• Effects of Ketamine on Neural Signatures of Parkinson's Disease and a Novel String-Pulling Behavior Quantification System

      Cowen, Stephen L.; Vishwanath, Abhilasha; Falk, Torsten; Fuglevand, Andrew (The University of Arizona., 2021)
      The first part of the thesis reviews my investigations into the neural mechanisms underlying Parkinson’s disease (PD) and levodopa-induced dyskinesia (LID) and their treatment with levodopa and ketamine. The second part of the thesis summarizes our group’s development of a novel string-pulling behavior for assessing motor function in rodents.Part 1: Parkinson’s disease (PD) is a debilitating neurodegenerative disorder with motor and cognitive deficits. While levodopa is the leading treatment for PD, in many individuals long-term administration leads to Levodopa-Induced Dyskinesias (LID), which are uncontrollable involuntary movements. These side-effects are as debilitating as PD. A common neurophysiological feature of LID is the emergence of 80-Hz brain oscillations in the motor cortex. Ketamine, an FDA approved drug used in treating depression among other disorders and has been shown to alleviate LID symptoms in an animal model of LID. We used a unilateral 6-hydroxydopamine-lesion PD rat model to investigate the neural systems-level mechanisms of the effects of ketamine on LID. We found that ketamine administration was followed by reduced 80-Hz LID oscillations, increased gamma oscillations (~50 Hz), and reduced burst firing in individual neurons. One interpretation of these results is that ketamine triggers gamma activity that competes with and consequently reduces 80 Hz activity. Part 2: The development of complex motor tasks and behavioral analysis is crucial for understanding the workings of motor neurons and study motor deficits. We developed a novel behavior quantification system for a bi-manual string-pulling task. The system has a unique “infinite loop” and the capacity to simultaneous record neural activity. This integrated string-pulling system allows for investigation into the neural systems of motor behavior in healthy and disease populations.
    • Exploring G-Protein-Coupled Receptors Regulation, Specificity and Controllability of Exosomes Release in the Neuronal Cell Line SH-SY5Y

      Falk, Torsten; Sadideen, Doraid; Sherman, Scott J.; McKay, Brian S. (The University of Arizona., 2016)
      Parkinson's disease is a neurodegenerative disease characterized by the buildup of aggregated and spread of misfolded alpha-synuclein. How the misfolded alpha-synuclein contributing to the toxicity and death of neuronal cells has been the focal point of research. The spread of alpha-synuclein has been attributed to many mechanisms, one of which is via cell-derived vesicles called exosomes. This project aims to examine the controllability of exosome release. SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with dopamine receptor 3-green fluorescent protein, G-protein receptor 143 or green fluorescent protein and treated with either dopamine or L-DOPA. Medium was harvested and subjected to ultracentrifugation and a silver stain and western blot were performed. There was no significant difference in the total protein in the exosome fraction lanes between the treatment groups or within them. Another aim was to test the specificity of exosomes. Exosomes isolated from SH-SY5Y or MCF-7 were labeled with Exo-Red dye and introduced to wells containing SH-SY5Y, MCF-7 and CHO-K1 cells at room temperature and -4C. At room temperature, exosomes were observed intercellular in all of the cell lines, however, they did not deliver their content. At -4C exosome uptake was halted and they remained on the surface of the cells. Exo-Red labeled SH-SY5Y exosomes were treated with proteinase K and were introduced to CHO-K1 cells at -4C and room temperature. CHO-K1 did not take up exosomes, suggesting exosomes contain one or more necessary proteins needed to interact with the cellular membrane to initiate internalization. CHO-K1 cells were treated with versene to examine the involvement of integrin proteins. Exo-Red labeled SH-SY5Y exosomes were trapped on the surface of CHO-K1 after versene treatment. Lastly, Exo-Red labeled SH-SY5Y exosomes were biotinylated and magnetically captured then introduced to SH-SY5Y and MCF-7 cells and a silver stain and a biotinylated blot were performed. MCF-7 bound more Exo-Red labeled SH-SY5Y exosomes.
    • Insights into the Mechanisms Involved in Protective Effects of VEGF-B in Neurons

      Falk, Torsten; Sherman, Scott; Caballero, Beatrice; St. John, Paul (The University of Arizona., 2016)
      Vascular endothelial growth factor-B (VEGF-B), when initially discovered, was thought to be an angiogenic factor, due to its intimate sequence homology and receptor binding similarity to the prototype angiogenic factor, VEGF-A. Studies demonstrated VEGF-B, unlike VEGF-A, did not play a significant role in angiogenesis or vascular permeability and has become an active area of interest because of its role as a survival factor in pathological processes in a multitude of systems, including the brain. By characterization of important downstream targets of VEGF-B that regulate different cellular processes in the nervous system and cardiovascular system, it may be possible to develop more effective clinical interventions in diseases such as Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and ischemic heart disease, which all share mitochondrial dysfunction as part of the disease. Here we summarize what is currently known about VEGF-B function in pathological processes, compare probable mechanisms of action and elude to its potential as a homeostatic protective factor to increase mitochondrial function in the setting of neurological disease and cardiovascular disease.
    • Integrated Behavior Quantification System for the Measurement of Movement Kinematics and Neural Activity in the String Pulling Task

      Cowen, Stephen; Jordan, Gianna; Falk, Torsten; Fuglevand, Andrew (The University of Arizona., 2021)
      There are many behaviors used in the study of motor disorders. Simple ones like the rotarod test are easy to implement and give a single measure of behavior. Others, like the center-out task, skilled reaching task, vermicelli handling test, and string pulling task are more complex. These often require manual segmentation of movement to identify motor patterns. We have developed an integrated hardware and software system capable of automatically controlling and quantifying behavior in the string pulling motor task. This system also integrates with neural recording systems to allow for the syncing of behavioral states to neural responses. Our system also streamlines the training of this behavior by allowing the automated shaping of animals. Key elements of the hardware included 3D-printed parts, a high-speed camera, a rotary encoder, an Arduino microcontroller, and custom circuits. Open-source deep-learning software (DeepLabCut) was used to record paw and head movements and custom software used this information to further categorize the phases and segments of each pull. Using this system, we were able to identify 97 neurons that were significantly responsive to right paw motion, 83 neurons that were significantly responsive to left paw motion, and others that were responsive to other components of the string pulling task. This system has applications in the study of motor control, motivation, and motor disorders such as Parkinson's Disease, Huntington's Disease, and strokes.
    • Regulation of the Endogenous Blood-Brain Barrier Transporter Organic Anion Transporting Polypeptide 1A4 (Oatp1a4) by Testosterone in an Immortalized Mouse Brain Endothelial Cell Line (bEnd.3)

      Ronaldson, Patrick T.; Nava, Raul; Falk, Torsten; Lynch, Ronald M. (The University of Arizona., 2021)
      The biochemical and physical properties of the blood-brain barrier (BBB) are known to regulate drug delivery to the central nervous system (CNS), making it incredibly challenging to treat neurological diseases. A viable strategy may be to target organic anion transporting polypeptides (OATPs in humans; Oatps in rodents), transporters that facilitate blood-to-brain drug uptake. Over the past several years, our laboratory has studied the involvement of OATPs/Oatps in the BBB transport of drugs that are effective in treatment of neurological pathologies such as cerebral hypoxia/reoxygenation stress and ischemic stroke. Using male and female Sprague-Dawley rats, we have shown that Oatp1a4, the primary drug transporting Oatp isoform at the rodent BBB, is critical for brain delivery of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors (i.e., statins). More recently, we have shown that Oatp1a4 functional expression is higher at the BBB in female Sprague-Dawley rats as compared to their male counterparts. Interestingly, this work also showed that Oatp1a4 protein expression and transport activity in brain micro-vessels from castrated male rats was the same as in female control rats or in ovariectomized females. This observation pointed towards a role for male gonadal sex hormones in the regulation of Oatp1a4 at the BBB. Therefore, we sought to determine the effect of testosterone on Oatp1a4 protein expression using a mouse brain micro-vessel endothelial cell line (bEND.3). Specifically, we studied the effect of testosterone in normoxic cells and in cells subjected to oxygen/glucose deprivation (OGD), an in vitro condition relevant to ischemic stroke. In normoxic (i.e., control) bEND.3 cultures, testosterone increased Oatp1a4 protein expression in a dose-dependent manner. In contrast, testosterone reduced Oatp1a4 protein expression in bEND.3 cells subjected to 8 h OGD but had no effect in these cultured mouse brain endothelial cells after 8 h OGD/24 h reoxygenation (i.e., OGD/R). Interestingly, testosterone treatment increased expression of the androgen receptor under both OGD and OGD/R conditions. Overall, these data provide the first evidence for differential regulation of Oatp1a4 protein expression by testosterone under normoxic, OGD, and OGD/R conditions. Further studies are required to evaluate the implications of these findings on transport of Oatp1a4 substrates (i.e., statins) and to determine the molecular machinery involved in altered Oatp1a4 expression in endothelial cells following exposure to testosterone.