Goffin, Darren; (2009) Dopamine receptor signalling to GABAa receptors: Regulation of cell-surface expression levels and gabaergic synapse formation. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

GABAa receptors are GABA-gated, Cl' permeable ion channels that play an important role in regulating neuronal excitability and development. There is increasing evidence that dopamine receptors can regulate the activity and function of GABAa receptors; yet very little is known about this during development. We have investigated this regulation using the embryonic striatum, an area of the brain that receives considerable dopaminergic input from the substantia nigra. Since the function of GABAa receptors can be modulated through changes in their cell-surface expression we have investigated the ability of dopamine receptors to regulate GABAa receptor surface levels in cultured embryonic striatal neurones. Changes in the surface expression of GABAa P2/3 subunits were assessed using a cell-surface ELISA procedure that utilises an antibody directed against the extracellular epitopes of these subunits. Activation of D1- or D2-like dopamine receptors caused a prolonged, dose-dependent decrease in the surface expression of (32/3 subunits. These decreases were due to an increase in dynamin-dependent endocytosis, as assessed by visualising receptor internalisation via immunocytochemistry and the use of dynamin inhibitory peptides in cell-surface ELISA experiments. DiR modulation involved PKA and PP2A pathways: the effect of DiRs was occluded PKA activation; and attenuated by inhibitors of PP2A. Additionally, DiR activation increased the phosphorylation of the MAP kinases, p42 and p44; and inhibition of the MAP kinase kinase, MEK, significantly attenuated D1R- mediated decreases in cell-surface expression. The D2R-mediated regulation was prevented by the presence of a PPl inhibitor. To assess whether the prolonged attenuation in GABAa receptor levels at the cell-surface is associated with changes in the development of GABAergic synapses, embryonic striatal cultures were treated with D1R or D2R agonists and the number, as well as the size, of presynaptic vesicular inhibitory amino acid transporter-1 (VIAAT-l)-positive terminals and postsynaptic P2/3-positive clusters of GABAa receptors were determined using immunocytochemistry and confocal microscopy. We demonstrate that both DiR and D2R activation decreased the size of (32/3 clusters without affecting the total number at the cell surface, leading to a concomitant decrease in their colocalization with VIAAT-1, suggesting a decrease in the number of GABAergic synapses. Furthermore, using FM1-43FX, which is only incorporated into active synapses, we found that there was a significant reduction in the number of functional GABAergic synapses following these treatments. We have also investigated the regulation of GABAa receptors by dopamine receptors in cultures of neural progenitor (NP) cells from the postnatal mouse hippocampus. These NP cells are capable of differentiating into new neurones that incorporate into existing neural networks. We have demonstrated that these cells express functional GABAa receptors that upon activation leads to an increase of intracellular Ca2+ levels via opening of L-type Ca2+ channels, as measured by changes in fluo-4 fluorescence. Activation of these receptors also caused a significant decrease in proliferation, as assessed by a decrease in BrdU incorporation; an effect that requires the entry of Ca through L-type calcium channels. Furthermore, while activation of D1Rs had no effect on proliferation when added alone, their activation abrogated the effects of GABAa receptor activation on proliferation. The effects of D1Rs occur by decreasing the ability of GABAa receptors to increase intracellular calcium levels and, as revealed by cell surface ELISA experiments, by decreasing the surface expression of GABAa receptors. D2R activation on the other hand showed no effect on the proliferation of these NP cells in the absence or presence of GABAa receptor activation. In summary, these results lead us to propose a novel role for D1Rs and D2RS in fine-tuning of GABAergic synaptogenesis in the developing striatum potentially through regulation of GABAa receptor cell-surface levels. Additionally, we have also shown that DiR activation can increase the proliferation of NP cells by preventing GABAa receptor-mediated inhibition of proliferation.

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