Theses and Dissertations at Montana State University (MSU)
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Item Improving the two-photon absorption properties of fluorescent proteins for neuroscience(Montana State University - Bozeman, College of Letters & Science, 2020) Molina, Rosana Sophia; Chairperson, Graduate Committee: Thomas Hughes; Yong Qian, Jiahui Wu, Yi Shen, Robert E. Campbell, Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Understanding the fluorescence change in red genetically encoded calcium ion indicators' in the journal 'Biophysical Journal' which is contained within this dissertation.; Tam M. Tran, Robert E. Campbell, Gerard G. Lambert, Anya Salih, Nathan C. Shaner, Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Blue-shifted green fluorescent protein homologues are brighter than enhanced green fluorescent protein under two-photon excitation' in the journal 'The Journal of physical chemistry letters' which is contained within this dissertation.; Jonathan King, Jacob Franklin, Nathan Clack, Christopher McRaven, Vasily Goncharov, Daniel Flickinger, Karel Svoboda, Mikhail Drobizhev, Thomas E. Hughes were co-authors of the article, 'An instrument to optimize fluorescent proteins for two-photon excitation' which is contained within this dissertation.Untangling the intricacies of the brain requires innovative tools that power basic research. Fluorescent proteins, first discovered in jellyfish, provide a genetically encodable way to light up the brains of animal models such as mice and fruit flies. They have been made into biosensors that change fluorescence in response to markers of neural activity such as calcium ions (Ca 2+). To visualize them, neuroscientists take advantage of two-photon excitation microscopy, a specialized type of imaging that can reveal crisp fluorescence images deep in the brain. Fluorescent proteins behave differently under twophoton excitation compared to one-photon excitation. Their inherent two-photon properties, namely brightness and peak absorption wavelength, limit the scope of possible experiments to investigate the brain. This work aims to understand and improve these properties through three projects: characterizing a set of red fluorescent protein-based Ca 2+ indicators; finding two-photon brighter green fluorescent proteins; and developing an instrument to screen for improved fluorescent proteins for two-photon microscopy. Analyzing nine red Ca 2+ indicators shows that they can be separated into three classes based on how their properties change in a Ca 2+-dependent manner. In one of these classes, the relative changes in one-photon properties are different from the changes in two-photon properties. In addition to characterizing, identifying and directly improving fluorescent proteins for enhanced two-photon properties is important. Presented here is a physical model of the light-absorbing molecule within the green fluorescent protein (the chromophore). The model predicts that green fluorescent proteins absorbing at higher energy wavelengths will be brighter under two-photon excitation. This proves to be the case for 12 blueshifted green fluorescent proteins, which are up to 2.5 times brighter than the commonly used Enhanced Green Fluorescent Protein. A way to directly improve fluorescent proteins is through directed evolution, but screening under two-photon excitation is a challenge. An instrument, called the GIZMO, solves this challenge and can evolve fluorescent proteins expressed in E. coli colonies under two-photon excitation. These results pave the way for better two-photon fluorescent protein-based tools for neuroscience.Item Determining function of the IKAP protein in the peripheral nervous system for targeted therapeutic intervention in familial dysautonomia(Montana State University - Bozeman, College of Letters & Science, 2017) Ohlen, Sarah Beth; Chairperson, Graduate Committee: Frances Lefcort; Magdalena L. Russell, Michael J. Brownstein and Frances Lefcort were co-authors of the article, 'BGP-15 prevents the death of neurons in a mouse model of familial dysautonomia' in the journal 'Proceedings of the National Academy of Sciences' which is contained within this thesis.Familial Dysautonomia (FD) is a recessive genetic disorder that leads to devastation of the peripheral nervous system and is the result of incomplete neurodevelopment and progressive neurodegeneration. The disorder is also marked by a continual loss of retinal ganglion cells that leads to blindness. Even with early identification and treatment, the disorder is ultimately fatal. FD is caused by mutation in the IKBKAP gene that leads to cell-type specific loss of the IKAP protein, also known as ELP1. IKAP functions as a part of the six-unit Elongator complex. The role of Elongator is unresolved, although data has accumulated that support Elongator function in tRNA modification and efficient translation of proteins and that its absence leads to cell stress and neurological impairment. We have a mouse model of FD in which mouse Ikbkap is deleted from the peripheral nervous system, and it recapitulates the death of autonomic and TrkA+ sensory neurons observed in FD patients. As we can culture TrkA+ neurons in vitro, while also studying this neuronal population in vivo, we have a system to investigate our goals of (1) determining cellular processes that go awry in absence of Ikap and (2) targeting these cell types and events to prevent their progressive death. We have determined that mitochondrial and cytoskeletal function are disrupted in Ikbkap -/-, TrkA+ neurons and show activation of stress signaling. Interestingly, disrupted mitochondrial function is an emerging hallmark common to most neurodegenerative diseases. We have identified that the compound, BGP-15, is able to restore aspects of mitochondrial function and stress signaling in vitro and can restore neuronal survival of TrkA+ neurons lacking Ikap in vitro and in vivo. BGP-15 also improves actin cytoskeletal function and target innervation. Additionally, we have determined that introduction of the C-terminal half of human IKAP is sufficient to increase neuronal survival in vitro. This smaller protein fragment is compatible with viral delivery to retinal ganglion cells and could be utilized for gene therapy, potentially preventing this neuronal death that lead to blindness. Our goals now are to further explore stress pathways common to many neurodegenerative disorders and optimize rescue strategies in vivo.Item If you build it, they will come: engineering the next generation of optical tools to image neural activity deep within the living brain(Montana State University - Bozeman, College of Letters & Science, 2017) Barnett, Lauren Marie; Chairperson, Graduate Committee: Thomas Hughes; Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Deciphering the molecular mechanism responsible for GCAMP6M's Ca 2+ dependent change in fluorescence' in the journal 'PLoSONE' which is contained within this thesis.; Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Making pKa-altering mutations in GCAMP6M changes the Ca 2+-dependent fluorescence response' submitted to the journal 'PLoSONE' which is contained within this thesis.; Jelena Platisa, Marko Popovic, Vincent A. Pieribone and Thomas Hughes were co-authors of the article, 'A fluorescent, genetically-encoded voltage probe capable of resolving action potentials' in the journal 'PLoSONE' which is contained within this thesis.; Lauren M. Barnett, Mikhail Drobizhev, Geoffrey Wicks, Alexander Mikhaylov, Thomas E. Hughes and Aleksander Rebane were co-authors of the article, 'Two-photon directed evolution of green fluorescent proteins' in the journal 'Nature Scientific Reports' which is contained within this thesis.To see the activity of large, integrated neural circuits functioning in real-time inside of a living brain, neuroscientists will need multiple genetically-encoded fluorescent activity sensors that can be individually targeted to specific cell types, are fast enough to resolve multiple action potentials, can be distinguished from one another and imaged deep within the brain. The goal of this work is to better understand and improve upon the most recent generations of genetically-encoded Ca 2+ and voltage sensors, and to expand biosensor utility in two-photon excitation, which will be necessary to image neural activity deep within the brain. Genetically-encoded Ca 2+ sensors measure the intracellular Ca 2+ release that occurs downstream of an action potential. The GCaMP6 series are the best Ca 2+ sensors available, however little is known about how they work. Measurements of four different states in GCaMP6m reveal that its large Ca 2+-dependent change in 470 nm excited fluorescence is due to a redistribution of the chromophore protonation state, from a neutral form excited at ~400 nm to an anionic form excited at ~470 nm, via a change in pK a. Making pK a-altering mutations in GCaMP6m changes the Ca 2+-dependent fluorescence response. This highlights the importance of Delta pK a and identifies key amino acid positions that will be important for improving GCaMP6m and GCaMP-like biosensors. A direct readout of an action potential would be ideal for capturing complex signal transduction in the brain. This will require a bright, fast voltage sensor. ElectricPk is the first genetically-encoded voltage sensor with a fluorescence response fast enough to resolve multiple action potentials in mammalian neurons. This design indicates it is possible to couple a fluorescence change with a very fast (~1 ms) voltage-dependent movement in the Ciona intestinalis voltage-sensitive phosphatase protein. Whether imaging a downstream Ca 2+ signal or a direct change in membrane potential, to image neuronal activity in deep brain tissue biosensors will need to be brightly fluorescent in two-photon excitation. The two-photon directed evolution of green fluorescent proteins presented here is a proof-of-principle design that shows a high-throughput screen focused on improving the two-photon properties of a fluorescent protein is possible.Item The role of FGF signaling in neural crest development(Montana State University - Bozeman, College of Letters & Science, 2014) Dunkel, Haley Ann Arthun; Chairperson, Graduate Committee: Frances LefcortThe mechanisms that stimulate Neural Crest Cell (NCC) migration and cessation into discrete sympathetic ganglia (SG) and dorsal root ganglia (DRG) are incompletely understood. In this study we investigated the role of Endothelial Cells (ECs) and the shared growth factors of the nervous and vascular system: fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in the development of the peripheral nervous system. We hypothesized that ECs impact neural crest patterning. Using live time-lapse microscopy, we found that NCCs and ECs interact extensively during neural crest migration and DRG development, including stimulating proliferation of Pax3+ neural progenitors. These studies also revealed that ECs of the intersomitic vessel perhaps act as a substrate for migrating NCCs while the perisomitic vessel acted as a boundary keeping NCCs in the forming DRG. In order to determine the role of ECs in PNS development we focally eliminated them with FGF receptor (FGFR) blocker Su5402. Su5402 focally reduced ECs and caused NCCs to remain dorsal. Given that FGFs effects could be direct or indirect on NCC, we sought to reduce FGF signaling solely in NCe. To this end we overexpressed a dominant negative FGFRl (DNFGFR1) and FGFR3 in ovo in NCCs. Our studies demonstrated that while blocking FGFR3 signaling did not impair NCC migration, NCCs expressing the DNFGFRl behaved similarly as did the Su4502 treated NCCs: they stayed dorsally at the expense of the SG and DRG and exhibited distinct morphologies. Stimulating FGFR signaling with the ligand FGF8 promoted migration in vitro and increased ventral migration of NCCs in vivo. To determine if the alterations in DNFGFRl + NCCs was due to changes in cell adhesion molecule function, we transfected NCC with DNFGFRl and gain and loss of function Ncadherin constructs. Surprisingly the fate of DNFGFR1+ NCCs was rescued by increasing expression of N-cadherin, while double transfections of DNFGFRl and DN-N-cadherin further impaired migration. While it is possible that N-cadherin and FGFRl directly interact, the exact interactions of FGFRl and N-cadherin need to be further investigated. FGFR3 was not found to function in NCC migration, but preliminary findings show it is required for TrkA+ neural differentiation.Item The Fronto-Parietal network and beyond : a study of the spatiotemporal patterns underlying visual working memory(Montana State University - Bozeman, College of Letters & Science, 2014) Dotson, Nicholas Monroe; Chairperson, Graduate Committee: Charles M. Gray; Rodrigo F. Salazar and Charles M. Gray were co-authors of the article, 'Spatiotemporal activity patterns reveal the interplay between integration and segregation during visual working memory' which is contained within this thesis.Working memory, an integral component of higher cognitive functions, involves the short-term retention and utilization of behaviorally relevant information when that information is no longer available in the environment. Tragically, individuals suffering from traumatic brain injuries, psychiatric disorders, and other neurological disorders often exhibit working memory deficits. The study of working memory may thus provide insight into the mechanisms underlying cognitive functions and the potential to alleviate major health problems. In order to understand cognitive processes, like working memory, several pieces of information must be considered: the cortical and sub-cortical areas involved, the manner in which these areas integrate, or share information, and the underlying dynamics of these integrative processes. These pieces form a hierarchical structure of investigation, from the individual areas to global principles of coordination. The objective of this study is to elucidate the relevant spatiotemporal patterns of oscillatory synchronization underlying visual working memory in the fronto-parietal network and across the brain. Relevant patterns for consideration are those that encode stimulus information, are modulated by the task, and those with distinct anatomical variations. The results of the studies presented in Chapters 2-4 provide extensive evidence that oscillatory synchronization is a mechanism for distributed integration. We show that the patterns of coherent activity 1) encode working memory items, 2) are indicative of the task period, 3) provide the potential for multiple functional networks, defined by the relative phase and, 4) are highly dynamic, with large fluctuations in magnitude and relative phase. Future studies will be necessary to further investigate the role of oscillatory synchronization. Efforts to perturb oscillatory activity in order to illustrate its utility, rather than simply correlating its activity with stimulus and task components, will be crucial. Finally, understanding the spatiotemporal activity patterns underlying working memory may ultimately allow for the identification of aberrant patterns, such as those brought on by disease, and allow for these patterns to be meaningfully interacted with - via neuroprosthetic devices.Item Analysis of the central nervous system in a mouse model of HSAN Type III(Montana State University - Bozeman, College of Letters & Science, 2013) Waller, Hannah Rose; Chairperson, Graduate Committee: Frances LefcortFamilial Dysautonomia (FD), also called Riley Day Syndrome, is a Hereditary Sensory and Autonomic Neuropathy (HSAN Type III) that is characterized by dysfunction of the sensory and autonomic nervous systems. The disease is caused by a severe reduction in levels of the protein IKAP as a result of a point mutation in Ikbkap mRNA which results in targeting of the mRNA for nonsense mediated decay. In humans, symptoms include autonomic crises, tachycardia, blood pressure lability, lack of overflow tears, decreased pain and temperature sensation, and scoliosis. Half of affected individuals die by age 40. Although FD has been traditionally classified as a disease of the autonomic nervous system, there have been notable effects observed in the central nervous system (CNS) as well, though many of these observations remain to be quantified. The presented study evaluated the impact of FD on the CNS using a mouse model where Ikbkap was deleted selectively from neurons of the CNS. For this model, a conditional knockout (CKO) strategy was employed because mice that are null for Ikbkap die by embryonic day 10.5, precluding their usefulness for analyzing FD in the adult CNS. For this study, morphological analyses and immunohistochemical staining were performed on the brain tissue. Affected mice were found to have a significant reduction in choline acetyltransferase (ChAT) positive neurons in the dorsal motor nucleus of the vagus nerve (DMNX) relative to controls, indicating potential decreased parasympathetic innervation of the nucleus in the heart and other target organs. Additionally, the size of the lateral ventricles and hippocampus relative to hemisphere size was significantly increased for the mutant mice. Further, the corpus callosum and lateral amygdaloid nucleus areas were significantly decreased relative to wild-type controls. Cortical layering was found to be normal in Talpha1tubulin-Cre/Ikbkap CKO mice. Taken together, these results suggest that the morphological differences are associated with increased cell death and decreased neurogenesis and cell differentiation. The neural and morphological findings presented in this study are the first data demonstrating perturbations in the CNS of a mouse model for FD and may explain some of the phenotypes observed in FD patients.Item Exploration of cat striate cortex during natural scene stimulation(Montana State University - Bozeman, College of Letters & Science, 2013) Baker, Jonathan Lee; Chairperson, Graduate Committee: Charles M. GrayThe mammalian visual system evolved to process and represent objects within the complexities of the natural world to appropriately guide behavior in order to ensure survival and reproduction. Visual neuroscience has long sought to understand the neural basis of these processes, but the complexity of both the visual world and the brain has traditionally required a highly reductionist approach. The classical approach has led to the development of models of visual perception that are largely based on the analysis of how single cells within the visual system respond to simple and parametrically defined visual stimuli. However our ultimate goal is to understand how the visual system operates in the natural world. The experiments and results contained in this thesis were conducted, in part, to explore the cat's visual system during the presentation of time varying natural scenes, i.e. movies, and to attempt to validate our current working models of the visual system. Novel large-scale recording methods were employed to record from and characterize responses of large populations of cat primary visual cortex neurons during classical as well as movie stimulation. Contrary to the current models and our general understanding of how visual cortex represents information, groups of adjacent neurons responded heterogeneously to the movies presented and individual responses were very brief and highly sparse in time. The diversity and dynamics of the spiking activity was also significantly correlated with fluctuations of the ongoing local field potentials, specifically within the gamma frequency band, ranging from 25 to 90Hz. These gamma band oscillations also exhibited rich spatiotemporal dynamics throughout the movie presentations. In conclusion, time-varying natural scenes evoke response dynamics within the cat primary visual cortex not typically observed under more classical stimulus regimes. Future experimentation and the construction of biologically feasible models of visual cortex should take into account the diversity of responses observed under more natural stimulus conditions.Item Mouse and stem cell models of frontotemporal dementia(Montana State University - Bozeman, College of Letters & Science, 2012) Orr, Miranda Ethel; Chairperson, Graduate Committee: George A. Carlson; Frances Lefcort (co-chair)Alzheimer's disease (AD) is the most prevalent brain disease in the United States, and an escalating health concern. AD patient brains acquire hallmark protein aggregates, referred to as senile A beta plaques and neurofibrillary tangles (NFTs), that coincide with brain cell loss and dementia. A subset of AD patients carry mutant genes. Our understanding of AD largely relies on model systems that express these gene variants. Mice engineered to express AD-mutant human genes develop A beta plaques, but fail to develop the tau-containing NFTs or cell loss. Mutant tau variants are required to induce NFTs and neuronal loss in mice, but AD patients carry normal tau genes. The inability of mouse tau to become a pathogenic protein in the presence of AD-mutant gene variants, and the general insufficiency of the current systems to recapitulate AD, inspired the research described here. To determine if species differences between mouse and human tau inhibit the progression of AD in mice, I utilized a well-characterized mouse model of a related disease, frontotemporal dementia (FTD). FTD mice carry mutant human tau and develop NFTs and cell loss. I ablated mouse tau in FTD mice and looked for signs of more severe pathology. I compared the FTD mice, with and without mouse tau, to FTD mice with and without wild type human tau to investigate potential tau species-specific differences. My studies indicated that wild type tau, mouse or human, dampened the pathological effects of FTD tau implying a general, not mouse-specific, effect of normal tau protein. Our data suggest that unknown factors, distinct from endogenous mouse tau, contribute to the inability of mice to model AD. The recent interest in patient-specific stem cell (SC) models to study disease necessitates a thorough evaluation of their ability to recapitulate key characteristics of disease, reproducibility, and longevity. I generated and characterized brain SC cultures from FTD fetal mice and compared them to those generated from mice with normal human tau. Significant genotype associated differences were discovered in the SC system and later verified in adult mice to reinforce the potential of patient-specific SC models to study disease.Item Analysis of the expression and function of chicken protocadherin 1 in neural crest cell migration and peripheral nervous system formation(Montana State University - Bozeman, College of Letters & Science, 2007) Bononi, Judy; Chairperson, Graduate Committee: Roger Bradley.The necessary steps of development from a single cell to a multi-celled functional organism are complex. Many molecules have been identified and their roles characterized in this process. One interesting population of cells includes the highly migratory neural crest cells (NCCs) unique to the vertebrate embryo and existing transiently during early embryonic development. The NCCs migrate along specific pathways at specific timepoints, stop at target locations, differentiate and give rise to a variety of cell types and tissues. Trunk NCCs must choose between two different migratory pathways: the ventral route, giving rise to neurons and glia of the dorsal root ganglia (DRG), sympathetic ganglia (SG), Schwann cells of the ventral root (VR); or the dorsolateral pathway, giving rise to melanocytes. Although many aspects of neural crest migration have been elucidated, cessation of migration and subsequent differentiation at target structures is not clearly defined. One family of molecules involved in various steps of NCC migration is the cell-cell adhesion molecules, the cadherins. To investigate the involvement of cadherins in NCC migration and differentiation during development using the avian model system, a combination of experiments and techniques including a library screen, in situ hybridization, in ovo electroporation, immunohistochemical and immunofluorescence staining as well as live time-lapse confocal imaging were performed. Results from these experiments produced the discovery and isolation of a novel molecule in the family of cadherin adhesion molecules, chicken protocadherin-1 (cPcdh1). Expression analysis showed cPcdh1 expressed in migrating NCCs, the DRG, SG and Schwann cells along the VR. A distinct expression pattern showed cPcdh1 along the periphery of the DRG, where crest cells are in an undifferentiated and mitotically active state. Further testing with deletion constructs and siRNA demonstrated when cPcdh1 function is inhibited, a greater percentage of cells migrate to the SG and VR at the expense of the DRG. Time-lapse confocal imaging showed cPcdh1 cells having an elongated cell shape with contact primarily being formed with neighboring cells along the periphery and longer cell-cell contact than observed in the control. Collectively, the results provide evidence for cPcdh1 involvement in NCC migration arrest and DRG formation.Item Characterization of the neural codebook in an invertebrate sensory system(Montana State University - Bozeman, College of Letters & Science, 2007) Aldworth, Zane Nathan; Chairperson, Graduate Committee: John P. Miller; Tomas Gedeon (co-chair)An outstanding problem in neuroscience is to describe the relationship between various stimulus sources in the environment and how they are represented by patterns of activity in nervous systems, a problem generically referred to as 'neural coding'. Most previous methods developed to address this problem have assumed a linear relationship between environmental stimuli and neural responses, and generally relied on measures of the mean state of the environment preceding neural activity to characterize the stimulus-response transformation. The goal of this thesis is to develop new methods of characterization that extend earlier work, and to demonstrate the utility of these new methods through application to an invertebrate sensory system. All applications of the methods developed in this thesis were carried out in the cercal system of crickets. The cercal system mediates the detection and analysis of low velocity air currents, and is implemented around an internal representation of air current direction that demonstrates the essential features of a continuous neural map. The stimulus feature selectivity, timing precision and coding characteristics of two bilateral pairs of primary sensory interneurons of the cercal system were characterized using three novel techniques. First, estimates of the cells' feature selectivity that take the natural variance in stimulus-response latency (i.e., spike 'jitter') into account were derived. Second, the cells' stimulusresponse relationship was probed for specific non-linear aspects that could constitute 'temporal' encoding. Third, an iterative stimulation paradigm was used to test and refine the predictions of the cercal system's stimulus selectivity. Compared to earlier characterization of this system, these new analytical procedures yield significantly different estimates of the stimulus feature selectivity of these cells. A 'code book' for the stimulus-response characteristics of these cells is presented, with emphasis on demonstrating instances where a cell represents different stimuli with distinct spike 'code-word' patterns.