Cannabinoid Mobilization in Neurons

描述(申请人提供)：这个项目旨在通过让学生参与德克萨斯大学布朗斯维尔分校(93%的西班牙裔社区)正在进行的内源性大麻素系统研究，提高他们的研究能力及其学生在神经科学方面的技能和兴趣。大麻素是大麻和大麻中的生物活性成分，通过作用于特定的受体--大脑大麻素受体(CB1R)来影响大脑。CB1R的天然配体是内源性大麻素(ECB)。ECB在神经系统的许多领域被认为是重要的信号分子，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，这使得了解ECB的合成过程变得非常重要。然而，启动ECB合成的生理刺激并不是很清楚。该项目的目标是研究欧洲央行生产的细胞机制。神经元去极化和去极化诱导的胞浆钙([Ca+]c)增加产生ECB，这种产生可以通过一种被称为DSI(去极化诱导的GABA能抑制)的现象来实时生物检测。PI的初步研究结果表明，兰尼定受体(RyR)的钙释放决定了DSI的大小。根据这一证据，推测RyR可能是神经元产生ECB所必需的和充分的关键分子。这一假说将通过实现以下特定目标来验证：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CB1R的解剖关系；以及3)确定电压门控Ca~(2+)通道和存储操作的Ca~(2+)进入在RyR依赖的ECB产生中的作用。实验在培养的海马片上进行，采用免疫组织化学、siRNA和EGFP转染、钙离子成像和膜片钳记录等技术。ECB及其受钙离子的调节是生物医学科学家非常感兴趣的话题。因此，鉴定细胞外钙信号通路和细胞外钙感受蛋白的产生，将有助于我们对潜在的神经信号系统有一个重要的了解，并为研究从基本突触传递到细胞外基质的病理生理作用开辟新的途径。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，并帮助他们确立了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.