Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
10 results
Search Results
Item Analysis of transport in the brain(Montana State University - Bozeman, College of Engineering, 2021) Ray, Lori Ann; Chairperson, Graduate Committee: Jeffrey Heys; Jeffrey J. Heys was a co-author of the article, 'Fluid flow and mass transport in brain tissue: a literature review' in the journal 'Fluids' which is contained within this dissertation.; Jeffrey J. Iliff and Jeffrey J. Heys were co-authors of the article, 'Analysis of convective and diffusive transport in the brain interstitium' in the journal 'Fluids and barriers of the CNS' which is contained within this dissertation.; Martin Pike, Jeffrey J. Iliff and Jeffrey J. Heys were co-authors of the article, 'Quantification of transport in the whole mouse brain' which is contained within this dissertation.Neurodegeneration is one of the most significant medical challenges facing our time, yet the gap between therapies and understanding of the inner workings of the brain is great. Impairment of waste clearance has been identified as one key underlying factor in the vulnerability of the brain to neurodegeneration, stimulating research towards understanding transport of molecules in the brain. Based on experimental findings, a unique-to-the-brain circulation has been proposed, the glymphatic system, where cerebrospinal fluid surrounding the brain moves into the brain along the periarterial space that surrounds cerebral arteries, flows through the interstitial space between brain cells, where cellular wastes reside, and carries waste out of the brain tissue along perivenous routes. However, current gaps in knowledge about the driving force for fluid flow have generated scientific skepticism, and an independent method for quantifying transport and demonstrating the presence or absence of convection is desirable. In this work, computational transport models are developed and used to analyze published experimental data to determine fundamental transport parameters for different aspects of the glymphatic circulation. Calculated transport parameters are compared to the known diffusivity of tracers through brain tissue to draw conclusions about the presence and significance of bulk flow, or convection. Based on these analyses, transport in the periarterial spaces surrounding major arteries is over 10,000 times faster than diffusion and in brain tissue, containing both periarterial and interstitial space, transport is around 10 times faster than diffusion alone (for characteristic transport lengths around 1 mm). Interstitial velocity is determined to be on the order of 0.01 mm/min, making convection in the interstitial spaces of the brain critical to the transport of large, slow-to-diffuse molecules implicated in neurodegeneration. Convection is demonstrated to be a significant mechanism of transport throughout the brain. Observations and analyses from this work contribute further evidence to a circulatory-like system in the brain with relatively rapid convection along periarterial space, branching throughout the brain tissue and slower convection across that tissue, in the interstitial spaces of the brain. Transport models developed in this work are demonstrated to be useful tools for gleaning further information from experimental data.Item Concussion education practices among high school coaches in Montana(Montana State University - Bozeman, College of Education, Health & Human Development, 2021) Hughes, Patrick Michael; Chairperson, Graduate Committee: Tricia SeifertConcussions in high school sports present a legitimate threat to athletes across the United States. In the absence of qualified healthcare providers, coaches are most often the individuals who are tasked with making sideline analyses of removing the injured athlete from participation. To help ensure the most optimal outcomes for these athletes, it is important to accurately determine the most effective ways of training coaches. This study analyzed high school coaches in the state of Montana to determine their familiarity with different types of concussion education programs. In addition, particular focus was placed on determining if coaches' familiarity of concussion education programs differed significantly between coaches in urban versus rural school settings and between coaches of different sports.Item Techniques for improving activity based biosensors: a Kuhl platform for engineering(Montana State University - Bozeman, College of Agriculture, 2020) Thomas, Merrilee Anne; Chairperson, Graduate Committee: Thomas Hughes and Susy C. Kohout (co-chair); Thomas E. Hughes was a co-author of the article, 'Optically activated, customizable, excitable cells Kuhl platform for evolving next gen biosensors' submitted to the journal 'PLOS One' which is contained within this dissertation.According to Kuhn, ''there are three classes of problems - determination of significant facts, matching of facts with theory, and articulation of that theory (Kuhn 2012).'' The current paradigm in molecular neuroscience is that there is a need for revolutionary tool development in neuroscience. Interestingly, the need for better tools in neuroscience is to answer neuroscience theories and provide the determination and articulation of those theories. Currently, the neuroscientist's toolbox is growing and the ways in which those tools are used is rapidly changing. Neuroscience underwent a revolution when we were able to take single-cell recording in vivo and then assign field properties to individual neurons based upon those responses (O'Keefe and Bouma 1969; O'Keefe and Dostrovsky 1971; Moser et al. 1995). Scientists became adept at imaging increasingly smaller regions of the functioning human brain (Price 2012). We have since been able to genetically encode and manipulate proteins and pathways while recording from them using fluorescence (Southern and Berg 1982; Chalfie 2009). In vitro and in vivo we have harnessed the use of light to stimulate or inhibit specific neurons or ligands (Boyden 2011; Adamantidis et al. 2007). These tools are just the beginning and by no means is this an exhaustive list. We introduce the Kuhl synthetic cell system that provides a customizable de-novo excitable cell. The Kuhl system is activated using a blue light photo activated cyclase bPAC. It can be used to create better tools to image the brain and can be used to screen multi-color fluorescent sensors. Interestingly, sensors that are within bPACs activation spectrum can be used in these synthetic cells. We show that both red and green Ca 2+ sensors can be imaged simultaneously, and both Ca 2+ and Voltage sensors can be screened in the Kuhl system. The Kuhl system has the potential to be used to screen for drug compounds and in theory, they could be used in studying pathways that are less understood, such as the mTOR pathway.Item Large-scale spatiotemporal cortical dynamics in visual short-term memory: from spiking activity to oscillations(Montana State University - Bozeman, College of Agriculture, 2020) Hoffman, Steven Joseph; Chairperson, Graduate Committee: Charles M. Gray and Jamie Mazer (co-chair); Nicholas M. Dotson was an author and Baldwin Goodell and Charles M. Gray were co-authors of the article, 'A large-scale semi-chronic microdrive recording system for non-human primates' in the journal 'Neuron' which is contained within this dissertation.; Nicholas M. Dotson and Charles M. Gray were co-authors of the article, 'The cortical local field potential exhibits distinct spatial gradients that vary with frequency and time during visual short-term memory' which is contained within this dissertation.; Dissertation contains a article of which Steven Joseph Hoffman is not the main author.Cognitive processes occur through coordinated activity via disparate cortical and subcortical brain structures. Although these structures may be widely separated, evolutionary pressures dictate that cognition must occur rapidly and efficiently. In order to capture these brain-wide activity patterns the tools for measuring them need to be similarly capable of measurements of both high spatial coverage, and high temporal resolution. Additionally, the measurements would ideally be of the activity of the fundamental units involved in cognition, that is the neurons, rather than an extrapolation of their activity via a different signal source. However, outside of the work presented here, current technologies are rare that allow both the requisite coverage and spatiotemporal resolution to achieve these measurements. The results of the studies presented in Chapters 2-4 provide both the tools for making such measurements, and the initial analyses of the neuronal dynamics during short-term memory. In Chapter 2 we present the technological and methodological process for recording neural activity (both action potentials and local field potentials) from across roughly a hemisphere of cortex in the macaque monkey performing a visual short-term memory task. In visual short-term memory a visual percept must be maintained then recalled when it is no longer present. This cognitive process is one we use nearly incessantly in every-day life. In Chapter 3 we found task dependent spiking activity during short-term memory is wide-spread, and that most areas display a balanced state of both increases and decreases in firing rate. Within these areas we found a hierarchically organized subset of cortical areas that also showed stimulus specific activity during the memory period of the task. In Chapter 4 we used spectral analysis to investigate the oscillatory make-up of neural activity across the recorded areas. We found within specific frequency bands there are different gradients of amplitude of spectral power across cortex. Additionally, we found that we could use a small number of spectrally derived variables in order to decode the brain area origin of the signal. This shows that areas have a characteristic spectral composition, that varies systematically across the cortical mantle.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 Exploring the feasibility of an automated biocuration pipeline for research domain criteria(Montana State University - Bozeman, College of Engineering, 2019) Anani, Mohammad; Chairperson, Graduate Committee: Indika KahandaResearch on mental disorders has been largely based on manuals such as the ICD-10 (International Classification of Diseases) and DSM-V (the Diagnostic Statistical Manual of Mental Disorders), which rely on the signs and symptoms of disorders for classification. However, this approach tends to overlook the underlying mechanisms of brain disorders and does not express the heterogeneity of those conditions. Thus, the National Institute of Mental Health (NIMH) introduced a new framework for mental illness research, namely, Research Domain Criteria (RDoC). RDoC is a research framework which utilizes various units of analysis from genetics, neural circuits, etc., for accurate multi-dimensional classification of mental illnesses. The RDoC framework is manually updated with units of analysis in periodic workshops. The process of updating the RDoC framework is accomplished by researching relevant evidence in the literature by domain experts. Due to the large amount of relevant biomedical research available, developing a method to automate the process of extracting evidence from the biomedical literature to assist with the curation of the RDoC matrix is key. In this thesis, we formulate three tasks that would be necessary for an automated biocuration pipeline for RDoC: 1) Labeling biomedical articles with RDoC constructs, 2) Retrieval of brain research articles, and 3) Extraction of relevant data from these articles. We model the first problem as a multilabel classification problem with 26 constructs of RDoC and use a gold-standard dataset of annotated PubMed abstracts and employ various supervised classification algorithms. The second task classifies general PubMed abstracts relevant to brain research using the same data from the first task and other unlabeled abstracts for training a model. Finally, for the third task, we attempt to extract Problem, Intervention, Comparison, and Outcomes (PICO) elements and brain region mentions from a subset of the RDoC abstracts. To the best of our knowledge, this is the first study aimed at automated data extraction and retrieval of RDoC related literature. The results of automating the aforementioned tasks are promising; we have a very accurate multilabel classification model, a good retrieval model, and an accurate brain region extraction model.Item Design and implementation of a real-time system to characterize functional connectivity between cortical areas(Montana State University - Bozeman, College of Engineering, 2017) Parsa Gharamaleki, Mohammadbagher; Chairperson, Graduate Committee: Brendan MumeyDespite a thorough mapping of the anatomical connectivity between brain regions and decades of neurophysiological studies of neuronal activity within the various areas, our understanding of the nature of the neural signals sent from one area to another remains rudimentary. Orthodromic and antidromic activation of neurons via electrical stimulation ('collision testing') has been used in the peripheral nervous system and in subcortical structures to identify signals propagating along specific neural pathways. However, low yield makes this method prohibitively slow for characterizing cortico-cortical connections. We employed recent advances in electrophysiological methods to improve the efficiency of the collision technique between cortical areas. There are three key challenges: 1) maintaining neuronal isolations following stimulation, 2) increasing the number of neurons being screened, and 3) ensuring low-latency triggering of stimulation after spontaneous action potentials. We have developed a software-hardware solution for online isolations and stimulation triggering, which operates in conjunction with two hardware options, Hardware Processing Platform (HPP) or a Software Processing Platform (SPP). The HPP is a 'system on a chip' solution enabling real-time processing in a re-programmable hardware platform, whereas the SPP is a small Intel Atom processor that allows soft real-time computing on a CPU. Employing these solutions for template matching both accelerates spike sorting and provides the low-latency triggering of stimulation required to produce collision trials. Recording with a linear tetrode array electrode allows simultaneous screening of multiple neurons, while the software package coordinates efficient collision testing of multiple user-selected units across channels. This real-time connectivity screening system enables researchers working with a variety of animal models and brain regions to identify the functional properties of specific projections between cortical areas in behaving animals.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 effects of alcohol exposure on the development of the rat superior colliculus(Montana State University - Bozeman, College of Letters & Science, 1999) Selong, Tiffany HardinItem Novel pharmaceutical combination confers protection from delayed cell death following transient cerebral ischemia(Montana State University - Bozeman, College of Letters & Science, 2009) Chapman, Courtney Myfanwy; Chairperson, Graduate Committee: A. Michael BabcockStroke is a leading cause of death and disability throughout the world; ischemia is the most common form of stroke. Medical procedures such as cardio-pulmonary bypass surgery can cause ischemic stroke can be caused. There are no treatments to limit neural impairment following stroke. The current research investigates neuroprotection offered by treatment with a novel drug combination consisting of Simvastatin TM, Gemfibrozil TM, Troglitazone TM, and Spironolactone TM. Animals were treated with the drug cocktail three weeks proceeding and one week subsequent to surgery. Ischemic insult was induced by clamping the carotid arteries for 5 min. Sham subjects underwent similar surgical procedures, but the carotids were not clamped. Twenty-four hrs following the surgical procedure locomotor activity was monitored in an open field for 5 min. Seven to fourteen days following ischemia or the sham procedure animals were sacrificed and sections containing the hippocampal CA1 region were mounted on slides and stained with cresyl violet. The CA1 region was rated on a 4-point scale for level of damage. Rodents generally show increased locomotor activity following transient global ischemia in an open field. In our study, ischemic animals that received vehicle demonstrated increased activity relative to the animals that received the drug treatment on all behavioral measures. Ischemic animals that received vehicle treatment had significantly more neural damage in the hippocampal CA1 region than ischemic animals receiving the drug. The appearance of neurons in the CA1 hippocampal regions of animals in the sham condition was not significantly different from ischemic animals in the drug treatment condition. It is concluded that the drug treatment is effective in offering neuroprotection during transient global ischemia. The next step is to characterize the biochemical mechanisms behind the neuroprotection conferred by the drug treatment. Contrasting the protein expression levels of animals receiving the vehicle treatment with animals receiving the drug treatment following an ischemic insult will assist in elucidating these pathways. Predictions are made regarding the biochemical mechanisms affected by the drug treatment based on previous research on the biochemical pathways affected by each pharmaceutical.