Clark Lindgren - US grants

9352801 Lindgren Over the next three years the biology department will implement a new curriculum designed to introduce students to the process of science more effectively. Courses will be shifted from content-based teaching to inquiry-based teaching, placing less emphasis on "the facts of biology" and more emphasis on "doing biology." The goal of this project is to increase both the quantity and the quality of laboratory experiences available to students in animal physiology, introductory biology, and advanced research. This will require the purchase of eight (8) physiological workstations. Each workstation will include a Macintosh IIsi computer, MacLab hardware and software, a printer, and miscellaneous physiological transducers and amplifiers. Students in Animal Physiology will use the workstations extensively to study a variety of physiological processes, such as nerve conduction, muscle contraction, and heart function. The workstations will not only increase the pedagogical effectiveness of individual laboratory exercises, but will also make it practical to introduce a significant component of independent research into this course. Students in the introductory course, Structure and Function of Organisms, will use the workstations primarily to study human physiology. As with the animal physiology course, the workstations will allow students to investigate a wide variety of questions through "hands on" experimentation. Finally, the workstations will be used to increase opportunities for students to participate in advanced research projects in neurophysiology. The MacLab system was selected on the basis of my past experience using other data acquisition systems and on the basis of a pilot test of MacLab by a student doing an independent research project at Grinnell this past summer. MacLab is ideal for use in both introductory courses and advanced courses because it is easy to operate but also possesses extensive capabilities for data acquisition, analysis, and presentation. ***y

A grant has been awarded to Grinnell College under the direction of Dr. Nancy Rempel-Clower to purchase a quantitative microscopy workstation for collaborative student/faculty research in neuroscience and developmental biology. The microscopy workstation consists of a research microscope equipped with confocal capability and a computerized system for neuroanatomical analysis and 3-dimensional reconstruction. This quantitative microscopy workstation will support research across both the biology and the psychology departments at Grinnell, and will be used to address a variety of research questions in the areas of neuroscience and developmental biology.

The quantitative microscopy workstation will enable investigations that will contribute to our understanding of basic questions about neural and developmental processes. The new equipment will be used for investigations of: 1) the neuroanatomical connections between the amygdala and prefrontal cortex in the rat; 2) the genetic basis of the development of the C. elegans pharynx; 3) lens formation in chicken embryos; 4) synaptic plasticity in the lizard neuromuscular junction; and 5) the neuronal and neurochemical substrates that regulate the structure of REM sleep.

The proposed activities using the microscopy workstation have wide-ranging broader impacts. Grinnell College strongly supports interdisciplinary research and teaching, which characterizes the use of the proposed microscopy workstation. The equipment acquired with this grant will be used across departments to meet the research needs of faculty and their students for sophisticated microscopic analyses. Students currently gain some experience in microscopy in several existing courses, and the new workstation provides a valuable opportunity for students to expand their microscopy skills in more specialized applications, training that will serve them well in future research endeavors. Grinnell College is a leader in training underrepresented groups in the sciences, and the faculty who will use the microscopy workstation have been integrally involved in programs to enhance science education for these students. The workstation acquired with this grant will significantly enhance the College's infrastructure by providing sophisticated tools for quantitative microscopic analysis for neuroscience and developmental biology applications, and will enrich the research training and learning of a broad cross-section of students.

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

With this award from the Major Research Instrumentation (MRI) program, the Chemistry Department at Grinnell College will acquire an ion trap gas chromatography mass spectrometer (GC-MS) and a time-of-flight (TOF) mass spectrometer for use in multi-departmental research projects spanning biology, chemistry, and neuroscience. The instrumentation will support a range of research activities, including 1) investigations to probe solution structure, ligand binding and protein dynamics by hydrogen/deuterium exchange mass spectrometry; 2) characterization of organoheterobimetallic complexes to investigate their chemical ionization pathways; 3) studies of gas phase cation small molecule reactivity thereby examining metal-metal cooperativity; 4) investigations to differentiate Rubisco activase proteins that have been implicated in heat-stress response in plants; 5) measurements of endocannabinoids (eCBs) or their metabolites at the lizard neuromuscular junction; 6) identification of residues of nicotinic receptors that comprise the binding site for an allosteric modulator; and 8) studies of herbicides and pharmaceutical contaminants and their degradation products in ground and surface waters.

Mass spectrometry (MS) is used to identify the chemical composition of a sample and determine its purity by measuring the mass of the molecular constituents in the sample after they are ionized and detected by the mass spectrometer. Chromatography is an isolation technique that precedes the mass spectrometry analysis. It separates a mixture into its several constituent chemicals which are then analyzed by the mass spectrometer. These are widely used analytical techniques to identify and quantify the chemical composition of a sample. It is important for undergraduate students to be trained in the technique using modern instrumentation. The instrumentation will be used by research students, in laboratory class instruction and in an outreach program involving middle school students.

DESCRIPTION (provided by applicant): Neurons communicate at chemical synapses. Synapses assemble neurons into networks of neurons and these networks create the remarkable abilities of the nervous system to perceive the world, process information, and direct behavior. Synapses also endow the nervous system with the ability to change over time - that is, to learn. Unconventional neuromodulators, such as nitric oxide, endocannabinoids, and prostaglandins, have been known about for many years;however, their participation in synaptic modulation at the vertebrate neuromuscular junction (NMJ), where a motor nerve synapses onto a muscle cell, is a more recent finding. Despite its apparent simplicity, the NMJ employs a surprisingly complex array of signaling pathways that modulate its activity. Using a muscle from the green anole lizard, which has been shown by my laboratory to undergo a biphasic modulation of neurotransmitter release that involves nitric oxide and the endocannabinoid 2-Arachidonoylglycerol (2-AG), will be used to address five questions regarding the mechanisms and/or roles of these unconventional neuromodulators. The first question will determine whether the synaptic depression mediated by 2-AG improves neuromuscular endurance by reducing ACh release during periods of intense, unremitting activity (e.g. exercise). If true, this will not only reveal a basic function of endocannabinoids at the NMJ, but may also have important implications for the therapeutic use of modulators of the endocannabinoid system (e.g. rimonabant). Nitric oxide (NO) is essential for synaptic modulation induced by the activation of muscarinic receptors at the NMJ. In fact, disruption of NO synthesis at the NMJs of humans with muscular dystrophy may contribute to fatigue. The second question will use a fluorescence-based technique for measuring NO to determine whether the muscle is the source of the NO that is essential for muscarinic-receptor dependent modulation at the NMJ. The third question follows closely from the second and will determine whether glutamate or the dipeptide N-acetylaspartylglutamate (NAAG) activates the synthesis of NO. NAAG has been implicated in numerous pathologies including pain, traumatic brain injury, schizophrenia and stroke. Although vertebrate motor neurons have long been known to express high levels of NAAG, the function of this peptide at the NMJ has not been rigorously explored. The final two questions will focus on the function of perisynaptic Schwann cells (PSCs), glial cells intimately associated with the pre- and postsynaptic elements of the NMJ. Preliminary work has implicated the prostaglandin PGE2-G in the second phase of muscarinic modulation at the NMJ. Activation of muscarinic ACh receptors is predicted to induce the synthesis of the enzyme cyclooxygenase (COX) in the PSCs, which will then convert 2-AG to PGE2-G. This prediction will be tested by measuring levels of COX mRNA using quantitative PCR and determining its location using fluorescence in situ hybridization. These studies, along with an electrophysiological investigation of NMJs at which the PSCs have been acutely ablated by a complement-mediated procedure, will further our knowledge of glial cell function in synaptic plasticity at the NMJ with likely implications for synaptic modulation in the peripheral and central nervous system. PUBLIC HEALTH RELEVANCE: Using electrophysiological, optical and molecular genetic approaches at the vertebrate neuromuscular junction this research will explore five fundamental hypotheses about the involvement of unconventional synaptic plasticity at the chemical synapse. The findings from this research will have direct relevance to the therapeutic use of modulators of the endocannabinoid system (e.g. rimonabant). It will also provide basic knowledge that may better inform our understanding of muscular dystrophy and numerous other diseases that are caused by the dysfunction of glial cells or the peptide N-acetylaspartylglutamate (NAAG).

Non Technical Abstract:
The LI-COR Odyssey Infrared Imaging System acquired through this award will enable highly quantitative investigations in diverse areas of research such as mitotic spindle function, muscle function, obesity, receptor function, and plant responses to heat stress. This instrument uses infrared (IR) technology for the highly sensitive and accurate measurement of biological molecules over several orders of magnitude, thus facilitating the detection of very low but biologically meaningful signals in a diverse group of applications, including protein and nucleic acid gels, protein arrays, In-Cell Westerns and tissue sections. Further, this instrumentation will be incorporated into undergraduate science education and research training programs in biology, biochemistry, neuroscience, and psychology.

Technical Abstract:
This highly sensitive imaging system will be used in a broad range of ongoing research projects, making previously qualitative assays quantitative. In particular, the Odyssey imaging system will be used to: a) examine the role of the protein myosin-10 in mitotic spindle function by facilitating dominant-negative analysis; b) investigate the involvement of high-fat-diet-induced changes in levels of the adipose hormone leptin in the hippocampus; c) analyze the contribution of the 3' untranslated region of the Rubisco activase gene to the stability of the Rubisco activase protein and thus to the heat stress response in plants; d) explore the role of perisynaptic glial cells at the neuromuscular junction by determining whether they express COX-2 in response to stimulation of their muscarinic acetylcholine receptors; and e) monitor the isolation and purification of neuronal nicotinic acetylcholine receptor subunits for use in studies to analyze drug-receptor interactions.

An award is made to Grinnell College to purchase a point-scanning confocal microscope system. Grinnell College has been an active participant and leader in efforts to improve science pedagogy by incorporating active learning techniques into the classroom, improving access to science for women and underrepresented groups, and integrating research experiences throughout the curriculum. This instrument will support these efforts by providing new research possibilities for faculty and their students, both in the research lab and in existing laboratory courses. Beyond this, the investigators will create and teach a new course dedicated to microscopy techniques. More specifically, they will incorporate the proposed instrument into an existing program that provides educational opportunities for inmates at a nearby medium security prison. Further, the co-PIs propose the development of a workshop for regional liberal arts colleges, to be hosted at Grinnell, the aim of which is to exchange best practices and work together to design experiments using fluorescence microscopy and/or confocal microscopy that are accessible to undergraduate students in a variety of settings. <br/><br/>This award will support a range of research and teaching/training activities in biology, biochemistry, chemistry, and physics including: (1) studying synaptic plasticity at the neuromuscular junction (NMJ); (2) detecting microtubule formation below the diffraction limit of light; (3) characterizing neurovascular patterning and guidance in the developing vertebrate heart; (4) deciphering the genetic control of Ca2+ levels in cell migration; (5) characterizing the spatiotemporal activity of various proteins in regulating mitotic spindle function; and (6) detecting allosteric conformational changes in nicotinic acetylcholine receptors. This award will enable Grinnell College faculty and students to address questions that are beyond their current capability. The results will be disseminated in publications in peer-reviewed journals and in presentations at scientific conferences throughout the world.<br/><br/>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.