapidly change the internal responsiveness of those cells to incoming sensory inputs and thus alter an organism's perception of the world around it. Determining if steroids work through such mechanisms will help us better understand basic hormone signaling mechanisms as well as the complex neurochemical processes that enable animals, including humans, to adjust their behavioral responses to constantly changing social environments. The experiments will contribute to the training of our nation's future scientists, educators and doctors through the hands-on research experiences that will be provided for students at an exclusively undergraduate institution. Students will participate in projects related to the award through a lab course run by the PI as well as through focused independent research experiences in the PI's lab. The PIs will recruit students from diverse socioeconomic and racial backgrounds to participate in the research. Additionally, the PI will promote STEM education on a Native American reservation in Maine through a neuroscience workshop.

Part 2: Although non-genomic androgen receptor signaling mechanisms have been well-documented in the peripheral tissue of a variety of vertebrates their rapid, central influences have not been thoroughly examined. This project will investigate a novel mechanism through which androgens may rapidly affect social behavior in male goldfish. The goals of this project are to assess whether 1) testosterone rapidly promotes courtship in competitive contexts through androgen receptors; 2) androgen receptors rapidly modulate courtship by influencing responses to pheromones; and 3) androgen receptors affect early olfactory processing and/or functional coupling across brain networks. By characterizing a novel mechanism through which androgens rapidly modulate the processing of social stimuli, new insight into the fundamental neuroendocrine signaling mechanisms that allow animals to rapidly adjust their behavioral responses to constantly changing social environments will be potentially revealed. This project will also have two major types of broader impact. First, it will directly enhance the scientific education of undergraduates at Bowdoin College by integrating the proposed research into an existing neuroscience laboratory course and through independent summer internships and honors projects. Second, the PIs will continue a 4-day workshop in northern Maine for Native American 7th grade students that is built around this project.