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Title: 5-HT1A receptor expression : studies in postmortem tissue and characterisation of a model system
Author: Bunn, Lindsey J.
Awarding Body: Sheffield Hallam University
Current Institution: Sheffield Hallam University
Date of Award: 2008
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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.
Supervisor: Dalton, Caroline ; Hall, Adrian ; Reynolds, Gavin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available