Genetic Elucidation of VTA Glutamate Neurons

描述（由申请人提供）：我的长期目标是在一个学术研究机构建立一个独立的实验室，在那里我将有自由和资源来促进我们对大脑的理解，特别是对成瘾的神经机制的理解。我的近期目标是在掌握新技能和新概念的同时，展现出实现独立所需的足智多谋和生产力，使我能够独立发展。具体来说，我将学习几种新方法，包括1)体外转运试验，2)神经元培养，3)切片电生理学，以及4)尿道追踪。我还将参加一些旨在提高我的技术素养或增强我的领导和管理能力的课程。我计划招聘一名或多名本科生助理，这既可以提供指导机会，又可以提高我的工作效率。在这个奖项的指导阶段结束时，我的目标是能够撰写一份极具竞争力的R01提案，这是资助独立学术实验室的关键机制。环境：本提案指导阶段的大部分培训将在加州大学旧金山分校（UCSF） Robert Edwards博士的指导下进行。罗伯特是泡转运蛋白生物物理学和细胞生物学前沿的世界知名研究者。Robert特别关注囊泡多巴胺和谷氨酸转运体的体外研究方法，这与本提案的职业发展和研究计划部分完全吻合。加州大学旧金山分校Mission Bay校区的生理学系是一个令人兴奋的环境，可以开展这项工作，有充足的合作机会，并可以获得进一步研究计划所需的标准和新兴技术。我还建议在Howard Fields博士的指导下进行1-2年的培训，他仍然隶属于UCSF，但他的实验室现在位于加利福尼亚州埃默里维尔附近的Ernest Gallo诊所和研究中心（EGCRC）。Howard是一位杰出的研究者，他的主要研究兴趣是阐明成瘾背后的神经回路。与霍华德合作，我的主要目标是学习如何通过小鼠中脑的急性切片，从荧光多巴胺或谷氨酸神经元中进行电生理记录。EGCRC是一个充满活力的研究环境，其主要任务是了解成瘾的神经机制，以治疗和预防药物滥用为目标。因此，我在EGCRC的时间将为我提供神经生理学方面的新技能，但也使我接触到一个研究团体的工作，这个研究团体广泛地分享了本提案中所描述的目标。研究计划：腹侧被盖区（VTA）多巴胺神经元对药物依赖的发生和发展起着至关重要的作用。然而，它们在药物成瘾中的确切作用仍不清楚。因此，了解药物滥用在大脑回路中产生导致成瘾的分子和化学变化的机制的关键一步是确定多巴胺神经元对正常大脑功能的贡献。新的证据表明，多巴胺神经元具有共同释放兴奋性神经递质谷氨酸的能力，而谷氨酸囊泡转运体（VGLUTs）是神经元释放谷氨酸所必需的。该项目的一个中心目标是使用一个经过基因工程处理的小鼠模型来缺乏多巴胺神经元特异性的谷氨酸囊泡转运蛋白（VGLUT2），以评估多巴胺神经元共同释放谷氨酸在大脑发育和功能中的作用。将使用电生理、解剖、生化和行为技术。重点将放在确定谷氨酸的共同释放是否有助于对药物滥用的行为反应，特别是奖励学习。此外，VGLUT2诱导囊泡酸化和促进囊泡多巴胺积累的潜力将在上述小鼠中进行评估。第二个目的是表征VTA中不表达多巴胺能标记物的第二群VGLUT2+神经元。这些神经元代表了一种未知的VTA神经元群，也可能在介导药物滥用反应中发挥重要作用。使用双转基因报告小鼠，将评估VGLUT2+神经元的电生理特性、药理反应和解剖投影，并与它们的多巴胺能邻居进行比较。最终目的是将表达Cre重组酶的病毒载体注射到条件VGLUT2敲除小鼠的VTA中，以评估所有VGLUT2+ VTA神经元在目标导向行为中的作用。本研究的总体目标是阐明谷氨酸释放VTA神经元的功能，特别是评估其在介导药物滥用的分子和行为反应中的作用。

DESCRIPTION (provided by applicant): 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 is 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.