Browsing by Subject "Dopamine"

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    The Anatomical Distribution Patterns, Physiological Effects, and Quantification of Biogenic Amines in the Central Nervous Systems of Araneae and Scorpiones (Arthropoda: Chelicerata)
    (2019-08) Auletta, Anthony
    The arthropod subphylum Chelicerata is one of the most diverse groups of organisms on the planet, and yet relatively little is known about the structural and functional organization of chelicerate central nervous systems (CNSs). To address this knowledge gap, I conducted a comparative study of biogenic amines in the CNSs of three representative chelicerates: the wolf spider Hogna lenta (Araneae: Lycosidae), the jumping spider Phidippus regius (Araneae: Salticidae), and the bark scorpion Centruroides sculpturatus (Scorpiones: Buthidae). In H. lenta and P. regius, I mapped the anatomical distribution of catecholaminergic neurons (i.e., those that produce dopamine [DA] or norepineprhine [NE]) in the CNS, using an antiserum against tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. TH immunoreactivity was detected throughout the spider CNS, including in the visual system, the arcuate body (a site of sensorimotor integration), and the neuromeres of the appendages and opisthosoma, thus suggesting that catecholamines play vital roles in many different behaviors and other physiological processes in spiders. Using similar immunocytochemical methods, I also described the distribution of catecholaminergic neurons in the ventral nerve cord (VNC) of C. sculpturatus, as well as neurons that contain octopamine (OA) and serotonin (5-hydroxytryptamine, 5-HT). Of particular note in the scorpion were clusters of large efferent TH-ir neurons, which exited the CNS to directly innervate the tissues of the book lungs, implying a role for catecholaminergic modulation of respiratory functions. These studies include the first description of catecholamines in any chelicerate taxon, and provide a much-needed foundation upon which future functional studies of biogenic amines in chelicerates can be based. Additionally, I utilized a combination of immunocytochemistry, quantitative chemistry, electrophysiology, and bioinformatics techniques to examine the possibility that NE is an endogenous signaling molecule in chelicerates, despite the widespread notion that invertebrates lack NE. Using ultra-performance liquid chromatography and mass spectrometry, I detected non-trace amounts of NE in the CNSs of both C. sculpturatus and H. lenta. Endogenous NE was localized to cells of the supraneural lymphoid glands in the scorpion, which implies a previously unrecognized secretory role for these structures. NE was also shown to elicit robust patterned electrophysiological activity in the terminal nerves of the scorpion, which was distinct from the patterns produced by other amines. Finally, I identified genes for distinct NE, OA, and DA receptors in the C. sculpturatus genome. Taken together, my results support the idea that NE is an endogenous and physiologically active modulator in scorpions, and possibly in the Chelicerata more broadly, thus challenging the idea that adgrenergic signaling is exclusive to the vertebrates. The implications of these findings are discussed in relation to the evolution of aminergic systems within the Arthropoda and the Bilateria as a whole.
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    Astrocyte-neuron signaling in the nucleus accumbens: implications for brain reward signaling
    (2019-05) Corkrum, Michelle
    Dopamine is one of the major reward signaling molecules in the brain. Dopaminergic transmission contributes to physiological states such as learning, memory encoding, movement and motivated behaviors; and, the disruption of dopamine signaling can contribute to neuropsychiatric diseases such as substance use disorders. The majority of research on reward signaling has focused on neurons; however, astrocytes are emerging as key components of brain information processing. Astrocytes are a subset of glial cell, one of the most abundant cell types in the brain. Although astrocytes are not electrically excitable, in response to brain activity, they demonstrate increases in intracellular calcium and the subsequent release of neuroactive substances, termed gliotransmitters. Therefore, my dissertation aimed to investigate the hypothesis that astrocytes respond to brain reward signaling with elevations in cytoplasmic calcium, and subsequently modulate neuronal activity in the nucleus accumbens, one of the major reward centers of the brain. Utilizing fiber photometry, I found that astrocytes in the nucleus accumbens respond to dopamine and amphetamine with cytoplasmic calcium elevations in vivo. To elucidate the cellular mechanisms of this phenomenon and the consequences of astrocyte calcium signals on neuronal activity, we conducted experiments applying multiphoton calcium imaging and whole-cell patch clamp electrophysiology in acute brain slices containing the nucleus accumbens core. We found that astrocytes respond to dopamine, amphetamine and opioids with intracellular calcium elevations and subsequently modulate neuronal activity, either through adenosine signaling in the case of dopamine and amphetamine or glutamatergic signaling in the case of opioid exposure. Furthermore, we demonstrate that astrocytes contribute to the acute psychomotor behavioral effects of amphetamine, illustrating astrocyte modulation of drug-related behaviors. Overall, the current body of work provides evidence that astrocytes actively contribute to brain reward processing via responding to dopamine and drugs of abuse with intracellular calcium increases and modulating neuronal and synaptic activity in the nucleus accumbens, one of the major nodes of the reward system.
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    DEVELOPING CHEMICAL TOOLS TO MAP MOLECULAR MECHANISMS THAT DRIVE DISEASE
    (2023-04) Hurben, Alexander
    Human health is impacted by molecular level events. Within this context, DNA encodes for cellular instructions and proteins execute these genetic protocols to ensure the cell’s components are functioning properly. Thus, the underlying biochemistry within the cell is tightly regulated to ensure vitality. However, when these processes become compromised or damaged, there is increased susceptibility towards developing cancer and neurodegenerative diseases. Often, small chemical changes to our DNA and proteins can be the culprits of such dysregulation. These seemingly minuscule modifications can impair the cell’s proper functions, which can lead to cell death or uncontrolled growth, and ultimately manifest as degenerative disease or cancer. Electrophilic small molecules can be responsible for chemically altering cellular machinery. Additionally, enzymes can make chemical changes such as post-translational modifications on proteins and epigenetic DNA modifications, which can have pronounced effects on cell function and can be equally damaging if not properly regulated. Understanding how molecular events influence health is of paramount importance in designing therapeutics that effectively prevent and treat disease, as well as discovering biomarkers which enable early detection. Despite immense research efforts from the scientific community, there is much remaining to learn about these microscopic processes. This is due to their inherent complexity and lack of technologies to study them. This thesis aims to contribute to addressing this problem by developing new chemical tools which advance our knowledge of the molecular mechanisms that drive disease. This work is composed of seven chapters which explore reactive dopamine metabolites linked to Parkinson’s disease, tools to study the biological implications of elevated intracellular methylglyoxal concentrations, and the development of small molecule epigenetic modulators to regulate aberrant DNA methylation. Chapters I, II, and III of this thesis explores how dysregulated dopamine may contribute to Parkinson’s disease initiation. Chapter I commences with a review of dopamine metabolism and the subsequent generation of reactive dopamine derived metabolites in neurons. This is followed by an overview of protein damaged induced by these metabolites and a review of chemical tools and techniques implemented to study dysregulated dopamine in various experimental systems. Next, Chapter II describes our efforts in designing and implementing a dopamine derived chemical probe to profile dopamine modified proteins which found that dopamine metabolites disrupt protein-folding pathways critical for maintaining healthy neurons. We also detail our development of photoactivatable dopamine probes in Chapter III. Collectively, this work improves the understanding of dopamine protein modification and by extension, molecular events that may contribute to Parkinson’s, which may inform future Parkinson’s therapeutic development. Chapter IV and Chapter V of this thesis focuses on the reactive metabolite methylglyoxal. Methylglyoxal is a sugar-derived metabolite produced naturally in all cells. This reactive compound forms adducts with DNA and proteins, thereby altering their function and influencing cell signalling. Consequently, methylglyoxal protein adducts are implicated in numerous diseases such as cancer, neurodegeneration, diabetes, and cardiovascular disease. In many of these diseases, the cellular processes that break down methylglyoxal become compromised, leading to elevated levels of this reactive molecule within cells. Existing chemical tools to investigate methylglyoxal biology are limited, leading to an incomplete understanding of its physiological and disease-causing roles. Here, we disclose a chemical tool that confers light-mediated release of a methylglyoxal probe within cell models. We use this chemical to identity of the resulting protein adducts. This work enables studying protein adducts induced by methylglyoxal in a controlled fashion to illuminate how this reactive compound impacts various disease states. We also detail our efforts in profiling proteins which undergo covalent DNA crosslinking in the presence of methylglyoxal in Chapter V. This effort is the first study to identify this type of methylglyoxal adduct at a proteome wide scale, which provides a list of candidate methylglyoxal derived DNA-protein cross to investigate in future work. Collectively, these efforts further our understanding of basic methylglyoxal biology and its role in disease progression. Finally, Chapters VI and VII of this thesis describe our efforts towards developing small molecule epigenetic modulators. Regulating gene expression is critical for keeping cells healthy. Over- or under-expressed genes can lead to cancer and other diseases. Accordingly, cells have many methods to control when specific genes are turned on or off in order to function properly; DNA methylation being an example. There are many proteins which control the addition and removal of DNA methyl marks across the genome to ensure appropriate gene expression. Mutations in, or dysfunction of, these proteins can initiate certain cancers. One essential group of proteins involved in removing DNA methylation marks is ten-eleven translocation (TET) methylcytosine dioxygenases (TET). Given TET’s central role in cancer development, theses protein represent a potential drug target. However, there is a paucity of small molecules which selectively inhibit their function without affecting other cellular processes. Thus, there is a need to develop potent and selective compounds which block TET-meditated DNA demethylation. Within Chapter VI, we show our efforts towards the development of novel small molecule TET inhibitors which led us to uncover that copper contamination is responsible for the activity of a reported TET inhibitor. In Chapter VII, we present work on a novel TET inhibitor scaffold which features a bifunctional cofactor-substrate mimetic design. This work has the potential to generate new anticancer therapeutics and improve our understanding of how TET and DNA methylation is linked to cancer development. Ultimately, the work in this thesis provides a novel suite of chemical tools for studying dopamine dysregulation, methylglyoxal metabolism, and TET function. These tools provide insights into cellular damage caused by dopamine and methylglyoxal adducts as well as probes for altering DNA methylation status. Such tools are critical for mapping molecular mechanisms that drive disease.
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    Dynamic regulation of R7BP (R7 Binding Protein) containing R7 RGS (R7 Regulators of G protein Signaling) protein complexes: role in controlling neuronal dopamine and opioid signaling in the striatum.
    (2010-02) Anderson, Garret R.
    G protein-coupled receptor (GPCR) signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior and nociception in mammals. One member of the R7 RGS family, RGS9-2 has been previously implicated as an essential modulator of signaling through neuronal dopamine and opioid G protein coupled receptors. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with R7BP (R7 RGS Binding Protein), which we have found to ultimately affect several critical properties of RGS9-2. First, it is this interaction with R7BP which is necessary for determining the subcellular targeting of RGS9-2 to the plasma membrane and to the specialized neuronal compartment of excitatory synapses, the postsynaptic density. Secondly, R7BP plays a selective role amongst the R7 RGS family in determining the proteolytic stability of RGS9-2. Further characterization of R7 RGS complexes in the striatum revealed that two equally abundant R7 RGS proteins, RGS9-2 and RGS7, are unequally coupled to the R7BP subunit which is present in complex predominantly with RGS9-2 rather than with RGS7. However, upon changes in neuronal activity the subunit composition of these complexes in the striatum undergoes rapid and extensive remodeling. Changes in the neuronal excitability or oxygenation status result in extracellular calcium entry, uncoupling RGS9-2 from R7BP, triggering its selective degradation. Concurrently, released R7BP binds to cytoplasmic RGS7 and recruits it to the plasma membrane and the postsynaptic density. These observations introduce activity dependent remodeling of R7 RGS complexes as a new molecular plasticity mechanism in striatal neurons and suggest a general model for achieving rapid posttranslational subunit rearrangement in multi-subunit complexes. The physiological consequence of this remodeling process appears to play a role in determining the signaling sensitivity to dopamine stimulation. Considering that upon the genetic elimination of RGS9, all available R7BP is funneled towards complex formation with RGS7, not only are RGS9 controlled GPCR signaling pathways affected, but those controlled by RGS7 as well. RGS9 knockout mice have an increased sensitivity to dopamine and opioid receptor stimulation and consequently display altered motor and reward behavior. The question arises as to the role of modulation of RGS7 function in controlling these behaviors. Since the function of RGS9-2 is controlled by its association with R7BP, we would predict that the elimination of R7BP would lead to similar alterations in striatal physiology for RGS9 controlled pathways. While at the same time, RGS7 would be largely unaffected by the elimination of R7BP, thus RGS7 controlled pathways would predictably remain unaltered. Using this rationale, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration consistent with the essential role of RGS9 in controlling these behaviors and the critical role played by R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not exhibit higher sensitivity to locomotor-stimulating effects of cocaine, suggesting a role for RGS7 in controlling dopamine sensitivity. Using a striatum-specific knockdown approach, we demonstrate that the sensitivity of motor stimulation to cocaine is indeed dependent on RGS7 function. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.
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    High-throughput automated detection and analyses of locomotor and hunting sequences in larval zebrafish unveil the role of a conserved dopaminergic diencephalospinal tract in locomotor development and goal-directed behavior
    (2015-03) Lambert, Aaron Mattthew
    The dopaminergic diencephalospinal tract (DDT), and its source orthopedia- specified dopaminergic (DAergic) population, is the most conserved part of the vertebrate DAergic system. The source somata of the DDT have widespread ascending and descending projections that span and have potential to integrate the entire rostro-caudal axis of the central nervous system, from telencephalon to spinal cord. Mammalian studies confirm that the extensive DDT network is multifunctional, even via its direct influence within the spinal cord. While specific mechanosensory and nociceptive functions of the DDT acting in the spinal cord in vivo have been elucidated in adult mammals, whether or not the DDT also exerts locomotor influences in the spinal cord in vivo, as well as whether the DDT plays an early role during development, has remained unknown despite suggestive in vitro studies. My thesis explored the role of the vertebrate DDT in locomotor development and goal-directed behavior in zebrafish larvae, a premiere model to elucidate the neural bases of such behaviors at organismal, systems, circuit, cellular, and subcellular levels in vivo. To this aim, I developed new methodologies for high-throughput, unbiased, automated detection and analyses of locomotion and goal-directed hunting. This approach was combined with peripheral nerve recordings of the neural locomotor output of the spinal cord and employed during: DAergic pharmacological perturbations, demarcated transections at varying locations of the nervous system, selective chemogenetic ablation of orthopedia neurons, and laser ablations of the DDT. Collectively, this thesis reveals that the DDT acts specifically through endogenous dopamine receptor 4 (D4R) signaling to mediate locomotor development and provide a multifunctional modulation of multiple locomotor parameters in a separable manner, by putatively influencing disparate neuronal targets concurrently. Moreover, this thesis elucidates that endogenous D4R signaling is crucial to goal-directed prey capture via a specific motor-centric role in shifting prey- directed motor strategies and providing precision of speed control during execution of advancing hunting maneuvers. From the collective elucidations of my thesis, I posit the existence of a modular organization subserving a versatile locomotor network, wherein separate neural modules are recruited spontaneously and during hunting and that the hunting module is further subdivided into separable orienting and advancing regimes. Integrated into this granular network, endogenous D4R signaling, perhaps through a widespread integrative impetus at disparate regions via the extensive DDT network, differentially influences multiple modules concurrently. These findings, integrated with the mammalian literature, suggest that the conserved vertebrate DDT is crucial for locomotor development, as well as motor planning and execution of goal-directed behavior.
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    Neural mechanisms of anxiety during opiate withdrawal:role of the ventral tegmental area and extended amygdala.
    (2011-07) Radke, Anna Kay
    Exposure to addictive drugs alters neural circuits involved in reward and motivation, executive control, habit formation, learning and memory, and negative affect, and all except the last are known to depend on changes in the mesolimbic dopamine system. Negative affective symptoms of withdrawal are common to all drugs of abuse and negatively reinforce drug taking behavior. Using potentiation of the acoustic startle reflex as a measure of anxiety during withdrawal from acute morphine exposure, the experiments detailed in this thesis tested the hypothesis that µ-opioid receptor-mediated activation of VTA dopaminergic neurons is responsible for triggering negative emotional symptoms of withdrawal via recruitment of the extended amygdala. These experiments demonstrate the emergence of a negative affective state that occurs during withdrawal from direct infusion of morphine into the ventral tegmental area (VTA), the origin of the mesolimbic dopamine system. Potentiation of startle during withdrawal from systemic morphine exposure requires a decrease in ì-opioid receptor stimulation in the VTA and can be relieved by systemic or intra-nucleus accumbens administration of a dopamine receptor agonist. Investigation of mechanisms downstream of dopaminergic signaling found a role for type 2 corticotropin-releasing factor receptors following the very first, but not subsequent, opiate exposures. Together these results suggest that transient activation of the VTA mesolimbic dopamine system triggers the expression of anxiety during opiate withdrawal, possibly via direct recruitment of the extended amygdala. This conclusion provides unique insight into the neural mechanisms responsible for negative reinforcement of drug taking during the earliest stages of dependence.