Cannabinoid mobilization in neurons (R15DA021683)

描述（由申请人提供）：该项目旨在提高PI的研究能力和技能和我们的本科生和硕士生在神经科学的兴趣，让他们参加PI的内源性大麻素系统正在进行的研究项目在德克萨斯大学布朗斯维尔（一个主要的教学机构与学术多元化93%的西班牙裔社区）。大麻素是大麻和印度大麻中的生物活性成分，通过作用于大脑大麻素受体（CB 1 R）来影响大脑。激活CB 1 R的天然分子是内源性大麻素（eCB）。eCB越来越多地被认为是许多神经生物学现象中的关键信号分子，包括突触可塑性，记忆，奖励学习，疼痛，神经保护和成瘾;所有这些都使得理解eCB合成的过程变得非常重要。PI已经证明，在海马中启动eCB产生的关键过程是从兰尼碱受体（RyR）释放钙离子。这一发现是通过人工去极化神经元发现的。因此，本发明的目的是鉴定激活海马神经元中的RyR以产生eCB的生理条件和内源性分子。Ghrelin是一种促食欲的脑肽激素。为什么是胃饥饿素？因为ghrelin和ghrelin受体参与了与奖励相关的记忆，而eCB也参与其中。此外，PI的初步数据表明，ghrelin受体的激活刺激蛋白激酶A（PKA），据报道，PKA可增强RyR的功能。该提议的具体目的是：1）确定生长素释放肽受体在海马中的定位，2）表征响应于生长素释放肽的细胞质钙动力学和eCB合成，和3）确定生长素释放肽调节海马突触可塑性的程度。采用膜片钳记录、钙离子成像、免疫组化、siRNA和eGFP转染等技术对培养的海马脑片进行实验。eCB及其钙调节是广泛的生物医学科学家非常感兴趣的话题。因此，识别生理条件， 启动脑eCBs的产生将为eCBs的有效神经元信号传导系统提供重要的理解。该项目还为学生提供了获得实验室研究经验的宝贵机会，这无疑提高了他们对公共卫生和疾病的认识，并帮助他们在生物医学研究领域建立自己的职业生涯。

DESCRIPTION (provided by applicant): This project is designed to enhance the PI's research capability and the skills and interests of our undergraduate and master's students in neuroscience by having them to participate in the PI's ongoing research project in the endogenous cannabinoid system at The University of Texas at Brownsville (a primarily teaching institution with an academically diverse 93% Hispanic community). Cannabinoid, the bioactive component in marijuana and hashish, affects the brain by acting on the brain cannabinoid receptor (CB1R). Natural molecules that activate CB1R are endocannabinoids (eCBs). eCBs are increasingly recognized as critical signaling molecules in many neurobiological phenomena including synaptic plasticity, memory, reward learning, pain, neuroprotection, and addiction; all of which makes understanding the process of eCB-synthesis a matter of great importance. The PI has demonstrated that a key process to initiate the production of eCBs in the hippocampus is a release of calcium ions from the ryanodine receptor (RyR). The discovery was made by artificially-depolarizing neurons. Therefore, the goal of the present proposal is to identify physiological conditions and endogenous molecule(s) that activate RyR in hippocampal neurons for the production of eCBs. Ghrelin, an orexigenic brain peptide hormone, is selected for investigation. Why ghrelin? Because ghrelin and the ghrelin receptor are involved in reward-related memories in which eCBs are also involved. In addition, the PI's preliminary data suggest the activation of the ghrelin receptor stimulates protein kinase A (PKA), which is reported to enhance the function of RyR. Specific Aims of this proposal are: 1) to determine the localization of the ghrelin receptor in the hippocampus, 2) to characterize cytoplasmic calcium dynamics and eCB-synthesis in response to ghrelin, and 3) to determine the extent to which ghrelin regulates hippocampal synaptic plasticity. Experiments are conducted in cultured hippocampal slices with the techniques of patch clamp recording, calcium imaging, immunohistochemistry, siRNA and eGFP transfection. eCBs and their regulation by calcium are topics of great interest to a wide range of biomedical scientists. Thus, the identification of physiological conditions that initiate the production of brain eCBs will provide a critical understanding for potent neuronal signaling systems of eCBs. The project also offers a valuable opportunity for students to obtain laboratory research experiences, which undoubtedly raise their awareness in public health and diseases, and aid them in establishing their professional careers in biomedical research.