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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

TOWARDS AN UNDERSTANDING OF PHARMACOLOGICALLY INDUCED INTRACELLULAR CHANGES IN NICOTINIC ACETYLCHOLINE RECEPTORS: A FLUORESCENCE MICROSCOPY APPROACH

Loe, Ashley M. 01 January 2016 (has links)
Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.
12

THE ANTINOCICEPTIVE EFFECTS OF ALPHA 7 NICOTINIC ACETYLCHOLINE RECEPTOR POSITIVE ALLOSTERIC MODULATORS IN DIFFERENT ANIMAL PAIN MODELS

Freitas, Kelen 29 May 2012 (has links)
The α7 nicotinic acetylcholine receptor (nAChR) subtype is abundantly expressed in the central nervous system (CNS) and in the periphery. Positive allosteric modulators (PAMs) of the α7 increase the response to an agonist and are divided into two types depending on whether they also decrease desensitization of the receptor (type II) or not (type I). Therefore, this study aims to investigate whether the enhancement of endogenous α7 nAChR function will result in a beneficial effect in nociceptive, inflammatory and chronic neuropathic pain models. While NS1738 and PNU-120596 were not active to reduce acute thermal pain, measured by hot-plate and tail-flick tests, only PNU-120596 dose-dependently attenuated paw-licking behavior in the formalin test. Our results with selective (MEK) inhibitor U0126 argues for an important role of extracellular signal-regulated kinase (ERK1/2) pathways activation in PNU-120596’s antinociceptive effects in formalin-induced pain. The α7 antagonist MLA, via intrathecal and intraplantar administration, reversed PNU-120596’s effects, confirming PNU-120596’s action through central and peripheral α7 nAChRs. Tolerance to PNU-120596 was not developed after chronic treatment of the drug. Furthermore, mixtures of PNU-120596 and choline, an endogenous α7 nAChR agonist, synergistically reduced formalin-induced pain, while interactions of non-antinociceptive doses of PNU-120596 and PHA-543613, a selective α7 nAChR agonist, or nicotine resulted in antinociception. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, -hypomotility and –antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via α7 nAChRs. In the carrageenan inflammatory test both NS1738 and PNU-120596 significantly reduced thermal hyperalgesia, while only PNU-120596 significantly reduced edema. Importantly, PNU-120596 reversed established thermal hyperalgesia and edema induced by carrageenan. In the chronic neuropathic pain (CCI) model, PNU-120596 had long-lasting (up to 6 hrs), dose-dependent anti-hyperalgesic and anti-allodynic effects after a single injection, while NS1738 was inactive. Subcutaneous and intrathecal administration of MLA reversed PNU-120596’s effects, suggesting the involvement of α7 nAChRs. Finally, PNU-120596 enhanced an ineffective dose of selective agonist PHA-543613 to produce anti-allodynic effects in the CCI model. Our results show a fundamental in vivo difference between type I and II α7 nAChR PAMs, and demonstrate type II’s potential for the treatment of chronic inflammatory pain.
13

α7 Nicotinic acetylcholine receptor-mediated calcium signalling in neuronal cells

Brown, Jack January 2014 (has links)
α7 nicotinic acetylcholine receptors (nAChR) are highly permeable to Ca2+ and are clinical targets for Alzheimer’s disease and schizophrenia. The aim of this work was to examine α7 nAChR-mediated Ca2+ signalling in neuronal cells using three different methods, and to evaluate the effects of the desensitizing agonist and prototypical smoking-cessation drug sazetidine-A on α7 nAChRs. Initial studies used 96-well plate assays with SH-SY5Y cells to characterize responses evoked by the α7 nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596. This was complemented by live-imaging of cortical cultures, where the compounds evoked robust Ca2+ responses from 12 % of cells. Co- application with Cd2+, ryanodine and xestospongin-C significantly inhibited these responses, suggesting the involvement of voltage-gated Ca2+ channels and Ca2+- induced Ca2+-release. CNQX and MK801 also significantly inhibited α7 nAChR mediated Ca2+ elevations, indicating a role for glutamate release. A high-content screening assay was developed to further examine these phenomena. Exploratory experiments using KCl, AMPA and NMDA validated a protocol that could be used to image Ca2+ elevations in large cell populations. Inconsistent responses to PNU-120596 and PNU2-282987 were also observed, reflecting the scarcity of α7 nAChRs in cortical cultures and the need for assay optimization. Combination with immunofluorescent labelling revealed α7 nAChR mediated Ca2+ elevations in a subpopulation of astrocytes and neurons, some of which were GABAergic. PNU-120596 potentiated the effects of sazetidine-A in SH-SY5Y cells (EC50 0.4 μM) eliciting responses in 14 % of cells in cortical cultures in a methyllycaconitine- sensitive manner, consistent with α7 nAChR activation. Pre-incubation with sazetidine-A concentration-dependently attenuated subsequent α7 nAChR-mediated responses in SH-SY5Y cells (IC50 476 nM) and cortical cultures, suggesting that α7 nAChRs could play a role in the behavioural effects of sazetidine-A. These comparative experiments enhance our understanding of α7 nAChR signalling and provide a new method to study them further.
14

Mild traumatic brain injury augments innate immune responses through neurokinin and cholinergic signaling

Hsieh, Terry 03 November 2016 (has links)
Pneumonia is the second leading cause of disability-adjusted life-years lost worldwide and the eighth leading cause of death in the United States. Traumatic brain injury (TBI) patients have classically been considered immunosuppressed, but recent research reported that mild head trauma patients have reduced incidence of pneumonia compared to blunt trauma patients. Using our mild TBI model followed by bacterial pneumonia, we investigated the effect of neuronal signaling on innate immune function. To test whether any mild injury primes host immune responses to pneumonia, we generated a mild tail trauma (TT) model. mTBI mice showed protection from bacterial pneumonia while TT mice did not. Using an FDA-approved neurokinin-1 receptor (NK1R) antagonist, aprepitant, we confirmed our previous findings that substance P (SP) is a key mediator of enhanced resistance to pneumonia. Blocking NK1R showed that mTBI-induced release of SP augments pulmonary neutrophil recruitment and microbicidal activity to pulmonary bacterial pathogens. In TT mice, NK1R agonism enhanced the same neutrophil functions, further supporting the hypothesis. No differences were found between mTBI and TT neutrophils’ ability to phagocytose, generate oxidative burst, or acidify phagosomes. However, neutrophils from mTBI mice produced more neutrophil extracellular traps in response to bacterial challenge. These studies show that neurokinin signaling in our model contributes to enhanced bacterial clearance. Cholinergic anti-inflammatory pathway signaling though the α7 nicotinic acetylcholine receptor (α7 nAChR) is also a critical component of improved survival. Blockade of α7 nAChR abrogated the mTBI survival benefit. Mimicking cholinergic signaling using α7 nAChR agonist recapitulated the mTBI reduced pro-inflammatory cytokine production and improved survival. No physiologic differences emerged within 24h following pneumonia, but mTBI and α7 agonist treated mice had significantly lower TNFα in bronchoalveolar fluid, suggesting reduced injurious pulmonary inflammation. However, replacing early TNFα during pneumonia did not increase mortality. Western blot analysis showed downregulation of HMGB1 release in mTBI mice, suggesting that vagal cholinergic signaling reduces late mediators of organ damage. Our experiments show that mTBI enhances resistance to pneumonia by activating the vagus nerve signaling through neurokinin and cholinergic pathways. Translation of these findings could be innovative solutions to fighting or preventing infections.
15

Investigating the Role of Nicotinic Acetylcholine Receptor Agonists in Lung Cancer Progression and Chemosensitivity in the Context of Treating Chemotherapy-Induced Peripheral Neuropathy

Kyte, Sarah L 01 January 2018 (has links)
While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms in the hands and feet, such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our collaborators in the laboratory of Dr. M. Imad Damaj have shown that nicotine, a nicotinic acetylcholine receptor (nAChR) agonist, and R-47, an α7 nAChR silent agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy in mice. With regard to cancer, this work demonstrates that nicotine and R-47 do not enhance A549 and H460 human non-small cell lung cancer cell viability, colony formation, or proliferation alone, and they do not attenuate paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, nicotine and R-47 do not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that targeting nAChRs may be a safe and efficacious approach for the prevention and treatment of CIPN in cancer patients.
16

Functions of the Cholinergic System in the Morbidities Associated with Alzheimer’s Disease and the Further Evaluation of Tools for the Molecular Imaging of this System

Quinlivan, Mitchell Owen Jeffrey January 2007 (has links)
Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.
17

Conotoxin overview and bioinformatic database setup

Chen, Shing-Hwei 28 November 2004 (has links)
Predatory shallow-water tropical marine snails within the genus Conus are estimated to consist of up to 700 species. These carnivorous mollusks have devised efficient venom harpoon-like radular teeth that allow them to predominantly incapacitate polychaete annelids (vermivores), in some cases fish (piscivores), or other mollusks (molluscivores) as an envenomation survival strategy for feeding, defense, and competitor deterrence. The venom of each Conus species contains a distinctive assortment of over 50 diversified disulfide-rich conotoxins with varied pharmacological specificities that selectively inhibit the function of ion channels (Ca2+, Na+, K+) or nicotinic acetylcholine receptors (nAChRs) involved in the animal neurotransmission. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing an exquisite selectivity to distinguish between channels / receptors and even particular their subtypes. Novel conotoxins detected in the molecular neurobiological approach, providing chemists and pharmacologists a vast library (>50,000 individual toxins) of conotoxins have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. In this article, we present the natural history of the Conus biology as well as the nomenclature, classification, structure, neurotoxicological mechanisms, post-translational modification, and pharmaceutical applications of conotoxins. In addition, we also set up the bioinformatic database and search engine about hitherto-identified name and distribution of Conus species and neuropharmacological mechanism, accession number, sequence, and 3D structure of conotoxins and provide researchers advantageous tools for further investigation.
18

Recurrent inhibitory network among cholinergic inerneurons of the striatum

Sullivan, Matthew Alexander 08 November 2012 (has links)
The striatum is the initial input nuclei of the basal ganglia, and it serves as an integral processing center for action selection and sensorimotor learning. Glutamatergic projections from the cortex and thalamus converge with dense dopaminergic axons from the midbrain to provide the primary inputs to the striatum. Striatal output is then relayed to downstream basal ganglia nuclei by GABAergic medium – sized spiny neurons, which comprise at least 95% of the population of neurons in the striatum. The remaining population of local circuit neurons is dedicated to regulating the activity of spiny projection neurons, and although spiny neurons form a weak lateral inhibitory network among themselves via local axon collaterals, feedforward modulation exerts more powerful control over spiny neuron excitability. Of the striatal interneurons, only one class is not GABAergic. These neurons are cholinergic and correspond to the tonically active neurons (TANs) recorded in vivo, which respond to specific environmental stimuli with a transient depression, or pause, of tonic firing. Striatal cholinergic interneurons account for less than 2 % of the striatal neuronal population, yet their axons form an extensive and complex network that permeates the entire striatum and significantly shapes striatal output by acting at numerous targets via varied receptor types. Indeed, the persistent level of ambient striatal acetylcholine as well as changes to that basal acetylcholine level underlie the major mechanisms of cholinergic signaling in the striatum, however regulation of this system by the local striatal microcircuitry is not well understood. This dissertation finds that activation of intrastriatal cholinergic fibers elicits polysynaptic GABAA inhibitory postsynaptic currents (IPSCs) in cholinergic interneurons recorded in brain slices. Excitation of striatal GABAergic neurons via nicotinic acetylcholine receptors (nAChRs) mediates this polysynaptic inhibition in a manner independent of dopamine. Moreover, activation of a single cholinergic interneuron is capable of eliciting polysynaptic GABAA IPSCs onto itself and nearby cholinergic interneurons. These findings provide an important insight into the striatal microcircuitry controlling cholinergic neuron excitability. / text
19

ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR REGULATION IN EXPERIMENTAL NEURODEGENERATIVE DISEASE

Charriez, Christina Margaret 01 January 2010 (has links)
The α7 nicotinic acetylcholine receptor (nAChR) is involved in learning and memory, synaptic plasticity, neuroprotection, inflammation, and presynaptic regulation of neurotransmitter release. Alzheimer’s disease (AD), a neurodegenerative disease characterized by diminished cognitive abilities, memory loss, and neuropsychiatric disturbances, is associated with a loss of nAChRs. Similarly, traumatic brain injury (TBI) may result in long term neurobehavioral changes exemplified by cognitive dysfunction. Deficits in α7 nAChR expression have previously been shown in experimental TBI and may be related to cognitive impairment experienced in patients following TBI. The purpose of this dissertation was to investigate changes in α7 nAChR expression in models of neurodegeneration and determine if allosteric modulation of the nAChR facilitates functional recovery following experimental TBI through changes in nAChRs. Experimental models employed include a transgenic mouse model of AD that overexpresses the amyloid precursor protein (APPswe mice) and the controlled cortical impact injury model of TBI in rats. Quantitative receptor autoradiography using α-[125I]-bungarotoxin and [125I]-epibatidine and in situ hybridization were used to investigate changes in nAChR density and mRNA expression, respectively. In the first study, the effects of aging and β-amyloid on α7 nAChR expression were evaluated in APPswe mice. Hippocampal α7 nAChR density was significantly upregulated in APPswe mice compared to wild-type mice. It is postulated that elevated Aβ levels bind to the α7 nAChR resulting in upregulation. In a second study, galantamine, a medication used in the treatment of AD, was administered subchronically following experimental TBI to determine if treatment could facilitate cognitive recovery and affect nAChR expression. Interestingly, the results indicate TBI interferes with agonist mediated upregulation of nAChRs, and galantamine did not improve function in a behavioral task of learning a memory. In a third study, the regulation of TBI related deficits in α7 nAChRs was examined 48 hours following injury. α7 nAChR deficits occurred with a reduction in α7 mRNA in several hippocampal regions and non-α7 nAChR deficits occurred with a reduction in α4 mRNA in the metathalamus. The results of these studies suggest AD and TBI may involve complex but parallel processes contributing to the regulation of α7 nAChRs.
20

Functions of the Cholinergic System in the Morbidities Associated with Alzheimer’s Disease and the Further Evaluation of Tools for the Molecular Imaging of this System

Quinlivan, Mitchell Owen Jeffrey January 2007 (has links)
Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

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