Induction of 5-HT1A receptor mRNA in differentiated SHSY-5Y neuroblastoma cells

1Lindsey J. Bunn, 2Gavin P. Reynolds, 1Adrian J. Hall and 1Caroline F. Dalton. 1Biomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam University, Sheffield, S1 1WB. 2Division of Psychiatry and Neuroscience, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL.

Serotonin (5-HT) is an important neurotransmitter involved in depression and other affective disorders (Lucki, 1998). The release of 5-HT in neurons is mediated by somatodendritic 5-HT1A autoreceptors (Ou, 2000). The presence of 5-HT1A receptor is thought to be increased in depressed patients, producing a reduction in the synthesis of 5-HT (Lemonde, 2003). The SHSY-5Y neuroblastoma cell line is a well characterised cell line model used in neurotransmitter studies. The aim of this study was to determine the presence of 5-HT1A receptor mRNA in this cell line at different time-points by real-time PCR, when differentiated with both nerve growth factor and aphidicolin or retinoic acid.

The SHSY-5Y cells were grown in DMEM containing 10% FCS and penicillin-streptomycin for different lengths of time (0, 3, 5, 7, 10 and 14 days) in the presence of either retinoic acid (RA, 10-5 M) or nerve growth factor (NGF, 100ng/ml) and aphidicolin (0.3μM) (LoPresti, 1992). After the indicated times cells were washed with phosphate buffered saline (PBS) and trypsinised, pelleted and resuspended in PBS. RNA was then extracted using SV total RNA isolation kit (Promega). cDNA was synthesised using Iscript cDNA synthesis kit (Biorad). For Real-time PCR relative expression ratios were normalised against housekeeping genes ubquitin C (UBC) and trytophan 5-monooxgenase activation protein zeta polypeptide (YHWAZ). Data are presented as means (±SEM). Values represent the relative fold change in 5-HT1A mRNA compared to undifferentiated cells (time point 0, control).

At day 5 following stimulation with RA a significant 17.2±6.4 fold increase (n=3, p<0.01) in 5-HT1A receptor mRNA was determined relative to the control, undifferentiated cells (n=3). However, after day 5 5-HT1A receptor mRNA levels declined and the change in expression observed was not significantly different relative to the control (7-days 1.5±0.2, 10-days 0.6±0.2, 14-days 4.1±1.5, n=3). Differentiation with NGF and aphidicolin significantly increased 5-HT1A mRNA levels at 10 days producing an 849.7±108 fold increase relative to control (n=3, p<0.01). However, by 14 days of stimulation with NGF and aphidicolin, 5-HT1A expression had significantly declined to a 1.6±0.1 fold (n=3) change in expression relative to control.

In conclusion, the results demonstrate that 5-HT1A receptor mRNA is expressed in the SHSY-5Y cell line when differentiated with either NGF and aphidicolin or RA. The differentiated SHSY-5Y cell line may provide a model that will be useful to investigate 5-HT1A receptor function.

Lemonde S et al., (2003) J Neurosci, 23 (25), 8788-99
LoPresti P et al., (1992) Cell Growth Differ, 627-35
Lucki I. , (1998) Soc Biol Psychiatr, 55, 225-33
Ou XM et al., (2000) J Biol Chem, 275, 8161-68