Medications that treat diseases such as Parkinson???s disease work by regulating dopamine transmission at synapses. Surprisingly, little is known about the mechanisms regulating dopamine release at synapses. In this thesis, we study mechanisms that regulate vesicle recycling in axons and dendrites of dopamine neurons. Key questions we addressed were: (1) Are vesicles in axons and dendrites associated with the same regulatory proteins, and thus by implication the same regulatory mechanisms, as in excitatory neurons; (2) Do vesicles undergo recycling, and (3) if so, are they characterised by a distinct pool size and rate of recycling. To study this, we cultured dopamine neurons and used immunocytochemistry to detect vesicular monoamine transporter 2 (VMAT2) and identify axons, dendrites and synaptic proteins, combined with labelling of recycling vesicles using FM 1-43. Vesicles in axons, but not in dendrites, were associated with presynaptic proteins such as Synaptophysin and Bassoon. We identified two kinds of presynaptic sites in axons: ???synaptic??? (located close to soma and dendrites??? and ???orphan???. The recycling vesicle pool size was smaller at orphan sites than at synaptic sites, and the initial rate of vesicle pool release was also lower at orphan sites. Both synaptic and orphan sites exhibited lower rates of vesicle pool release compared to hippocampal synapses, suggesting functional differences in presynaptic physiology between dopamine neurons and hippocampal neurons. In somatodendritic regions, VMAT2 was localised to the endoplasmic reticulum, Golgi, endosome, and large dense-core vesicles, suggesting that these vesicles might function as a part of the regulated secretory pathway in mediating dopamine release. None of the synaptic vesicle proteins we studied were detected in these regions, although some preliminary evidence of vesicle turnover was detected using FM 1-43 labelling. This thesis provides a detailed analysis of neurotransmitter release mechanisms in dopamine neurons. Our data suggests that presynaptic release of dopamine is mediated by mechanisms similar to those observed in excitatory neurons. In somatodendritic regions, our data suggests that VMAT2 is localised to organelles in secretory pathways, and that distinct mechanisms of release might be present at somatodendritic sites to those present in presynaptic sites. This thesis provides novel methods for analysing vesicle recycling in dopamine neurons, which provides the basis for further studies examining presynaptic function of dopamine neurons in normal brain function, disease, and therapeutic approaches.
| Identifer | oai:union.ndltd.org:ADTP/257588 |
| Date | January 2007 |
| Creators | Daniel, James, St. Vincent Clinical School, UNSW |
| Publisher | Awarded by:University of New South Wales. St. Vincent Clinical School |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright James Daniel, http://unsworks.unsw.edu.au/copyright |
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