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BUNN, Lindsey J. (2008). 5-HT1A receptor expression : Studies in postmortem tissue and characterisation of a model system. Doctoral, Sheffield Hallam University (United Kingdom)..
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Abstract
Serotonin (5-HT) neurotransmission is involved in the psychopharmacology of several psychiatric disorders including, depression, anxiety disorders and schizophrenia. The release of 5-HT in neurons is mediated by somatodendritic 5-HT[1A] autoreceptors. The presence of 5-HT[1A] receptor is thought to be increased in depressed patients, producing a reduction in the synthesis of 5-HT. A common single nucleotide polymorphism in the promoter region of 5HT[1A] receptor C-1019G is also associated with depression and suicide. The nuclear DEAF-1 related (NUDR) protein represses the 5-HT[1A] promoter region hence regulating both 5-HT[1A] transcription and receptor expression. The project involved undertaking a post-mortem study to determine any association between the 5-HT[1A] promoter polymorphism and the expression of 5-HT[1A] receptor mRNA and receptor density in control human hippocampal brain tissue. This was achieved by genotyping human brain tissue for the 5-HT[1A] receptor polymorphism C-1019G and 5-HT[1A] receptor mRNA levels were quantified using real-time PCR. Radioligand binding was used to determine B[max] and Kd quantifying 5-HT[1A] receptor density. The SHSY-5Y neuroblastoma cell line is a well characterised cell line model used in neurotransmitter studies when differentiated. The 5-HT[1A] receptor couples to Gi proteins inhibiting AC activity and hence mediating a variety of intracellular changes such as, decreasing cAMP leading to decreased Ca[2+] levels. The SH-SY5Y cell line study investigated whether the 5-HT[1A] agonist 8-OH-DPAT, inhibited forskolin stimulated Ca[2+] release in the SHSY-5Y cell line and whether the 5-HT[1A] antagonist p-MPPI reversed this effect using flow cytometry. The post-mortem study showed that the G-1019 allele had significantly higher 5-HT[1A] expression compared to the C allele in control hippocampal tissue. Radioligand binding data demonstrated that control samples with a GG or G/C genotype had a significantly higher 5-HT[1A] receptor density compared to samples with a CC genotype. SH-SY5Y cells differentiated with RA for 5 days or NGF and aphidicolin for 10 days had significantly increased 5-HT 1A receptor mRNA levels compared to undifferentiated cells. Western blots and immunocytochemistry confirmed the presence of the 5-HT[1A] receptor in this cell line. An increase in NUDR expression was observed at the same time there is an increase in 5-HT[1A] receptor expression in SH-SY5Y cells treated with RA or NGF and aphidicolin. Flow cytometry showed that 8-OH-DPAT efficiently diminished forskolin-stimulated increase in intracellular Ca[2+] in RA differentiated cells. 5-HT also a 5-HT[1A] agonist had a similar effect. SH-SY5Y cells treated with both p-MPPI and 8-OH-DPAT demonstrated that cells treated with p-MPPI at higher concentrations significantly increased forskolin-stimulated intracellular Ca[2+] levels and therefore effectively reversed the agonistic effect of 8-OH-DPAT. The findings presented in the post-mortem study are novel and the SH-SY5Y cell line study demonstrates that this cell line when differentiated with either RA or NGF and aphidicolin is a useful cell-line model system for studying the 5-HT[1A] receptor.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Contributors: | Thesis advisor - Dalton, Caroline [0000-0002-1404-873X] Thesis advisor - Hall, Adrian Thesis advisor - Reynolds, Gavin [0000-0001-9026-7726] |
| Additional Information: | Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 2008. |
| Research Institute, Centre or Group - Does NOT include content added after October 2018: | Sheffield Hallam Doctoral Theses |
| Depositing User: | EPrints Services |
| Date Deposited: | 10 Apr 2018 17:18 |
| Last Modified: | 03 May 2023 02:07 |
| URI: | https://shura.shu.ac.uk/id/eprint/19409 |
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