My long-term goal is to establish an independent laboratory at an academic research institution where I will have the freedom and resources to contribute to our understanding of the brain, and in particular, the neural mechanisms underlying addiction. My near-term goals are to demonstrate the resourcefulness and productivity necessary to achieve independence while mastering new skills and concepts that will allow me to thrive independently. Specifically, I will train on several new methodologies including 1) in vitro transport assays, 2) neuronal culture, 3) slice electrophysiology, and 4) tract tracing. I will also enroll in several classes designed to improve my technical literacy or enhance my ability to provide strong leadership & management. I plan to recruit one or more undergraduate student assistants which will both provide a mentoring opportunity and increase my efficiency. By the end of the mentored phase of this award, my goal is to be in a position to write a highly competitive R01 proposal, the crucial mechanism for funding an independent academic laboratory. Environment: Most of the training during the mentored phase of this proposal will occur at the University of California San Francisco (UCSF) under the direction of Dr. Robert Edwards. Robert is a world-renowned investigator at the forefront of vesicular transporter biophysics and cell biology. Robert's particular focus on in vitro approaches to the study of vesicular dopamine and glutamate transporters meshes perfectly with both the career development and research plan sections of this proposal. The physiology department at the Mission Bay campus of UCSF is an exciting setting to pursue this work with ample opportunity for collaboration and access to both standard and emerging technologies necessary to further this research plan. I also propose to spend 1-2 years training under the co-mentorship of Dr. Howard Fields who remains affiliated with UCSF but whose lab is now located at the Ernest Gallo Clinic and Research Center (EGCRC) in nearby Emeryville CA. Howard is a distinguished investigator whose main research interest is in unraveling the neural circuitry underlying addiction. Working with Howard my main goal will be to learn how to make electrophysiological recordings from fluorescent dopamine or glutamate neurons in acute slices made through the mouse midbrain. The EGCRC is a vibrant research environment where the primary mission is centered on understanding the neural mechanisms of addiction with the goal of treating and preventing drug abuse. Thus my time at the EGCRC will provide me with new skills in neurophysiology but also expose me to the work of a research community that broadly shares the goals described in this proposal. Research Plan: Dopamine neurons of the ventral tegmental area (VTA) are of fundamental importance to the initiation and development of drug dependence. However, their precise role in drug addiction remains unclear. Thus, a crucial step toward understanding the mechanisms by which drugs of abuse produce the molecular and chemical changes in brain circuitry that lead to addiction is to define the contribution of dopamine neurons to normal brain function. Emerging evidence suggests that dopamine neurons have the capacity to co-release the excitatory neurotransmitter glutamate and vesicular glutamate transporters (VGLUTs) are necessary for neurons to release glutamate. A central aim of this project is to use a mouse model that has been genetically engineered to lack the vesicular glutamate transporter (VGLUT2) specifically from dopamine neurons to assess the role of glutamate co-release by dopamine neurons on brain development and function. Electrophysiological, anatomical, biochemical & behavioral techniques will be employed. Emphasis will be placed on determining whether glutamate co-release contributes to the behavioral responses to drugs of abuse and, in particular, reward learning. In addition, the potential for VGLUT2 to induce vesicular acidification and promote vesicular dopamine accumulation will be assessed using the mice described above.
A second aim i s to characterize a second population of VGLUT2+ neurons in the VTA that do not express dopaminergic markers. These neurons represent an uncharacterized population of VTA neurons that may also play an important role in mediating responses to drugs of abuse. Using double-transgenic reporter mice, the electrophysiological properties, pharmacological responses, and anatomical projections of the VGLUT2+ neurons will be assessed and compared with their dopaminergic neighbors.
The final aim will be to inject viral vectors engineered to express Cre recombinase into the VTA of conditional VGLUT2 knockout mice to assess the role of all VGLUT2+ VTA neurons in goal-directed behaviors. The overall goal of this proposal is to elucidate the function of glutamate-releasing VTA neurons, and in particular, assess their role in mediating the molecular and behavioral responses to drugs of abuse.
- Relevance: A more rigorous understanding of the mechanisms by which drugs of abuse mediate their actions is critical to achieving the means to treat and prevent drug addiction. A central front in this struggle is the mesolimbic dopamine system. This proposal is designed to evaluate the contribution of glutamate release by both dopamine and non-dopamine VTA neurons in mediating the molecular and behavioral responses to drugs of abuse.
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