Cannabinoid Mobilization in Neurons

描述（由申请人提供）：该项目旨在通过让学生参与PI在德克萨斯大学布朗斯维尔分校正在进行的内源性大麻素系统研究来增强PI的研究能力以及学生在神经科学方面的技能和兴趣（学术多元化，93%是西班牙裔社区）。布朗斯维尔。大麻素是大麻和印度大麻中的生物活性成分，通过作用于特定的受体，即大脑大麻素受体（CB 1 R）来影响大脑。CB 1 R的天然配体是内源性大麻素（eCB）。eCB在神经系统的许多领域中越来越被认为是关键的信号分子，因为eCB调节许多重要的神经生物学现象，包括神经传递，记忆和学习，奖励和动机以及疾病中的神经保护，这使得理解它们的合成过程变得非常重要。然而，启动eCB合成的生理刺激还没有得到很好的理解。本项目的目标是研究eCB产生的细胞机制。神经元去极化和去极化诱导的细胞质钙（[Ca 2 +]c）增加产生eCB，这种产生可以通过称为DSI（去极化诱导的GABA能抑制抑制）的现象在真实的时间内进行生物测定。PI的初步研究结果表明，钙离子从兰尼碱受体（RyR）释放决定了DSI的大小。根据这一证据，假设RyR可能是神经元中产生eCB所必需和足够的关键分子。该假设将通过实现以下特定目的进行检验：1）分离和表征RyR介导的[Ca 2 +]c信号并确定其在eCB产生中的作用; 2）确定RyR的细胞定位及其与CB 1 R的解剖学关系; 3）确定电压门控Ca 2+通道和钙库操纵的Ca 2+内流在RyR依赖性eCB产生中的作用。采用免疫组织化学、siRNA和eGFP转染、钙离子成像和膜片钳记录技术对培养的海马脑片进行实验。eCB及其通过Ca 2+的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Ca 2+信号通路和Ca 2+传感蛋白的产生eCBs将提供一个重要的理解，为有效的神经元信号系统，并开辟新的途径，调查的现象，从基本的突触传递的病理生理作用的eCBs。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，并帮助他们建立生物医学科学的专业目标。

DESCRIPTION (provided by applicant): This project is designed to enhance the PI's research capability and the skills and interests of her students in neuroscience by having them to participate in the PI's ongoing research in the endogenous cannabinoid system at The University of Texas at Brownsville (an academically diverse 93% Hispanic community). Cannabinoids, the bioactive components in marijuana and hashish, affect the brain by acting at specific receptors, the brain cannabinoid receptors (CB1R). Natural ligands for CB1R are endogenous cannabinoids (eCBs). eCBs are increasingly recognized as critical signaling molecules in many areas of the nervous system because eCBs modulate many important neurobiological phenomena including neurotransmission, memory and learning, reward and motivation, and neuroprotection in diseases, which makes understanding the process of their synthesis a matter of great importance. However, the physiological stimuli that initiate the synthesis of eCBs are not well understood. The goal of this project is to investigate the cellular mechanisms of eCB production. Neuronal depolarization and the depolarization-induced increase in cytoplasmic calcium ([Ca2+]c) produce eCBs, and this production can be bio-assayed in real time by a phenomenon called DSI (depolarization-induced suppression of GABAergic inhibition). The PI's preliminary findings suggest that Ca2+ release from the ryanodine receptor (RyR) determines the magnitude of DSI. From this evidence, it is hypothesized that RyR may be a key molecule that is necessary and sufficient for the production of eCBs in neurons. This hypothesis will be tested by accomplishing the following Specific Aims: 1) To isolate and characterize RyR-mediated [Ca2+]c signal and determine its role in the eCB production; 2) To identify the cellular localization of RyRs and their anatomical relationships with CB1R; and 3) To determine the roles of voltage-gated Ca2+ channels and store-operated Ca2+ entry in the RyR-dependent production of eCBs. Experiments are conducted in cultured hippocampal slices with the techniques of immunohistochemistry, siRNA and eGFP transfection, Ca2+ imaging, and the patch clamp recording. eCBs and their regulation by Ca2+ are topics of great interest to a wide range of biomedical scientists. Thus, the identification of Ca2+ signaling pathways and Ca2+ sensing proteins for the production of eCBs will provide a critical understanding for potent neuronal signaling systems, and open new avenues of investigations for the phenomena ranging from basic synaptic transmission to pathophysiological roles 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 goals in biomedical sciences.