Research Projects (Abstract)
This new REU site, Research Experiences in Neuroscience for Undergraduates from Rural and Tribal Colleges, will provide hands-on research experience in neuroscience for up to 10 students from tribal and rural colleges, or for other interested students. All students, including those with scholarships provided through the North Dakota Association of Tribal Colleges, will be paired with faculty mentors and perform their research at the University of North Dakota. The primary objectives of this program are to (1) foster academic and social independence, (2) promote an interest in research and science careers, and (3) encourage professional development for the student participants. To begin the program, students will receive one week of training in cell, molecular and imaging techniques. The students will develop their own research projects with their faculty mentor (see list of faculty mentors and their research activities below). Students will attend weekly professional development sessions that will include instruction in oral and written presentations, data analysis and proper research conduct. Students will be expected to present their findings at an end of session symposium. This will prepare them for professional scientific presentations at a regional meeting. Salary, room and board and childcare will be provided for all students. By providing childcare assistance we are incorporating an innovative approach to offer research opportunities for a demographic that has been historically and grossly underrepresented. It is our hope that participating American Indian and rural students will choose to pursue graduate degrees in the sciences and return to their communities to help strengthen science education, research and literacy.
REU Site Activities
The research activities being conducted by REU faculty mentors at UND:
- Diane C. Darland
- Van A. Doze
- Peter J. Meberg
- Timothy Bigelow
- Tristan Darland
- Colin Combs
- Dane Crossley
- Thad A. Rosenberger
Diane C. Darland Laboratory Student Projects:
Project #1: Vascular endothelial growth factor function in neural development.
Neurovascular interactions can impact the development of the CNS as well as the plasticity of developing neurons. Several factors have been identified that show overlapping function in the nervous and vascular systems. Among these is vascular endothelial growth factor A (VEGF). VEGF has been identified as a potent regulator of angiogenesis and has recently been shown to impact neural proliferation and neuronal survival in vivo. The angiogenesis-inducing effects of VEGF are largely mediated via activation of the VEGFR2 homodimer; however, VEGF has also been shown to act via formation of a co-receptor complex of VEGFR2 and neuropilin 1. This project is designed to test the hypothesis that VEGF regulates neuronal stem cell proliferation and differentiation via the VEGFR2-neuropilin pathway. The first component of this project is to determine the expression pattern of VEGF and VEGFR2 during developmental neurogenesis in the CNS. The second component of this project is to examine developmental neurogenesis in VEGF-expressing mice that lack VEGF-mediated signaling via the VEGFR2-neuropilin heterodimer.
Project #2: Vascular endothelial growth factor (VEGF) isoform regulation of neuronal survival and differentiation.
Neurogenesis requires the coordinated regulation of neural stem cell proliferation, initiation of differentiation with exit from the cell cycle, and acquisition of differentiated properties associated with a given cellular function. Neurogenesis in the brain occurs in concert with development of the CNS vasculature. Therefore, the coordinated regulation of heterotypic cell-cell interactions may play a role in regulating neurogenesis and neural differentiation. One factor that may impact neural and vascular cell interactions is VEGF, a potent angiogenesis factor that has more recently been implicated in neuronal survival and axon pathfinding. The goal of this project is to test the hypothesis that perictye-derived VEGF promotes contact-dependent differentiation of neurons and that perturbation of this molecular pathway disrupts neuronal differentiation. The first component of this project will involve the development of a tri-culture Transwell™ system consisting of primary brain endothelial cells and myofibroblasts cultured together in capillary-like tubes on one side of a Transwell™ membrane with primary neurons cultured on the other side. The second component of this project involves disrupting VEGF signaling in the tri-cultures and assessing the impact on neuronal survival, proliferation and differentiation.
Van A. Doze Laboratory Student Projects:
Adrenergic Modulation of Hippocampal Interneurons
Dr. Doze’s lab is examining the role of the endogenous neurotransmitter norepinephrine (NE) in the brain and its effects on the physiological functions of interneurons. The excitatory networks of higher cortical structures, such as the hippocampus, consist primarily of relay neurons called pyramidal cells. The activity of these cells is controlled by powerful local circuit inhibitory interactions, mediated by a small number of fast-spiking cells called interneurons that use the inhibitory neurotransmitter g-amino-butyric acid (GABA). These interneurons comprise a diverse group of cells, each presumably specialized for a distinct form of synaptic inhibition based on their individual firing behavior and the region of the postsynaptic cell where their terminals synapse. The hippocampus in particular receives a dense projection of NE-containing fibers, many of which form direct contacts with GABAergic neurons, suggesting that NE may regulate synaptic inhibition through direct actions on interneurons. However, the effects of NE on interneurons are largely unknown.
NE has been implicated in regulating the sleep-wake cycle, promoting vigilance, and enhancing learning and memory, but our understanding of how different subsets of NE-responsive interneurons modulate these neural processes is incomplete. Recent evidence suggests that NE may differentially regulate at least two discrete, functionally defined, subpopulations of inhibitory GABAergic interneurons in the hippocampus. To test this hypothesis, the following specific aims have been set forth: Project #1 characterize the electrophysiological effects of NE on different subtypes of hippocampal interneurons, Project #2 identify the particular receptors mediating these NE responses, and Project #3 determine the connectivity, neurochemical and synaptological profile of those interneurons affected by NE. In addition to extending our basic understanding of how NE modulates interneuron function, the information derived from this project may yield important insights into the mechanisms underlying certain cognitive and behavioral states.
Undergraduate participants will work in the laboratory, alongside undergraduate and graduate students at UND. In addition to basic lab procedures, all students will be trained to handle rodents, dissect brains and prepare brain slices via methods approved by the Institutional Animal Care and Use Committee at UND and to, depending on which aim of this project they choose to work on (aim #1, 2 or 3), will learn to use a variety of lab equipment and techniques. These equipment and techniques include: field and whole-cell electrophysiological recordings, infrared video-imaging, single cell real time RT-PCR analysis, intracellular dye-filling, immunohistochemistry, morphological reconstruction, and fluorescent microscopy.
Peter J. Meberg Laboratory Student Projects:
Actin Cytoskeleton in Neuronal Function
Dr. Meberg’s research is focused on the regulation and role of the actin cytoskeleton in neuronal development and synaptic plasticity. Recent work has focused on the role of two closely related actin binding proteins in neurons, actin depolymerizing factor (ADF) and cofilin, that are primarily localized to growth cones and dendritic spines. ADF and cofilin are inhibited by phosphorylation. Active ADF and cofilin increase actin turnover through F-actin severing and increasing the rate of actin monomer loss from the minus end of actin filaments. Students would have projects related to one of the following research areas:
Project #1: ADF/cofilin activity and growth cone dynamic. Overexpression of ADF/cofilin increases neurite outgrowth, and extracellular signals that affect growth cone navigation require signaling through ADF/cofilin. Students would inhibit or overexpress active forms of ADF/cofilin and do live imaging of growth cones.
Project #2: Regulation of ADF/cofilin activity by synaptic activity. Seizures induce the loss of dendritic spines (ref) and sprouting of mossy fiber axons in the hippocampus. Dr. Meberg and his students discovered that seizure induction in rats results in the redistribution of actin filaments in the hippocampus and dephosphorylation/activation of ADF and cofilin (manuscript in preparation). These results indicate that ADF/cofilin may contribute to axon sprouting by altering actin dynamics. Student projects could include investigations into the signal pathways upstream of ADF/cofilin dephosphorylation and in vitro models to study seizures, such as hippocampal slices (in collaboration with Dr. Doze).
Project #3: Proteomic screen to discover other cytoskeleton regulating proteins involved in seizure effects on cytoskeleton. ADF and cofilin are not the only proteins likely involved in mossy fiber sprouting and spine loss after seizures. Currently the Meberg lab is screening for other candidate regulators of the cytoskeleton by comparing levels of protein phosphorylation between control and seizure animals. Several likely candidates have already been identified by 1d gel electrophoresis and mass spectroscopy, and it is anticipated that several more will be identified on 2d gels in the next few months. For each candidate identified, the following will need to be done: a) Verify that the protein is indeed changed in phosphorylation by using western blots; b) Examine the intracellular and tissue distribution of the protein in neurons; and c) determine how changes in phosphorylation/ activity of the protein influence synaptic and morphological properties of the neurons.
Timothy Bigelow (Electrical Engineering)
Medical ultrasound has been successfully used in cardiac applications to image blood flow for many years. Recently, imaging blood flow using ultrasound has been significantly enhanced by the use of ultrasound contrast agents. Ultrasound contrast agents are lipid encapsulated microbubbles on the order of a few microns in diameter that are injected into the blood stream. These microbubbles are very effective at scattering the ultrasound waves producing contrast in the ultrasound image allowing for the quantification of blood flow dynamics even for very slow flow such as the perfusion of blood through capillaries. In addition to imaging, ultrasound contrast agents have also shown great potential in animal experiments for enhancing drug delivery to cells, performing gene therapy, and enhancing heating and subsequent cell death when performing ultrasound thermal ablation. Due to the past successes of contrast agents in blood flow imaging as well as the possible therapeutic benefits, there is a strong desire to extend the use of contrast agents to the fetus. Regrettably, the use of ultrasound contrast agents under certain ultrasound exposure conditions has resulted in vascular damage in adult animals. Therefore, any potential damage to the developing vascular system of the fetus needs to be understood before extending the use of contrast agents to the human fetus. Depending upon the interest of the student, the following projects are potentially available.
- Obtain images of blood perfusion in the developing fetal brain in an animal model and assess the clinical potential for such images.
- Expose the developing fetal brain to varying levels of ultrasound after injection of an ultrasound contrast agent and determine the thresholds for vascular damage.
Zebrafish are currently the only vertebrate model organism amenable to forward genetics, in which mutagenesis, extensive screening for various phenotypes and genetic analysis are used to identify novel genes, or novel functions for known genes in biological processes of interest. I have two main projects in my developing research program; both involve characterizing mutations generated in zebrafish. The first set of mutants has defects in the ability to generate new neurons in the peripheral retina. These defects may represent genes important in adult neurogenesis which is prevalent in fish, but only limited in mammals. The second set of mutants display deviant behavioral responsiveness to cocaine, in short the drug has abnormal addictive potential in these fish. I am currently trying to identify the defective genes in these two sets of mutants and trying to understand their cellular context. While genetic means have been used to map the mutations, I would like to employ proteomic and microarray methods to complement genetic approaches in identifying the defective genes. I am also interested in developing new addiction related behavioral tests to serve as screening tools for additional genetic studies. My long term goal is to find novel genes and molecular pathways linked to retinal neural stem cell regulation and response to cocaine.
One of our research goals is to determine the mechanisms by which inflammatory activation of brain glial cells contributes to neurodegeneration. Currently, our main interest is the process by which a specific type of glia, microglia, contribute to the pathophysiology of Alzheimer's disease (AD). AD is a neurodegenerative disorder characterized by progressive dementia and an accumulation of extracellular senile plaques and intracellular neurofibrillary tangles in the brain. In addition, diseased brains exhibit a profoundly increased microglial and astrocyte activation phenotype. One prevailing theory is that this “gliosis” contributes to the neuron loss that is observed during disease.
Microglia are the resident immune effector cells of the brain and their activation state can likely contribute to both degeneration and regeneration. This dichotomy provides the opportunity to modulate their phenotype to promote their regenerative function while limiting their degenerative behavior. For example, it is now becoming clearer that many of the neurodegenerative diseases that afflict our brains result, in part, from aberrant microglial responses. In addition to AD, Parkinson’s disease, amyotropic lateral sclerosis, and multiple sclerosis are other examples of chronic nervous system diseases in which microglial hyper-reactivity likely contributes to the cell death that occurs. We are working to understand specifically what goes wrong with the microglia in these conditions. Once we identify the nature of the pathologic response, we work to stop or reverse it in an effort to promote cell survival in the brain. This approach allows us to identify and propose novel therapeutic agents for treating these diseases. We routinely use in vitro primary cell culture models of disease to first define our molecular targets for therapeutic intervention. Next, we verify the efficacy of our approach through in vivo whole animal rodent models of disease. Currently we are pursuing ongoing projects related to Alzheimer’s disease, Parkinson’s disease, cerebrovascular disease, and multiple sclerosis.
Jara JH, Singh BB, Floden AM, Combs CK. Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death. J Neurochem. 2007 Mar;100(5):1407-20.
Austin SA, Floden AM, Murphy EJ, Combs CK. Alpha-synuclein expression modulates microglial activation phenotype. J Neurosci. 2006 Oct 11;26(41):10558-63.
Neural Pharmacology of fetal vascular function.
Neural regulation of vascular function is surprisingly understudied in most fetal vertebrates. This is amazing given the major selective pressures that occur during critical developmental periods of the vertebrate life cycle. The focus of my research is to understand how neural and neuropharmacological control of cardiovascular function develops in vertebrates and the impact the developmental environment can have on that maturation process. This project will focus on the neuropharmacology of a unique fetal vascular bed, the chorio-allantois, in fetal chickens at two points of development. This vascular bed contains the majority of the fetal blood volume for long periods of time and thus must be tightly regulated to maintain normal cardiovascular function. While the potential contribution of the chorio-allantois (CA) vascular bed to fetal cardiovascular function is clear, the neuropharmacology is unknown. An assessment of cholinergic and adrenergic control is therefore critical. This project will utilize a perfused isolated CA vascular technique to investigate the change in sensitivity to adrenergic and cholinergic stimulation, two key components of autonomic nervous control. These studies will be carried out on fetal day 15 and 19, which represent the transition in gas exchange methods in the animal. Collectively the study will provide important information needed to expand our understanding of the regulatory overlap between the nervous system and the cardiovascular system during early vertebrate development.
Inflammation induced tissue injury is a major and primary contributing factor to the pathogenesis of a variety of human brain diseases including; cerebral ischemia, spinal cord injury, multiple sclerosis, AIDS-related dementia, and Alzheimers disease. Inflammation induces the production, by activated microglia and astrocytes, of the pro-inflammatory cytokines (e.g. IL-1, TNFa, etc.). These cytokines bind to receptors that, among other things, couple to “effector” enzymes such as phospholipases A2 and phospholipases C, provoking pathological lipid-mediated signaling cascades. Induction of these cascades is known to disrupt membrane phospholipid metabolism, increase the expression of enzymes found in the inflammatory cascades, and ultimately result in altered cellular homeostasis and cell death. Unfortunately, little is known concerning the relative contribution that specific inflammation-induced signaling pathways play in the progression of neurodegenerative events associated with these diseases.
Our long-term goal is to understand the extent by which alterations in lipid-mediated signal transduction contribute to the progression of injury associated with neuroinflammation and to use this knowledge to develop therapeutic strategies to treat these diseases. Studies in our laboratory include: identifying lipid-mediated signaling pathways that contribute to the progression of inflammatory events in the central nervous system, identifying the specific roles phospholipases A2 and C play in the progression of disease, and to distinguish the role that ether phospholipid metabolism has in normal and injured brain.
