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Influence of cellular environments on PET ligand binding : an application to imaging endogenous neurotransmitter release

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Title: Influence of cellular environments on PET ligand binding : an application to imaging endogenous neurotransmitter release
Authors: Quelch, Darren
Item Type: Thesis or dissertation
Abstract: The ability to image endogenous neurotransmitter release will vastly increase our understanding of a variety of neuropsychiatric disorders. Current published work in this field is mainly limited to measuring dopamine release in vivo. Recently, there have been increasing efforts to enable imaging the release of other neurotransmitters such as serotonin, opioid peptides and GABA. The changes in binding observed during endogenous release studies are generally assumed to take place at the cell surface by competition of radioligand with a neurotransmitter molecule. This has been termed The Occupancy Model. However, an Internalisation Hypothesis has been proposed given that many positron emission tomography (PET) ligand target proteins are susceptible to agonist induced endocytosis. In this thesis, studies are presented which investigate further the Internalisation Hypothesis and the influence these processes may have on imaging release of other neurotransmitters, such as opioid peptides. In vitro radioligand binding studies are presented investigating the effect of different buffers, representative of conditions experienced by a receptor following endocytosis, on the binding of three D2/3 receptor PET ligands. A decreased ability to bind, driven by a reduction in affinity, was observed in sub-cellular environments compared with those found at the cell surface. The contribution of each cellular compartment to overall striatal binding was also determined. The majority (~65%) of the total striatal signal was shown to result from membrane bound D2/3 receptors, a small (~30%) contribution was derived from the microsomal D2/3 receptors, and the cytosolic fraction contributes negligibly to the overall signal. The potential of imaging endogenous opioid peptide release using an experimental pharmacological challenge was investigated. The effects of cellular environments on two radioligands known to label opioid receptors in vivo were characterised and the affinity of a range of endogenous opioid peptides at [11C]carfentanil and [3H]diprenorphine labelled sites determined. [11C]Carfentanil and [3H]diprenorphines sensitivity to endogenous opioid peptide release by acute amphetamine challenge was explored following radioligand injection in rodents, microdissection and uptake assessment, ex vivo autoradiography and sub-cellular fractionation combined with in vitro radioligand binding. The effects of acute amphetamine challenge on opioid receptor trafficking in the striatum and hypothalamus was investigated using dual labelling fluorescence confocal microscopy. These studies demonstrate that [11C]carfentanil maybe more sensitive than [3H]diprenorphine to endogenous opioid peptide release following acute amphetamine challenge. The reduction in [11C]carfentanil binding observed in the hypothalamus was accompanied by increased association of µ receptors with EEA-1, an early endosomal marker. Collectively, these data suggest an agonist-induced internalisation process may contribute to the observed signal change with these PET ligands in vivo. Lastly, the effects of different cellular environments on the binding parameters of three distinct protein targets was investigated; namely the GABAA ionotropic receptor, the serotonin transporter and the D1 G-protein coupled receptor. These data demonstrate that the effects of different cellular environments are both protein target and radioligand specific and that the effects of agonist induced internalisation on radioligand binding is not specific to D2/3 G-protein coupled receptors, but multiple PET radioligand protein targets.
Content Version: Open Access
Issue Date: Sep-2013
Date Awarded: Dec-2013
URI: http://hdl.handle.net/10044/1/18308
DOI: https://doi.org/10.25560/18308
Supervisor: Tyacke, Robin
Nutt, David
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (Great Britain)
Department: Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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