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.
|Date of Award||1 Apr 2014|
|Supervisor||Sue Wonnacott (Supervisor)|
- nicotinic acetylcholine receptor (nachr)