Signaling in the nervous system relies on the transfer of chemical signals from one neuron to the next. There are two main classes of these signals: fast neurotransmitters and neuromodulators. Fast neurotransmitters are released from specialized release sites and bind directly to receptors on the opposing post-synaptic membrane to exert an instant change in the membrane potential of the post-synaptic cell. In contrast, neuromodulators are released slowly and diffuse through the extracellular space. They bind to receptors on several cells at once to exert long-lasting changes in a population of neurons. The mechanisms in the secretory pathway for neuromodulators are not well understood. Extensive research from many laboratories on the release of fast neurotransmitters has found that a complex of proteins at the presynaptic membrane, known as the active zone, morphologically docks and functionally primes synaptic vesicles for rapid and precise release. Here I focus on release mechanisms of dopamine, a neuromodulator critical for movement, reward, and emotion. We have recently found that an active zone-like complex of proteins is required for unexpectedly rapid dopamine release. The requirement of a release site strongly suggests that dopamine vesicles are positioned close to their future sites of release and are rendered release ready, reminiscent of the docking and priming of synaptic vesicles. I hypothesize that the specialized, active zone-like release site in dopamine neurons both docks and primes dopamine vesicles to allow for fast exocytosis upon action potential triggering. I will dissect these two processes on a functional and structural level.
In aim 1, I will characterize the role of the priming protein, Munc13 in dopamine release. My preliminary data suggests that Munc13 is essential for dopamine release. I will use carbon fiber amperometry, super resolution and confocal microscopy, and mouse genetics to systematically characterize the localization and function of Munc13 in dopamine neurons.
In aim 2, I will characterize vesicle docking in dopamine axons and in mutant mice that lack potential docking proteins. To unambiguously identify dopamine terminals, I will employ conditional tagging of vesicles with horseradish peroxidase (HRP) for cell-type identification in electron microscopic images. I will then use serial EM to 3D-reconstruct striatal dopamine axons. In summary, the experiments proposed here will contribute to a novel mechanistic understanding of the dopamine secretory pathway. Our finding that dopamine release occurs rapidly and precisely signals the beginning of a paradigm shift for dopamine transmission. The proposed work expands on dissecting the make- up and function of the rapid exocytotic machinery for dopamine. Precise understanding of dopamine secretion will also provide new insights into how dopamine signaling may break down in neurological disease.

Public Health Relevance

The nervous system relies on the transmission of chemical messengers, or neurotransmitters, from one cell to another. Dopamine is a neurotransmitter that is critical for proper nervous system function and defective dopamine signaling is the cause of several neurological disorders. My work aims to understand how the release of dopamine is regulated to ensure its precise transmission.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Sieber, Beth-Anne
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Harvard Medical School
Schools of Medicine
United States
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