Neurobiology of Psychostimulant and Opiate Addiction

精神兴奋剂和阿片成瘾的神经生物学

基本信息

批准号：
7733834
负责人：
Toni Shippenberg
金额：
$ 97.41万
依托单位：
NATIONAL INSTITUTE ON DRUG ABUSE
依托单位国家：
美国
项目类别：
财政年份：
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/7733834
关键词：
Abstinence Acute Address Affect Affinity Agonist Alkaloids Analgesics Anhedonia Animal Model Anxiety Attenuated Behavior Behavioral Binding Biochemical Biotinylation Brain Brain Chemistry Brain region Cell surface Cells Chronic Clinical Cocaine Condition Corpus striatum structure Dependence Depressed mood Development Dopamine Dopamine D2 Receptor Dopamine Receptor Dorsal Drug Addiction Drug abuse Drug usage Dynorphins Endorphins Enkephalins Exhibits Exocytosis Failure G-Protein-Coupled Receptors Genes Genus Mentha Glutamates Goals Habits Human Image Imaging Techniques Impulsivity In Vitro Incentives Iodides Lead Learning Left Life Ligands MAP Kinase Gene MAPK14 gene Maintenance Mediating Microdialysis Mitogen-Activated Protein Kinase 3 Monitor Moods Morphine Motivation Mus Mutation Nerve Neurobiology Neurons Neurosciences Neurotransmitters Nucleus Accumbens Opiate Addiction Opiates Opioid Opioid Receptor Pain Pathogenesis Perfusion Pertussis Toxin Pharmaceutical Preparations Phase Phosphatidylinositols Phosphorylation Phosphotransferases Play Prefrontal Cortex Presynaptic Terminals Proteins Quinpirole Receptor Activation Receptor Signaling Recruitment Activity Recurrent disease Regulation Relapse Relative (related person)Rewards Role Serotonin Spinal Cord Stimulus Synapses Synaptic Cleft System Techniques Testing Thinking Time Tissues Tribes Withdrawal Work addiction base behavioral sensitization cellular imaging conditioning delta opioid receptor dopamine D3 receptor dopamine transporter dopaminergic neuron driving behavior drug craving drug of abuse dysphoria extracellular in vivo monoamine mu opioid receptors neuroadaptation neurochemistry neuronal cell body neurotransmission novel prevent protein protein interaction psychostimulant receptor research study response salvinorin A serotonin transporter time use transmission process uptake

项目摘要

Psychostimulants increase transmission of the neurotransmitter, dopamine, in the nucleus accumbens and prefrontal cortex. This action contributes to the rewarding effects of these agents and the initiation of drug abuse. Following continued drug use, enduring changes in brain chemistry are observed within the nucleus accumbens, prefrontal cortex and other regions of the prefrontal-cortico-striatal loop, a circuit that controls incentive motivation, learning and impulsivity. Increasing evidence suggests that these neuroadaptations lead to the dysregulation of behavior that characterizes addiction. Psychostimulants enhance dopamine transmission by inhibiting the dopamine transporter, a protein that clears dopamine released into the synaptic cleft. By inhibiting dopamine clearance, synaptic and extracellular neurotransmitter concentrations are increased. We and others have shown that D2 and D3 dopamine receptors receptors regulate extracellular dopamine concentrations in the nucleus accumbens and striatum. Extracellular dopamine concentrations are decreased in response to D2/D3 receptor agonists and increased in response to antagonists. Our in-vivo and in-vitro studies provided evidence that D2/D3 receptors regulate extracellular dopamine concentrations by affecting both transporter mediated uptake and release. Due to the lack of ligands selective for these receptor sub-types and the presence of both D2 and D3 receptors in the brain regions examined, the role of each in the regulation of dopamine dynamics could not be delineated. Using live-cell imaging techniques, our studies have provided the first direct demonstration that D2 receptors regulate dopamine transporter activity in heterologous expression systems. We monitored dopamine transporter function in real time using the high affinity, fluorescent dopamine transporter substrate, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). Addition of D2/D3 agonists (e.g., quinpirole, PD128907) to cells co-expressing these proteins induced a rapid and concentration-dependent increase in ASP+ accumulation. The increase in uptake was pertussis toxin-sensitive indicating a Gi/Go dependent mechanism. D2 receptor activation induced phosphorylation of extracellular related kinase (ERK1/2) and Akt, a major target of phosphoinositide 3-kinase (PI3K), confirming that the D2 receptor signals through these kinases. ERK 1/2 inhibition prevented the effects of D2/D3 receptor agonists on ASP+ accumulation. PI3K inhibition was without effect indicating that D2 receptor stimulation upregulates DAT function via an ERK1/2-dependent and PI3K-independent mechanism. Biochemical and imaging studies revealed that the dopamine transporter and D2 receptor are in close physical proximity providing a cellular basis for the interaction of these two proteins. Mutation studies revealed that the N-terminus of the dopamine transporter is not required for D2/DAT interactions. We have completed studies assessing the role of D3 receptors in DAT regulation. In cells co-expressing D3 receptors and the dopamine transporter, D3 receptor activation rapidly increased ASP+ uptake. D3 receptor stimulation, like D2 receptor stimulation, activated ERK 1/2 and PI3K. In contrast, however, to D2 receptor stimulation, inhibition of either kinase prevented the increase in uptake evoked by D3 receptor activation. Inhibition of other kinases (e.g. p38 MAPK) was without effect. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression as well as increased dopamine transporter exocytosis. In contrast, following prolonged D3 receptor activation, dopamine transporter function is down-regulated and transporter cell surface expression is reduced. K-opioid receptor (KOPr) agonists decrease extracellular dopamine concentrations in the nucleus accumbens and dorsal striatum. Acute administration of the potent KOPr agonist, salvinorin A, an alkaloid found in the mint leaf and used by Indian tribes for its psychotomimetic actions, produces similar effects. We previously showed that synthetic KOPr agonists decrease dopamine concentrations in the nucleus accumbens by increasing dopamine transporter function and inhibiting release. Using the technique of no-net flux microdialysis under transient conditions we examined whether local perfusion of salvinorin A produces similar effects in the striatum. In contrast to what we have observed in the nucleus accumbens, we found no effect of salvinorin A on dopamine uptake. However, dopamine release was markedly depressed. Although these findings suggest that salvinorin A may not modulate dopamine uptake, our ex-vivo studies demonstrate marked changes in striatal uptake following brief incubation (e.g., 5 min) of striatal tissue with this KOPragonist or synthetic agonists. Therefore, we hypothesize that the failure to observe regulation of uptake in-vivo may be due to the longer duration of receptor activation used in in-vivo studies. On-going studies are examining this issue since identification of the mechanisms by which salvinorin modulates dopamine transmission is important for understanding the increase in recreational use of this opiate as well as its psychotomimetic and dysphoric effects. Cocaine binds with high affinity to the dopamine transporter as well as to other monoamine transporters. Using heterologous systems we have found that KOPr agonists also modulate the serotonin transporter. In contrast, however to the dopamine transporter, KOPr agonist regulation of the serotonin transporter varies as a function of the duration of receptor activation. Whereas transient receptor activation increases serotonin transporter function, more prolonged receptor activation decreases function. These effects are observed in response to synthetic agonists and in response to salvinorin A. Our previous studies have shown that KOPr agonists attenuate several behavioral and neurochemical effects of cocaine. We hypothesize that the cocaine-antagonist-like effects of these agents result at least in part from their ability to regulate dopamine and serotonin transport. On-going studies seek to elucidate the cellular mechanisms by which KOPr systems regulate monoamine transporters and whether blockade of this regulation attenuates the cocaine-antagonist like actions of KOPr agonists. The repeated use of morphine and other mu opioid receptor (MOPr)agonists leads to tolerance and dependence. These effects limit the clinical use of opiates in the treatment of pain and are also thought to play an important role in the maintenance of opiate addiction, once developed. The repeated intermittent administration of morphine can result in an enhancement of its behavioral effects (e.g., sensitization), a phenomenon implicated in drug-craving. Studies in the spinal cord have suggested that delta opioid receptors (DOPr) may contribute to the development of analgesic tolerance and to opiate dependence. Whether DOPr play a role in mediating the rewarding effects of opiates or the development of sensitization is unknown. We addressed this issue using mice which lack the gene encoding the DOPr. Our studies revealed that mice lacking DOPr exhibit an augmentation of context-dependent sensitization and that tolerance is reduced relative to wildtype mices. The conditioned rewarding effects of morphine were also reduced in these mice reduced and similar findings were obtained in response to pharmacological blockade of DOPr in wildtype mice. These findings indicate that the endogenous DOPr system is recruited in response to both repeated and chronic morphine administration and that this recruitment serves an essential function in the development of tolerance, behavioral sensitization, and the conditioning of opiate reward.

精神刺激物增加了伏隔核和前额叶皮层中神经递质，多巴胺的传播。这项行动有助于这些药物的有益影响和滥用药物的启动。持续使用药物后，在伏隔核，前额叶皮层和前额叶皮质 - 纹状体环的其他区域内观察到脑化学的持久变化，该电路控制了激励动机，学习和冲动。越来越多的证据表明，这些神经适应导致表征成瘾的行为失调。精神刺激剂通过抑制多巴胺转运蛋白来增强多巴胺的传播，多巴胺转运蛋白可以清除释放到突触裂隙中的多巴胺。通过抑制多巴胺清除，突触和细胞外神经递质浓度增加。我们和其他人表明，D2和D3多巴胺受体受体调节伏隔核和纹状体中细胞外多巴胺浓度。响应D2/D3受体激动剂，细胞外多巴胺浓度降低，并响应拮抗剂而增加。我们的体内和体外研究提供了证据，表明D2/D3受体通过影响转运蛋白介导的摄取和释放来调节细胞外多巴胺浓度。由于缺乏对这些受体亚型选择性的配体，并且在检查的大脑区域中都存在D2和D3受体的存在，因此无法描述每个受体的大脑区域中每个受体的子类型的作用。使用活细胞成像技术，我们的研究提供了第一个直接证明D2受体在异源表达系统中调节多巴胺转运蛋白活性。我们使用高亲和力，荧光多巴胺转运蛋白底物，4-（4-二乙基氨基酯）-n-甲基吡啶胺（ASP+）实时监测多巴胺转运蛋白的功能。将D2/D3激动剂（例如奎因螺激而PD128907）添加到共表达这些蛋白质的细胞中，诱导了ASP+积累的快速浓度依赖性增加。摄取的增加是百日咳毒素敏感的，表明GI/GO依赖性机制。 D2受体激活诱导细胞外相关激酶（ERK1/2）和AKT的磷酸化，这是磷酸肌醇3-激酶（PI3K）的主要靶标，证实了通过这些激酶的D2受体信号。 ERK 1/2抑制阻止了D2/D3受体激动剂对ASP+积累的影响。 PI3K抑制无效，表明D2受体刺激通过ERK1/2依赖性和PI3K独立的机制上调DAT功能。生化和成像研究表明，多巴胺转运蛋白和D2受体处于近距离接近，为这两种蛋白质相互作用提供了细胞基础。突变研究表明，D2/DAT相互作用不需要多巴胺转运蛋白的N末端。我们已经完成了评估D3受体在DAT调节中的作用的研究。在共表达D3受体和多巴胺转运蛋白的细胞中，D3受体激活迅速增加了ASP+摄取。 D3受体刺激，例如D2受体刺激，激活ERK 1/2和PI3K。但是，相反，对于D2受体刺激，抑制任何一种激酶，都阻止了D3受体激活引起的摄取的增加。抑制其他激酶（例如p38 MAPK）无效。生物素化实验表明，摄取的迅速增加与细胞表面增加和细胞内表达降低以及多巴胺转运蛋白胞吐作用有关。相反，随后D3受体激活，多巴胺转运蛋白的功能被下调，转运蛋白细胞表面表达降低。 K-阿片受体（KOPR）激动剂降低了伏伏核和背纹状体中细胞外多巴胺浓度。在薄荷叶中发现的生物碱salvinorin A的急性给药，印度部落用于其精神病作用，会产生相似的作用。我们先前表明，合成的Kopr激动剂通过增加多巴胺转运蛋白的功能和抑制释放来降低伏隔核中多巴胺的浓度。使用瞬态条件下的无网络通量微透析技术，我们检查了萨氏素蛋白A的局部灌注是否在纹状体中产生相似的作用。与我们在伏隔核中观察到的相反，我们发现萨尔维诺蛋白A对多巴胺的摄取没有影响。但是，多巴胺释放显着抑郁。尽管这些发现表明萨尔维诺蛋白A可能不会调节多巴胺的摄取，但我们的前体研究表明，在短暂孵育（例如5分钟）与该kopragonistist或合成激动剂的短暂孵育（例如5分钟）之后，纹状体摄取发生了明显变化。因此，我们假设未观察到体内摄取的调节可能是由于体内研究中使用的受体激活持续时间较长。正在进行的研究正在研究此问题，因为鉴定了萨维那诺蛋白调节多巴胺传播的机制对于理解这种阿片类药物的娱乐使用及其精神病和烦躁不安的效果的增加至关重要。可卡因与多巴胺转运蛋白以及其他单胺转运蛋白具有高亲和力结合。使用异源系统，我们发现Kopr激动剂还调节5-羟色胺转运蛋白。但是，对于多巴胺转运蛋白而言，羟色胺转运蛋白的Kopr激动剂调节随被受体激活持续时间而变化。瞬时受体激活增加了5-羟色胺转运蛋白的功能，而延长的受体激活会降低功能。这些作用是对合成激动剂的响应观察到的，并响应萨维那诺素A。我们的先前研究表明，Kopr激动剂会衰减可卡因的几种行为和神经化学作用。我们假设这些药物的类似可卡因抗抗酸剂的作用至少部分是由于它们调节多巴胺和5-羟色胺转运的能力。正在进行的研究旨在阐明KOPR系统调节单胺转运蛋白以及该调节的阻断是否会减弱可卡因 - 抗抗糖剂的作用，例如Kopr激动剂的作用。吗啡和其他MU阿片受体（MOPR）激动剂的反复使用导致耐受性和依赖性。这些效果限制了阿片类药物在治疗疼痛中的临床用途，并且一旦开发，也被认为在维持鸦片成瘾中起着重要作用。反复间歇性地给出吗啡可能会导致其行为效应（例如敏化）的增强，这是一种与药物抗药有关的现象。脊髓的研究表明，三角洲阿片受体（DOPR）可能有助于镇痛耐受性的发展和鸦片依赖性。 DOPR是否在介导鸦片的奖励作用或敏化的发展中发挥作用，尚不清楚。我们使用缺乏编码DOPR的基因的小鼠解决了这个问题。我们的研究表明，缺乏DOPR的小鼠表现出对上下文依赖性敏化的增强，并且相对于野生型使用，耐受性会降低。在这些小鼠中，还降低了吗啡的条件奖励作用，并响应于野生型小鼠的DOPR的药理阻断而获得了相似的发现。这些发现表明，内源性DOPR系统是针对重复和慢性吗啡给药的响应而募集的，并且该募集在耐受性，行为敏化和阿片反应条件的发展中起着至关重要的作用。