Since classical transmitters are made in the cytoplasm, release by exocytosis requires transport into secretory vesicles, and we have identified three families of proteins responsible for this activity. One family includes transporters for monoamines and acetylcholine, a second transporters for the inhibitory transmitter GABA and a third transporters dedicated to glutamate. In the process, the work has implicated vesicular transport in protection against the neural degeneration associated with Parkinson's disease. We have also identified transport proteins involved in the glutamine-glutamate cycle that regenerates the glutamate required for both excitatory and inhibitory transmission. We are now using a variety of approaches to understand the function of these proteins and their role in neurotransmitter release. We use biophysical approaches including electrophysiology and pH imaging as well as flux assays to study the mechanism of neurotransmitter transport into secretory vesicles. In addition, we use genetic manipulation in mice to test the physiological relevance of mechanisms identified in vitro , and to understand their role in behavior.

In the course of these studies, we have made several observations about another major function of the nerve terminal, the recycling of synaptic vesicle membrane. We found that individual synaptic vesicle proteins contain their own, distinct signals which determine how they recycle. Vesicular glutamate transporter 1 (VGLUT1) interacts directly with a component of the endocytic machinery, and this influences the rate and pathway of recycling during stimulation. A distinct pathway appears to mediate the bulk endocytosis that follows prolonged stimulation. We are now pursuing the possibility that these two pathways regenerate synaptic vesicles with distinct properties. In addition, previous work has demonstrated the release of dopamine and other monoamines from dendrites as well as axon terminals. Consistent with this, vesicular monoamine transporter 2 (VMAT2) localizes to dendrites and axons, and we have identified the sequences responsible for sorting to regulated secretory vesicles in dendrites. We will now use this information to understand the biogenesis of this secretory pathway which is responsible for the release of all neural peptides and many growth factors as well as hormones such as insulin. We will also explore the role of this pathway in neurons by genetic manipulation in vivo .

We also wish to understand the pathogenesis of Parkinson's disease (PD), which appears to take place at the nerve terminal. VMAT2 protects against an exogenous toxin in an animal model of PD, and perhaps against the endogenous transmitter dopamine, which is intrinsically toxic. We are thus very interested in the possibility that a defect in the mechanisms protecting against dopamine toxicity can cause PD. Interestingly, the presynaptic protein a -synuclein also has an important role in Parkinson's, and we are pursuing its physiological role at the nerve terminal through a combination of biochemistry and optical imaging.