Dissecting the Composition of Striatal Dopamine Release Machinery

Abstract

There are two general modes of neurotransmission. In synaptic transmission, neurotransmitter-filled vesicles are rapidly released, and the signal is sensed by the target cell in a spatially and temporally precise manner. The precision for exocytosis is garnered by a highly organized protein machine known as the active zone. In contrast, neuromodulation often relies on a process known as volume transmission in which release is followed by widespread transmitter diffusion in the extracellular space and modulation of many target cells. While much is known about the proteins that regulate fast neurotransmission, we know much less about the mechanisms of neuromodulator release and reception. Dopamine is a typical neuromodulator that is thought to rely on volume transmission, however it also codes for processes that occur on rapid timescales. The protein machinery responsible for dopamine release is not well understood. My thesis work addresses this question using both a targeted and an unbiased approach to characterize dopamine release sites. In Chapter 2, I adapt a striatal synaptosome purification method to investigate the localization of several known release site proteins in dopamine axons. I find that dopamine neurons contain clusters of release site proteins typically found in fast synapses and that removal of these proteins causes changes in release site and axonal structure. In Chapter 3, I take an unbiased approach to generate a dopamine neuron-specific release site proteome in order to determine its overall composition. I show that dopamine release sites contain both proteins previously attributed to fast synapses as well as proteins without known synaptic function, introducing avenues for future investigation. Finally, in Chapter 4, I ask how the release site structure is affected when dopamine release is ablated by removal of release site scaffolding proteins or removal of the calcium sensors that trigger the release of dopamine-filled vesicles. I find that removal of RIM, a scaffolding protein, causes widespread disruption of dopamine release sites, while removal of the calcium sensor synaptotagmin-1 does not strongly disrupt their composition. My three-pronged approach provides a comprehensive characterization of the release machinery that mediates dopamine release. It establishes a broad proteomic description of the dopamine release sites in both control mice and mice in which dopamine release is ablated, suggests that these release sites are surprisingly sparse in the dopamine axonal arbor, and helps establishing that known release site proteins that are typically found in the secretory machines of fast synapses are also present and clustered in dopamine axons.