Baines, Abigail (2011) Adenosine receptor trafficking : implications for epilepsy. PhD thesis, University of Warwick.

Abstract

Adenosine receptors (ARs) modulate many cellular and systems-level processes in the mammalian CNS. However, little is known about the trafficking of ARs in neurons, despite their importance in controlling seizure activity and in neuroprotection in cerebral ischemia. To address this I examined the agonist-dependent internalisation of C-terminal GFP-tagged A1R, A2AR and A3R in primary hippocampal neurons and compared findings to CHO cells. Furthermore, I developed a novel super-ecliptic pHluorin (SEP)-tagged A1R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A1R in real-time. I demonstrate the differential trafficking of ARs in neurons: the A3R internalised rapidly, with the A1R internalising more slowly, and with little evidence of appreciable A2AR trafficking over the time-course of the experiments. These findings were consistent with trafficking data in CHO cells and previous literature. Futhermore, the novel SEP-A1R construct revealed the time-course of internalisation and recovery of cell surface expression to occur within minutes of agonist exposure and removal, respectively. These observations reveal the labile nature of cell surface expression of neuronal adenosine A1R and A3Rs. Given the high levels of adenosine in the brain during seizures, internalisation of the inhibitory A1R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states. To test this I monitored trafficking of A1R in response to two seizure conditions, kainic acid and Mg2+free-/high K+. I observed following acute Mg2+free-/high K+ treatment an increase in dendritic puncta consistent with A1R internalisation. In addition to monitoring changes to GFP-tagged AR constructs I attempted to elucidate the effect of agonist exposure on native A1R. Electrophysical recordings revealed that localisation of ARs may impact on receptor regulation, since no evidence of internalisation was observed. This suggested that post-synaptic A1R may be more resistant to internalisation as previously noted in the literature. This study provides an initial study into the regulation of adenosine receptors in hippocampal neurons and has developed tools that will provide useful in further studies to elucidate the regulation of ARs during pathological conditions.

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