Actions Of Endogenous /Exogenous Cannabinoids On Brain R

内源性/外源性大麻素对大脑 R 的作用

• 批准号: 6987926
• 负责人: Carl R Lupica
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6987926
• 关键词: biophysics; brain cell; brain electrical activity; calcium; cannabinoids; confocal scanning microscopy; dopamine; electrophysiology; flash photolysis; gamma aminobutyrate; laboratory rat; marijuana abuse; membrane channels; neurons; neurotransmitter transport; nucleus accumbens; pharmacokinetics; potassium; psychopharmacology; reinforcer; sectioning; synapses; tegmentum; tissue /cell preparation

The main psychoactive component of marijuana is known as delta9-tetrahydrocannabinol (THC). In addition, it has recently been discovered that endogenous substances are synthesized in the brain that can activate cannabinoid receptors, and these substances are referred to as endocannabinoids. All drugs, both natural and synthetic, that act at receptors for this substance are known collectively as cannabinoids (CBs). Cannabinoid drugs obtained by the smoking or ingestion of marijuana are used illicitly presumably because they are reinforcing or rewarding to humans. The objectives of this study are to gain knowledge about the underlying mechanisms through which cannabinoids alter brain cell function, and ultimately the mechanisms that produce the pleasurable effects of these drugs that sustain their illicit use. Cannabinoids act in discrete brain areas to produce their effects. The focus of this laboratory is to examine the effects of drugs of abuse on the electrical activity of individual nerve cells (neurons) and the ways in which these neurons communicate with each other via synaptic connections. Therefore, one of our goals is to identify specific electrically conducting ion channels whose activity is modified by CBs. To achieve these goals we will utilize rat brain slices acutely obtained from discrete brain areas, perform whole-cell electrophysiological recordings, and, using cellular anatomical techniques, morphologically reconstruct the neurons from which we record. The brain slice technique permits continuous and visually clear access to these areas of relatively intact nerve cell circuits while allowing perfusion with control saline or various solutions of drugs. In addition, because of the excellent access to the neurons in this preparation we can, through the use of both biophysical techniques and ion channel blocking agents, identify the ion channels acted upon by abused drugs. In the present study we will examine cannabinoid actions on nerve cells and their connections in an area of the midbrain known as the ventral tegmental area (VTA). This brain area and its connections have been strongly implicated in the reinforcing and rewarding actions of virtually all abused drugs. We will also examine the effects of these drugs in the nucleus accumbens, a brain area that is also known to be involved in the rewarding effects of CBs. Cannabinoids are known to be abused and are self-administered by laboratory animals. However, their actions in the VTA have only been recently investigated. It is known that injection of CBs into anesthetized rats causes an increase in firing rate of nerve cells in the VTA, and that activity of these nerve cells is important in producing pleasurable sensations. However, the mechanism by which this increase in firing is incompletely understood. Therefore, the present study will be one of the first to examine the mechanisms of action of cannabinoids on neurons in this important region of the brain. Therefore, these studies will allow greater understanding of the neural substrates that support the abuse of this class of drugs, as well as describe the basic mechanisms of drug abuse in general. It is hoped that these studies will produce knowledge that can be used to develop treatments for the abuse of CBs. Brain slice preparations will be used in conjuction with confocal microscopy to answer basic questions regarding the synthesis and release of endocannabinoids in VTA and NAc neurons. Previously, we demonstrated that by blocking a potassium ion channel (sK) that normally keeps DANs relatively quiescent, we can increase their level of spontaneous activity, and this results in endocannabinoid release that inhibits GABA synaptic inputs to DANs, that arise from neurons in the nucleus accumbens. We have recently followed these studies up by demonstrating that other manipulations that can increase DAN activity, such as glutamatergic excitation of these neurrons, can also promote endocannabinoid release. Therefore, these studies are the first to demonstrate this novel circuit that both THC, and endocannabinoids can act upon. Based on our data, we hypothesize that activation of cannabinoid receptors will result in a decrease in inhibitory GABA release onto DANs, and that this will increase the output of the DANs, and increase dopamine release in the nucleus accumbens. A manuscript detailing these results has been submitted for review at the Journal of Neuroscience. As the next step in these ongoing studies, we have set up a confocal microscope equipped to perform calcium imaging in brain slices, and to further examine the role of endocannabinoids in regulating neurotransmitter release in dopamine-containing neurons (DANs) in the VTA. The basic strategy that will be used is to evoke the release of calcium from intracellular stores using the technique of flash photolysis, which causes the uncaging of a molecule such as phosphatidylinositol. We will then assess whether this rise in intracellular calcium can subsequently result in an increase in the release of endocannabinoid that will then inhibit the release of the inhibitory neurotransmitter GABA. The rise in intracellular calcium levels will be monitored using ratiometric calcium detection techniques and the level of calcium will be related to the degree of synaptic inhibition that can be reversed by cannabinoid receptor antagonist application. These studies will be the first to directly relate rises in intracellular calcium with the release of endocannabinoids in the mammalian CNS.

大麻的主要精神活性成分被称为delta9-tetrahydrocanbinol（THC）。此外，最近已经发现，内源性物质是可以激活大麻素受体的大脑中合成的，这些物质被称为内源性大麻素。所有天然和合成的药物都在该物质上起作用受体，统称为大麻素（CBS）。通过吸烟或摄入大麻获得的大麻素药物可能是非法使用的，因为它们正在增强或有益。这项研究的目的是获取有关大麻素会改变脑细胞功能的潜在机制的知识，并最终产生这些药物的愉悦作用的机制。大麻素在离散的大脑区域作用以产生其作用。该实验室的重点是检查滥用药物对单个神经细胞（神经元）的电活动的影响，以及这些神经元通过突触连接相互通信的方式。因此，我们的目标之一是确定特定的电导导电的活性由CBS修改。为了实现这些目标，我们将利用从离散的大脑区域急性获得的大鼠脑切片，执行全细胞电生理记录，并使用细胞解剖技术，从形态上重建我们记录的神经元。大脑切片技术允许连续和视觉清晰地进入这些相对完整的神经细胞回路的区域，同时允许使用控制盐水或各种药物溶液进行灌注。此外，由于在这种制剂中可以很好地访问神经元，我们可以通过使用生物物理技术和离子通道阻断剂来识别滥用药物作用的离子通道。在本研究中，我们将检查对神经细胞的大麻素作用及其在被称为腹侧段区域（VTA）的中脑区域的连接。这个大脑区域及其联系在几乎所有被滥用的药物的加强和有益的行动上都与之相关。我们还将检查这些药物在伏隔核中的作用，伏隔核是大脑区域，也涉及CBS的奖励作用。已知大麻素被滥用，并由实验动物自我管理。但是，他们在VTA中的行动直到最近才进行了调查。众所周知，将CBS注射到麻醉大鼠中会导致VTA中神经细胞的点火速率增加，并且这些神经细胞的活性对于产生愉悦的感觉很重要。但是，射击增加的机制尚不完全理解。因此，本研究将是最早检查大脑在这个重要区域中神经元作用机理的研究之一。因此，这些研究将允许对支持滥用这类药物的神经底物有更多的了解，并描述一般药物滥用的基本机制。希望这些研究能够产生可用于开发滥用CB的治疗方法的知识。脑切片制剂将与共聚焦显微镜结合使用，以回答有关VTA和NAC神经元中内源性大麻素的合成和释放的基本问题。以前，我们证明，通过阻止通常保持DAN相对静止的钾离子通道（SK），我们可以增加它们的自发活性水平，这导致内源性大麻素释放释放，从而抑制DAN的GABA突触输入，从而抑制了Neurons concumbens neurons cy concumbens中的Neurons产生。最近，我们遵循了这些研究，证明了其他可以增加DAN活性的操作（例如对这些神经元的谷氨酸能激发）也可以促进内源性大麻素的释放。因此，这些研究是第一个证明THC和内源性大麻素可以作用的新型电路的研究。基于我们的数据，我们假设大麻素受体的激活将导致抑制性GABA释放到DANS上，这将增加DAN的输出，并增加Accumbens核中多巴胺的释放。详细介绍这些结果的手稿已在《神经科学杂志》上进行审查。作为这些正在进行的研究的下一步，我们建立了一个配备了脑切片中钙成像的共聚焦显微镜，并进一步研究了内源性大麻素在调节VTA中多巴胺神经元（DAN）中神经递质释放中的作用。将使用的基本策略是使用闪光光解的技术从细胞内储存中释放钙，这会导致分子（例如磷脂酰肌醇）的渗透。然后，我们将评估细胞内钙的这种升高是否会随后导致内源性大麻素的释放增加，从而抑制抑制性神经递质GABA的释放。细胞内钙水平的升高将使用比率钙检测技术来监测，并且钙的水平将与突触抑制程度有关，而突触抑制程度可以通过大麻素受体拮抗剂的应用来反转。这些研究将是第一个将细胞内钙与哺乳动物CNS中内源性大麻素释放的直接升高相关的研究。