Neurobiology of Psychostimulant and Opiate Addiction

精神兴奋剂和阿片成瘾的神经生物学

• 批准号: 8336484
• 负责人: Toni Shippenberg
• 金额: $ 176.1万
• 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/8336484
• 关键词: Abstinence; Address; Affect; Agonist; Amygdaloid structure; Anhedonia; Animal Model; Animals; Anxiety; Attenuated; Behavior; Behavioral; Brain; Brain Chemistry; Brain region; Calcium; Cell surface; Cells; Chronic; Cocaine; Cocaine Dependence; Consensus; Corpus striatum structure; Coupled; Cues; Data; Dopamine; Drug Addiction; Drug abuse; Drug usage; Dynorphins; Endorphins; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Environment; Equilibrium; Exposure to; G-Protein-Coupled Receptors; GABA Receptor; Genes; Glutamate Decarboxylase; Glutamates; Goals; Human; Hyperactive behavior; Image; Imaging Techniques; Immunohistochemistry; Impulsivity; Incentives; Interneurons; Label; Lead; Learning; Life; Ligands; Location; Maintenance; Medial; Mediating; Microdialysis; Moods; Morphine; Motivation; Motor Activity; Mus; N-Methyl-D-Aspartate Receptors; Neurobiology; Neurons; Neurosciences; Neurotransmitters; Nucleus Accumbens; Opiate Addiction; Opioid; Opioid Receptor; Pathogenesis; Peptides; Perfusion; Pertussis; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Physiological; Point Mutation; Prefrontal Cortex; Preparation; Presynaptic Terminals; Proteins; Psychostimulant dependence; Rattus; Receptor Activation; Recurrent disease; Regulation; Relapse; Relative (related person); Rewards; Role; Saline; Serotonin; Site; Slice; Stimulus; Synapses; Synaptic Cleft; System; Techniques; Testing; Time; Up-Regulation; Ventral Tegmental Area; Withdrawal; Work; addiction; adverse outcome; awake; brain behavior; cellular imaging; clinically relevant; cocaine exposure; craving; delta opioid receptor; dopamine transporter; dopaminergic neuron; driving behavior; drug craving; drug of abuse; dysphoria; endogenous opioids; extracellular; gamma-Aminobutyric Acid; immunoreactivity; in vivo; kappa opioid receptors; kinase inhibitor; monoamine; mu opioid receptors; neuroadaptation; neurochemistry; neuronal cell body; neurotransmission; novel; prevent; protein protein interaction; psychostimulant; receptor; receptor upregulation; response; salvinorin A; serotonin transporter; therapy development; trafficking; transmission process; tyrosyl-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-phenylalanyl-phenylalanine

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. These neuroadaptations are thought to lead to the dysregulation of behavior that characterizes addiction. Psychostimulants enhance dopamine transmission by inhibiting the dopamine transporter (DAT), a protein that clears dopamine released into the synaptic cleft. By inhibiting dopamine clearance, synaptic and extracellular neurotransmitter concentrations are increased. We previously provided evidence that synthetic k- opioid receptor agonists inhibit dopamine transmission in the nucleus accumbens and striatum by decreasing release and facilitating DAT function. At present, however, the cellular mechanism(s) mediating the interaction of kappa opioid receptors with DAT is unknown. We have used heterologous expression systems and synaptosomal preparations to address this issue. Live cell imaging of cells co-expressing the k- opioid receptor and DAT revealed that activation of k-opioid receptors by synthetic agonists produces a rapid, pertussis sensitive up-regulation of DAT function. Similar effects are observed in response to salvinorin A, a naturally occurring, high potency k- opioid receptor agonist. By examining the effects of agonists in the presence of various kinase inhibitors and TAT-peptides, we have identified a critical role of a specific kinase in mediating the k-opioid receptor upregulation of transporter function. Consistent with the presence of consensus sites for this kinase in a restricted location of the transporter and its effects on phosphorylation, we have found that truncation or single point mutation of DAT, so as to remove these sites, results in a loss of k-opioid receptor regulation of transport and decreased transporter phosphorylation. Inhibitors of this kinase microinjected into the nucleus accumbens attenuate the behavioral effects of k- opioid receptor agonists suggesting that activation of this kinase contributes to the pharmacological actions of these agents. On-going studies are examining the physiological significance of k-opioid receptor regulation of DAT regulation to mesolimbic dopamine transmission and the behavioral effects of k- ligands. K- opioid receptor agonists regulate serotonin release. However, questions exist as to whether they may also modulate serotonin transport. This question is of clinical relevance since k-opioid receptor ligands are in development for the treatment of cocaine addiction and the serotonin transporter (SERT) is a substrate of cocaine. To begin to address this issue, we developed and validated a fluorescent imaging technique which enables quantification of SERT function and trafficking in single cells and in real time. Using this technique, we have shown that k-opioid receptor agonists, down-regulate SERT function and cell surface expression. Analogous effects are observed in synaptosomal preparations of the accumbens. In contrast, to DAT regulation by k-opioid agonists, SERT regulation requires calcium and activation of CAM kinase. Using Cre-SERT and Cre-DAT mice with flox-kappa opioid receptor mice, the physiological significance of k-opioid receptor regulation of monoamine transporter regulation will be probed. Mu opioid receptors (MOR) are enriched in the ventral tegmental area (VTA). The VTA is a critical site mediating the rewarding effects of MOR agonists. MOR activation therein increases dopamine transmission in the nucleus accumbens. Morphological data suggest that MOR are located on non-dopaminergic neurons in the VTA. Intracellular recordings in slice preparations of the VTA revealed that morphine increases the firing rate of dopamine neurons but inhibits the firing rate of non-dopaminergic neurons. Although the identity of the non-dopaminergic neurons was not definitively determined, these findings led to the hypothesis that activation of MOR on GABA neurons inhibits their activity, thereby, decreasing GABA release and disinhibiting VTA dopamine neurons. However, in a slice preparation, connectivity of functional circuits is not preserved. Therefore, questions exist as to whether MOR activation affects GABA release in the awake animal. Futhermore, studies examining the functional interplay of GABA, glutamate and dopamine in the VTA are lacking. We have addressed this issue in the awake animal using microdialysis in combination with sensitive analytical techniques for the simultaneous quantification of these transmitters. Our studies show that MOR activation in the VTA increases extracellular DA concentrations. GABA concentrations were decreased whereas glutamate concentrations in the VTA were unaltered. In contrast, no change in dopamine was observed in mice lacking the gene encoding MOR. However, in these animals, basal GABA overflow was significantly increased, and glutamate overflow was decreased. These data provide the first direct demonstration of tonically active MOR systems in the VTA that regulate basal glutamatergic and GABAergic transmission in this region. We hypothesize that increased GABAergic neurotransmission following MOR deletion is due to the elimination of a tonic inhibitory influence of MOR on GABA neurons in the VTA, whereas decreased glutamatergic transmission is a consequence of intensified GABA tone on glutamatergic neurons and/or terminals. As a consequence, somatodendritic dopamine release is unaltered. These findings indicate a critical role of VTA MOR in maintaining a balance between excitatory and inhibitory inputs to dopaminergic neurons. Furthermore, they provide suggestive evidence that VTA MOR may modulate vulnerability to drugs of abuse by regulating GABA and glutamatergic inputs to dopaminergic neurons.Animals exposed to a cocaine-paired environment demonstrated an augmented locomotor activity and increased mPFC GABA levels in the cocaine-paired environment. Dual labeling of cFos and glutamic acid decarboxylase 67 (GAD67) immunoreactivity in mPFC neurons revealed significantly greater co-localization of these proteins following exposure to the cocaine-associated environment relative to pseudo-conditioned rats or rats exposed to the saline-associated environment indicating that the conditioned neurochemical response to the cocaine-paired environment is associated with activation of intrinsic mPFC GABA neurons. BLA inactivation prevented the increase in locomotor activity and the augmentation of mPFC GABA transmission produced by cue exposure. Intra-mPFC application of the AMPA/KA receptor antagonist, NBQX, produced similar effects. These findings indicate that exposure to a cocaine-associated environment increases mPFC GABA transmission by enhancing excitatory drive from the BLA and activation of AMPA/KA receptors on mPFC GABA neurons. Intra-VTA perfusion of the DOR agonist, DPDPE, increased somatodendritic and NAc DA overflow. These effects were associated with a decrease in VTA GABA overflow. Glutamate overflow was unaltered. VTA GABAA receptor blockade increased basal DA levels in the VTA and prevented the DA increase produced by DPDPE. Analogous to DPDPE, intra-VTA perfusion of the DOR antagonist, TIPP-psi, increased DA in the VTA and NAc. In contrast to the agonist, the antagonist perfusion enhanced GABA and glutamate overflow. Upon blockade of both GABA and NMDA receptors, DPDPE decreased VTA DA levels. The present findings provide the first demonstration of direct regulation of GABA and glutamate neurotransmission by DORs in the VTA.

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。这些神经适应被认为会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（DAT）来增强多巴胺的传播，这是一种清除释放到突触裂隙中的多巴胺的蛋白质。通过抑制多巴胺清除，突触和细胞外神经递质浓度增加。我们先前提供了证据表明，合成K-阿片类受体激动剂通过降低释放和促进DAT功能来抑制伏隔核和纹状体中多巴胺的传播。然而，目前，介导Kappa阿片受体与DAT的相互作用的细胞机制尚不清楚。我们已经使用异源表达系统和突触体制剂来解决此问题。共表达K-阿片类受体和DAT的细胞的活细胞成像表明，合成激动剂对K-阿片类受体的激活产生了dat功能的快速，百日咳敏感的上调。响应萨尔维诺蛋白A（一种天然存在的高效力K-阿片类药物受体激动剂）也观察到了类似的影响。通过在存在各种激酶抑制剂和TAT肽的情况下检查激动剂的作用，我们已经确定了特定激酶在介导K-阿片类受体上调转运蛋白功能中的关键作用。与该激酶在转运蛋白的受限位置及其对磷酸化的影响中的共识位点的存在一致，我们发现DAT的截断或单点突变，以消除这些位点，从而导致K-阿普病受体调节的转运和转运蛋白磷酸化的损失。这种激酶对伏隔核的这种激酶的抑制剂减弱了K-阿片类受体激动剂的行为作用，这表明该激酶的激活有助于这些药物的药理作用。正在进行的研究正在研究DAT调节对中唇多巴胺传播的K-阿片受体调节的生理意义和K-配体的行为效应。 K-阿片类药物受体激动剂调节5-羟色胺释放。但是，存在有关它们是否也可能调节5-羟色胺运输的问题。 这个问题具有临床相关性，因为K-阿片受体配体正在开发可卡因成瘾，而5-羟色胺转运蛋白（SERT）是可卡因的底物。为了开始解决这个问题，我们开发了并验证了一种荧光成像技术，该技术可以实时量化SERT功能和运输。使用这种技术，我们已经表明K-阿片类受体激动剂下调SERT功能和细胞表面表达。在伏隔族的突触体制剂中观察到类似的效应。相比之下，要由K-阿片类动力学家进行DAT调节，SERT调节需要CAM激酶的钙和激活。使用与Flox-kappa阿片受体小鼠的Cre-Sert和Cre-Dat小鼠，将探测K-阿片受体调节单胺转运蛋白调节的生理意义。 MU阿片受体（MOR）富含腹侧段区域（VTA）。 VTA是一个关键的站点，介导了MOR激动剂的奖励作用。其中的MOR激活增加了伏隔核中多巴胺的传播。形态学数据表明，MOR位于VTA中的非多巴胺能神经元上。 VTA切片制剂中的细胞内记录表明吗啡会增加多巴胺神经元的发射速率，但抑制非多巴胺能神经元的发射速率。尽管未明确确定非多巴胺能神经元的身份，但这些发现导致了以下假设：MOR对GABA神经元的激活抑制其活性，从而减少GABA释放并消除抑制VTA多巴胺神经元。但是，在切片准备中，无法保留功能电路的连通性。因此，存在有关MOR激活是否会影响醒着动物中GABA释放的问题。 Futhermore，研究了VTA中GABA，谷氨酸和多巴胺的功能相互作用的研究。我们已经使用微透析与敏感的分析技术一起解决了清醒动物的问题，以同时定量这些发射机。我们的研究表明，VTA中的MOR激活会增加细胞外DA浓度。 GABA浓度降低，而VTA中的谷氨酸浓度未改变。相反，在缺乏编码MOR的基因的小鼠中未观察到多巴胺的变化。但是，在这些动物中，基底GABA溢出显着增加，谷氨酸溢流减少了。这些数据提供了VTA中音调的MOR系统的首次直接证明，该系统调节该区域的基底谷氨酸能和GABA能传播。我们假设MOR缺失后的GABA能神经传递增加是由于消除了VTA中MOR对GABA神经元的强直性抑制作用，而谷氨酸能传播的降低是GABA张力加剧对Gaba tone的增强对谷氨酸能神经元和/或术语的结果。结果，母源性多巴胺释放并没有改变。这些发现表明，VTA MOR在维持多巴胺能神经元的兴奋性和抑制投入之间保持平衡的关键作用。此外，它们提供了暗示性的证据，表明VTA MOR可能通过调节对多巴胺能神经元的GABA和谷氨酸能输入来调节对滥用药物的脆弱性。暴露于可卡因型环境的动物表现出增强的运动活性，并增加了MPFC Gaba级别的Cocaine Enigender in Cocaine Enigender。 CFO和谷氨酸脱羧酶67（GAD67）在MPFC神经元中的免疫反应性的双重标记表明，暴露于与可卡因相关的环境相关的可卡因相关环境中，这些蛋白质的共定位明显更大，相对于伪造的大鼠或与盐水相关的环境相关的环境，表明与盐水相关的环境相关的环境，以至于与盐水相关的环境相关的环境，以使盐水与盐水相关的环境相关，以使盐水伴随着盐水序列的环境环境与固有的MPFC GABA神经元的激活有关。 BLA失活阻止了运动活性的增加和通过提示暴露产生的MPFC GABA传播的增强。 AMPA/KA受体拮抗剂NBQX的MPFC内应用施用产生了相似的作用。这些发现表明，接触可卡因相关的环境通过增强BLA的兴奋性驱动以及MPFC GABA神经元上AMPA/KA受体的激活来增加MPFC GABA的传播。 DOR激动剂DPDPE的VTA灌注增加了体内源性和NAC DA溢出。这些影响与VTA GABA溢出的减少有关。谷氨酸溢流没有改变。 VTA GABAA受体阻滞增加了VTA中的基础DA水平，并防止了DPDPE产生的DA增加。 类似于DPDPE，DOR拮抗剂TIPP-PSI的VTA内部灌注增加了VTA和NAC中的DA。与激动剂相反，拮抗剂灌注增强了GABA和谷氨酸溢流。在阻断GABA和NMDA受体时，DPDPE降低了VTA DA水平。目前的发现提供了DOR在VTA中直接调节GABA和谷氨酸神经传递的首次演示。