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.

精神兴奋剂会增加伏隔核和前额皮质中神经递质多巴胺的传递。这种作用有助于这些药物的奖励作用和药物滥用的开始。持续吸毒后，在伏核、前额皮质和前额皮质纹状体环路（控制激励动机、学习和冲动的环路）的其他区域内观察到大脑化学物质的持久变化。这些神经适应被认为会导致成瘾特征的行为失调。精神兴奋剂通过抑制多巴胺转运蛋白（DAT）来增强多巴胺传输，多巴胺转运蛋白是一种清除释放到突触间隙的多巴胺的蛋白质。通过抑制多巴胺清除，突触和细胞外神经递质浓度增加。我们之前提供的证据表明，合成的 k-阿片受体激动剂通过减少释放和促进 DAT 功能来抑制伏隔核和纹状体中的多巴胺传递。然而，目前介导κ阿片受体与DAT相互作用的细胞机制尚不清楚。我们使用异源表达系统和突触体制剂来解决这个问题。共表达 k-阿片受体和 DAT 的细胞的活细胞成像揭示，合成激动剂对 k-阿片受体的激活产生快速、百日咳敏感性的 DAT 功能上调。在对 Salvinorin A 的反应中观察到类似的效果，salvinorin A 是一种天然存在的高效 k-阿片受体激动剂。通过检查激动剂在各种激酶抑制剂和 TAT 肽存在下的作用，我们确定了特定激酶在介导 k-阿片受体转运蛋白功能上调中的关键作用。与该激酶在转运蛋白的限制位置中存在共有位点及其对磷酸化的影响一致，我们发现 DAT 的截断或单点突变，以去除这些位点，导致 k-阿片类药物的损失转运的受体调节和转运蛋白磷酸化的降低。将该激酶的抑制剂显微注射到伏隔核中，减弱了k-阿片受体激动剂的行为效应，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 k-阿片受体调节 DAT 调节对中脑边缘多巴胺传递的生理意义以及 k-配体的行为效应。 K-阿片受体激动剂调节血清素的释放。然而，关于它们是否也可以调节血清素运输仍存在疑问。 这个问题具有临床意义，因为 k-阿片受体配体正在开发用于治疗可卡因成瘾，而血清素转运蛋白 (SERT) 是可卡因的底物。为了开始解决这个问题，我们开发并验证了一种荧光成像技术，该技术能够实时量化单细胞中的 SERT 功能和运输。使用这种技术，我们已经证明 k-阿片受体激动剂可以下调 SERT 功能和细胞表面表达。在伏隔核的突触体制剂中观察到类似的效果。相比之下，与 k-阿片类激动剂对 DAT 的调节不同，SERT 调节需要钙和 CAM 激酶的激活。使用 Cre-SERT 和 Cre-DAT 小鼠与 flox-kappa 阿片受体小鼠，将探讨 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) 富含于腹侧被盖区 (VTA)。 VTA 是调节 MOR 激动剂奖赏效应的关键部位。其中 MOR 的激活增加了伏隔核中的多巴胺传递。形态学数据表明，MOR 位于 VTA 中的非多巴胺能神经元上。 VTA 切片制备物的细胞内记录显示，吗啡会增加多巴胺神经元的放电率，但会抑制非多巴胺能神经元的放电率。尽管非多巴胺能神经元的身份尚未明确确定，但这些发现得出这样的假设：GABA 神经元上的 MOR 激活会抑制其活性，从而减少 GABA 释放并解除对 VTA 多巴胺神经元的抑制。然而，在切片制备中，功能电路的连接性未被保留。因此，关于 MOR 激活是否影响清醒动物中 GABA 的释放存在疑问。此外，还缺乏检查 VTA 中 GABA、谷氨酸和多巴胺功能相互作用的研究。我们已经使用微透析结合灵敏的分析技术在清醒的动物中解决了这个问题，以同时量化这些递质。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 中谷氨酸浓度未改变。相比之下，在缺乏编码 MOR 的基因的小鼠中，没有观察到多巴胺的变化。然而，在这些动物中，基础 GABA 溢出显着增加，而谷氨酸溢出减少。这些数据首次直接证明了 VTA 中的强直活性 MOR 系统调节该区域的基础谷氨酸能和 GABA 能传递。我们假设MOR缺失后GABA能神经传递增加是由于MOR对VTA中GABA神经元的强直抑制影响的消除，而谷氨酸能传递减少是谷氨酸能神经元和/或末梢上GABA张力增强的结果。因此，体细胞树突多巴胺的释放没有改变。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性输入之间的平衡中发挥着关键作用。此外，他们提供了暗示性证据，表明 VTA MOR 可能通过调节 GABA 和多巴胺能神经元的谷氨酸输入来调节对药物滥用的脆弱性。暴露于可卡因配对环境的动物在可卡因配对环境中表现出运动活性增强和 mPFC GABA 水平增加。 mPFC 神经元中 cFos 和谷氨酸脱羧酶 67 (GAD67) 免疫反应性的双重标记显示，相对于假条件大鼠或暴露于盐水相关环境的大鼠，暴露于可卡因相关环境后这些蛋白质的共定位显着增强，表明对可卡因配对环境的条件性神经化学反应与内在 mPFC GABA 神经元的激活有关。 BLA 失活阻止了运动活动的增加以及线索暴露产生的 mPFC GABA 传输的增强。 mPFC 内应用 AMPA/KA 受体拮抗剂 NBQX 产生了类似的效果。这些发现表明，暴露于可卡因相关环境可通过增强 BLA 的兴奋性驱动和 mPFC GABA 神经元上 AMPA/KA 受体的激活来增加 mPFC GABA 传输。 VTA 内灌注 DOR 激动剂 DPDPE，增加体细胞树突和 NAc DA 溢出。这些效应与 VTA GABA 溢出的减少有关。谷氨酸溢出未改变。 VTA GABAA 受体阻断增加了 VTA 中的基础 DA 水平，并阻止了 DPDPE 产生的 DA 增加。 与 DPDPE 类似，VTA 内灌注 DOR 拮抗剂 TIPP-psi，可增加 VTA 和 NAc 中的 DA。与激动剂相反，拮抗剂灌注增强了 GABA 和谷氨酸溢出。在阻断 GABA 和 NMDA 受体后，DPDPE 降低了 VTA DA 水平。目前的研究结果首次证明了 VTA 中的 DOR 对 GABA 和谷氨酸神经传递的直接调节。