Physiology and Pharmacology of Brain Reward Circuits

大脑奖励回路的生理学和药理学

• 批准号: 9555584
• 负责人: Carl R. Lupica
• 金额: $ 58.89万
• 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/9555584
• 关键词: 2-arachidonylglycerol; AM 251; Action Potentials; Acute; Address; Affect; Agonist; Anatomy; Anxiety Disorders; Area; Bilateral; Brain; CNR1 gene; Cannabinoids; Cell physiology; Cells; Cocaine; Cocaine Dependence; Cues; Data; Dependency; Disease; Disinhibition; Drug Addiction; Drug abuse; Electric Stimulation; Electrophysiology (science); Endocannabinoids; Ethanol; Exposure to; Feedback; Fire - disasters; Fluorescence; Food; Glutamates; Goals; Heroin; Human; Image; Infusion procedures; Ingestion; Injectable; Injection of therapeutic agent; Intake; Ion Channel; Knowledge; Laboratories; Link; LoxP-flanked allele; Major Depressive Disorder; Marijuana; Measurement; Measures; Mediating; Mental Depression; Mental disorders; Midbrain structure; Moods; Motivation; Mus; Neurons; Nicotine; Nucleus Accumbens; Peripheral; Pharmaceutical Preparations; Pharmacology; Phase; Physiological; Physiology; Post-Traumatic Stress Disorders; Presynaptic Terminals; Process; Property; Proteins; Psyche structure; Rattus; Rewards; Role; Self Administration; Signal Transduction; Slice; Smoking; Stimulus; Synapses; Syndrome; Techniques; Testing; Tetrahydrocannabinol; Ventral Tegmental Area; Water; Work; addiction; alertness; biological adaptation to stress; brain cell; calcium indicator; cannabinoid drug; cannabinoid receptor; cocaine use; design; dopaminergic neuron; drug of abuse; experimental study; extracellular; fluorescence imaging; gamma-Aminobutyric Acid; in vivo; induced pluripotent stem cell; information processing; inhibitor/antagonist; insight; lipoprotein lipase; marijuana use; mesolimbic system; novel; paired stimuli; patch clamp; pleasure; presynaptic; protein expression; psychostimulant; receptor; response; reward circuitry; stress disorder; synaptic inhibition

The main psychoactive component of marijuana is known as delta9-tetrahydrocannabinol (THC). In addition, endogenous substances are synthesized in the brain that can activate cannabinoid receptors, and these substances are referred to as endocannabinoids (eCB). 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 because they are reinforcing or rewarding to humans through interaction with the brain's "reward circuitry". One of the objectives of these studies is 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 use. The primary focus of this laboratory is to examine the mechanisms through which abused drugs alter the electrical activity of neurons and the ways in which these neurons communicate with each other via synaptic connections. Therefore, one of our goals is to identify specific ion channels whose activity is modified by abused drugs such as marijuana, nicotine, heroin, and cocaine. To achieve these goals we utilize rat brain slices acutely obtained from discrete brain areas involved in processing information regarding pleasurable and unpleasant environmental stimuli. We utilize whole-cell electrophysiological recordings, and cellular anatomical techniques to reconstruct the neurons from which we record. In these ongoing studies we are examining the mechanisms through which these drugs affect neurons and their connections in the ventral tegmental area (VTA). This brain area and its connections are strongly implicated in the reinforcing and rewarding actions of all abused drugs, as well as in mediating the rewarding effects of natural environmental stimuli, such as food, water, etc. The VTA is also involved in processing information regarding the physiological stress responses, mood and affect, and mental alertness. Because of its central role in these processes, the VTA is a brain area that contributes to disorders such as addiction, psychiatric stress disorders, clinical depression, and psychiatric anxiety disorders. Our recent work in the VTA is designed understand the mechanisms through which eCBs modulate VTA circuitry during psychostimulant exposure. Many studies show that antagonism of cannabinoid CB1Rs can block self-administration of several abused drugs. Furthermore, the increase in DA concentration that is observed in the nucleus accumbens (NAc) following systemic nicotine, cocaine or ethanol administration is greatly reduced by systemic administration of the CB1R antagonist/partial agonist SR141716A2. This drug also decreases DA release in the NAc in response to the presentation of reward-associated cues, suggesting that eCBs are important for DA increases in the mesolimbic system by unconditioned and conditioned stimuli associated with drug abuse. There is also evidence that infusion of CB1R antagonists directly into the VTA can reduce the rewarding effect of peripheral nicotine, and reduce the release of DA in the VTA following systemic cocaine administration in rats. Data from our laboratory and others also show that eCBs are released from dopamine neurons in the VTA. Collectively, these data suggest that these eCBs act within the VTA on CB1Rs to increase the release of DA in the NAc during the intake of abused drugs, or during exposure to drug-paired stimuli. Although CB1Rs are located on both GABAergic and glutamatergic axon terminals within the VTA, we hypothesize that it is the inhibition of GABA release and disinhibition of DA neurons by THC that is responsible for its rewarding effects. We further hypothesize that the increase in DA neuron activity by abused drugs causes release of the eCB, 2-aracchidonoylglycerol (2-AG), which then augments DA neuron excitation through inhibition of GABA release, in an eCB-driven positive feedback loop. For these reasons we are conducting experiments to determine whether abused drugs trigger eCB release in the VTA, and what mechanisms might underlie this action. The most sensitive physiological measure of eCB function that we have found in the VTA are synaptic GABAB-receptor-mediated IPSCs, activated by electrical stimulation. These responses are strongly inhibited by presynaptic CB1Rs, and are tonically inhibited by the eCB, 2-AG, since its amplitude increases in the presence of CB1R antagonists, or when 2-AG synthesis is inhibited by inclusion of a diacylglycerol lipase-alpha; (DGL-alpha) inhibitor, THL, in the whole-cell pipettes. Additionally, phasic, activity-dependent 2-AG release can be observed when DA neurons are transiently depolarized to fire action potentials, and this is also mediated by an increase in 2-AG release. Our recently acquired data show that GABA-B IPSCs in VTA DA neurons are inhibited by cocaine (10 microM), and this is reduced by the CB1R antagonist AM251 (2 microM). The effect of cocaine is partly mediated by 2-AG released from the recorded neuron, and from surrounding cells, since THL blocks the effect more strongly when applied extracellularly, versus intracellularly. These data suggest that cocaine stimulates the release of 2-AG in the VTA to reduce GABA release onto DA neurons. We hypothesize that the reduction of inhibition by cocaine increases the excitability of midbrain DA neurons to augment release of DA in projection areas like the NAc, as described in in vivo studies. Because this cocaine action may be important for its rewarding and addicting properties, we are performing additional experiments to determine the mechanism(s) by which cocaine releases 2-AG in the VTA. Thus, in the next fiscal year we will address this issue more directly by examining the effects of cocaine on Ca2+ dynamics in VTA DA neurons in brain slices, using confocal imaging of fluorescence emitted by genetically-encoded calcium indicator (GECI) proteins. We use the floxed GECIs GCaMP5 (AAV2/1.hSynap.Flex.GCaMP5G) or GCaMP6 (AAV1.CAG.Flex.GCaMP6f)61, injected bilaterally into the VTA of THCre expressing rat lines. Our preliminary data in THCre mice indicate excellent expression of these proteins, permitting measurement of fluorescence triggered by baseline Ca2+ oscillations, or during cocaine application. In most VTA DA neurons (80%), acute cocaine (10 M) reduces Ca2+ fluorescence, and this is blocked by D2 antagonist. However, in approximately 20% of the imaged VTA DA neurons, we observe a significant increase in Ca2+ signal during cocaine application, consistent with an increase in VTA DA neuron single unit activity with cocaine in vivo. We hypothesize that the cocaine-induced increase in the activity of these DA neurons causes release of 2-AG through a Ca2+-dependent mechanism, and that these cells will show greater inhibition of synaptic GABA by 2-AG. Thus, the DA neurons showing the largest cocaine-induced increase in Ca2+ signal will also show the largest increase in GABA-B IPSCs upon CB1R antagonism. To test this we will identify VTA DA neurons showing increased or decreased Ca2+-induced fluorescence following cocaine application in brain slices from THCre rats that have received intra-VTA injections of the AAV-GCaMP6 construct. We will then perform patch clamp recordings from these 2 groups of cells, record GABAB IPSCs, and evaluate the magnitude of the cocaine-induced inhibition of the synaptic response, and determine the dependence of the cocaine inhibition with a neutral CB1R antagonist (PIMSR1, 1 M). These experiments should establish a link between cocaine use and eCB release in the VTA, and identify a novel mechanism in which cocaine dependency may be disrupted.

大麻的主要精神活性成分被称为delta9-tetrahydrocanbinol（THC）。另外，内源性物质是在大脑中合成的，可以激活大麻素受体，这些物质被称为内源性大麻素（ECB）。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）。使用大麻或摄入大麻获得的大麻素药物是因为它们通过与大脑的“奖励电路”相互作用来增强或奖励人类。这些研究的目标之一是获得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维持其使用的愉悦作用的机制。该实验室的主要重点是检查滥用药物改变神经元的电活动以及这些神经元通过突触连接相互通信的方式的机制。因此，我们的目标之一是确定特定的离子渠道，其活动被大麻，尼古丁，海洛因和可卡因等滥用药物所修饰。为了实现这些目标，我们利用大鼠脑切片从涉及处理有关愉悦和不愉快的环境刺激的信息的离散大脑区域敏锐地获得。 我们利用全细胞电生理记录和细胞解剖技术来重建我们记录的神经元。在这些正在进行的研究中，我们正在研究这些药物影响神经元及其连接在腹侧段区域（VTA）的机制。该大脑区域及其联系极大地牵涉到所有受滥用药物的加强和有益的作用，以及介导自然环境刺激的有意义的效果，例如食物，水等。VTA还参与了处理有关生理压力反应，情绪和情感和心理和精神警觉和精神警觉的处理信息。 由于其在这些过程中的核心作用，VTA是一个大脑区域，会导致诸如成瘾，精神胁迫障碍，临床抑郁症和精神焦虑症等疾病。 我们在VTA中设计的最新工作理解了ECB在心理刺激暴露期间调节VTA电路的机制。 许多研究表明，大麻素CB1R的拮抗作用可以阻止几种滥用药物的自我管理。 此外，全身施用CB1R拮抗剂/部分激动剂SR14171716A2，全身性尼古丁，可卡因或乙醇给药后在伏隔核（NAC）中观察到的DA浓度的增加大大降低了。 该药物还响应于奖励相关线索的提示而降低了NAC中的DA释放，这表明通过与药物滥用相关的无条件和条件刺激，ECB对中左右系统的DA很重要。 也有证据表明，直接将CB1R拮抗剂直接注入VTA可以减少周围尼古丁的奖励作用，并在大鼠全身可卡因后减少VTA中DA的释放。 我们实验室和其他人的数据还表明，ECB是从VTA中的多巴胺神经元释放的。 总的来说，这些数据表明，这些ECB在cb1rs上作用于VTA内，以增加滥用药物摄入期间NAC中DA的释放，或者在暴露于药物配对刺激期间。 尽管CB1R位于VTA内的GABA能和谷氨酸能轴突末端，但我们假设是抑制了GABA释放的抑制和通过THC抑制DA神经元的抑制作用，这是其奖励效应的原因。 我们进一步假设，滥用药物的DA神经元活性的增加导致欧洲央行，2-阿酰基二烯丙基甘油（2-AG）的释放，然后通过抑制ecb驱动的阳性反馈循环来增强DA神经元的激发。 由于这些原因，我们正在进行实验，以确定是否滥用药物会触发VTA中的欧洲央行释放，以及哪些机制可能是该行动的基础。 我们在VTA中发现的最敏感的欧洲央行功能的生理度量是突触GABAB受体受体介导的IPSC，并被电刺激激活。 这些反应受到突触前CB1R的强烈抑制，并且由于CB1R拮抗剂的存在或通过纳入二酰基甘油脂肪酶脂肪酶-alpha抑制2-AG合成而受到欧洲央行，2-AG的调节抑制； （DGL-Alpha）抑制剂THL，在整个细胞移液器中。 另外，当DA神经元瞬时去极化为火灾动作电位时，可以观察到阶段，活性依赖性的2AG释放，并且这也是由2AG释放的增加介导的。 我们最近获得的数据表明，可卡因（10 microM）抑制了VTA DA神经元中的GABA-B IPSC，并且CB1R拮抗剂AM251（2 microM）降低了这一点。可卡因的作用部分由从记录的神经元释放的2AG介导，以及周围细胞的效果，因为当细胞外应用时，THL会更强烈地阻断效果，而不是细胞内的。 这些数据表明可卡因刺激VTA中2-AG的释放，以减少GABA释放到DA神经元上。 我们假设可卡因减少可卡因的抑制作用增加了中脑DA神经元在NAC等投影区域增强DA释放的兴奋性，如体内研究中所述。 由于这种可卡因作用对于其奖励和成瘾性能可能很重要，因此我们正在执行其他实验，以确定可卡因在VTA中释放2-AG的机制。 因此，在下一个财政年度，我们将使用可卡因对脑切片中VTA DA神经元中Ca2+动力学的影响进行更直接地解决此问题，并使用遗传编码的钙指示剂（GECI）蛋白发出的荧光进行共焦成像。 我们使用Floxed Gecis GCAMP5（AAV2/1.HSYNAP.FLEX.GCAMP5G）或GCAMP6（AAV1.CAG.FLEX.GCAMP6F）61，双侧注射到THCRE表达大鼠线的VTA中。 我们在THCRE小鼠中的初步数据表明这些蛋白质表达出色，允许测量由基线Ca2+振荡触发的荧光，或者在可卡因施用期间触发的。 在大多数VTA DA神经元（80％）中，急性可卡因（10 m）降低了Ca2+荧光，并且由D2拮抗剂阻止。 但是，在成像的VTA DA神经元中，大约有20％的人观察到可卡因应用过程中Ca2+信号的显着增加，这与可卡因在体内使用可卡因的VTA DA神经元单位活性的增加一致。 我们假设可卡因诱导的这些DA神经元的活性增加会通过Ca2+依赖性机制释放2AG，并且这些细胞将通过2-AG显示出更大的抑制突触GABA。 因此，显示可卡因诱导的Ca2+信号增加的DA神经元也将显示CB1R拮抗时GABA-B IPSC的增加。为了测试这一点，我们将确定可卡因在接受AAV-GCAMP6构建体注射的THCRE大鼠中可卡因施用可卡因后，可卡因在脑切片中可卡因施用后，可卡因在大脑切片中施用可卡因后，VTA DA神经元显示了VTA DA神经元。 然后，我们将从这两组细胞中执行斑块夹记录，记录GABAB IPSC，并评估可卡因诱导的突触反应抑制的大小，并确定与中性CB1R拮抗剂（PIMSR1，1 m）中可卡因抑制的依赖性。 这些实验应在VTA中的可卡因使用与欧洲央行释放之间建立联系，并确定可卡因依赖性可能被破坏的新机制。