Contributions of Glial Glutamate Transport and NMDA Receptors in Nicotine Relapse

胶质细胞谷氨酸转运和 NMDA 受体在尼古丁复吸中的作用

基本信息

批准号：
9301729
负责人：
Cassandra D Gipson-Reichardt
金额：
$ 2.1万
依托单位：
ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-15 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9301729
关键词：
Adult Affect Alkaloids Animals Award Biological Assay Brain Brain region Caliber Ceftriaxone Cells Cessation of life Chemosensitization Cues Data Dendritic Spines Dependency Development Down-Regulation Drug Addiction Electrophysiology (science)Exposure to Glutamate Transporter Glutamates Head Health Intravenous Learning Measures Mediating Mentors Methods Microinjections Morphology N-Methyl-D-Aspartate Receptors N-Methylaspartate National Research Service Awards Neurobiology Neuroglia Neurons Nicotine Nicotine Dependence Nucleus Accumbens Outcome Pharmaceutical Preparations Pharmacotherapy Phase Procedures Property Proteins Rattus Relapse Research Risk Factors Role Saline Self Administration Self-Administered Slice Small Interfering RNA Smoke Smoking Smoking Behavior Synapses Synaptic plasticity Techniques Tissues Tobacco Tobacco Dependence Tobacco smoking Training Treatment Efficacy Up-Regulation Vertebral column Western Blotting cigarette smoking design drug abstinence drug development drug reward innovation knock-down neurobiological mechanism neurochemistry neuropsychiatric disorder nicotine abuse novel novel therapeutics patch clamp prevent programs protein function receptor reduce tobacco use research study skills treatment group uptake

项目摘要

DESCRIPTION (provided by applicant): Nicotine abuse and addiction represents a large health liability. Indeed, cigarette smoking-related illness results in an estimated 6,000,000 deaths per year worldwide, yet 20% of adults currently smoke, and among those who attempt to quit, >90% relapse. Nicotine, the primary active alkaloid in tobacco, is self-administered by animals and produces cellular adaptations in brain regions associated with drug reward, such as the nucleus accumbens. Due to the cue dependency of smoking behavior, exposure to nicotine-associated cues is a risk factor for relapse. Here, I examine the role of glial glutamate transport and NMDA receptors in nicotine relapse vulnerability. I have found that nucleus accumbens core glutamatergic mechanisms are involved in nicotine relapse, including increased synaptic strength (measured as increased spine diameter and AMPA currents) and accompanying protein changes (including a decrease in the glial glutamate transporter, GLT1, and increases in the AMPA subunit GluA1 and NMDA subunit GluN2B). During the proposed award period, I will explore the mechanisms mediating cued nicotine reinstatement and a possible neuron-glia interaction underlying relapse vulnerability. In the K99 aims, I propose to functionally characterize nicotine-mediated down-regulation of GLT1, and to determine its role in reinstated nicotine seeking. To do this, I will learn glutamate uptake and whole cell patch clamp electrophysiology strategies, as well as employ my Western blot and intracranial microinjection skills I acquired during my F32 NRSA. I will also use antisense vivo morpholinos and ceftriaxone to examine the impact of up- or down-regulated GLT1 on reinstatement of nicotine seeking. I will employ these techniques during the R00 period to further characterize the role of GluN2B in cue-reinstated nicotine seeking, and to explore a potential neuron-glia interaction mediating nicotine relapse. During the R00 period, I will electro physiologically determine if the unregulated GluN2B receptors I found in nicotine-extinguished animals are extra synaptic and necessary for cued nicotine seeking. I will accomplish this with an innovative set of techniques including a coagonist degradation procedure using whole cell patch clamp, and administration of siRNA constructs to down regulate GluN2B in nicotine-extinguished animals to determine if normalizing this protein inhibits cued nicotine seeking in nicotine-extinguished animals. This will indicate a key role of glutamate overflow and activation of extra synaptic NMDA receptors in relapse vulnerability. Next, I will determine if restoring GluN2B with siRNA or GLT1 with ceftriaxone indirectly restores GLT1 or GluN2B, respectively, indicating a neuron-glia interaction in cue-induced nicotine relapse. Finally, I will examine if restoring GluN2B or GLT1 prevents the rapid, transient synaptic plasticity I previously found during cued nicotine reinstatement. These experiments have the potential to reveal novel neurobiological mechanisms of nicotine addiction, and could contribute to the development of novel therapeutic options aimed at reversing nicotine-induced neurobiological alterations.

描述（由申请人提供）：尼古丁滥用和成瘾会带来巨大的健康负担。事实上，全球每年约有 6,000,000 人死于与吸烟相关的疾病，但目前有 20% 的成年人吸烟，而在尝试戒烟的人群中，超过 90% 的人会复发。尼古丁是烟草中的主要活性生物碱，由动物自行施用，并在与药物奖赏相关的大脑区域（例如伏核）中产生细胞适应。由于吸烟行为的线索依赖性，接触尼古丁相关线索是复吸的危险因素。在这里，我研究了神经胶质谷氨酸运输的作用和 NMDA 受体在尼古丁复吸易感性中的作用。我发现伏核核心谷氨酸能机制与尼古丁复吸有关，包括突触强度增加（以脊柱直径和 AMPA 电流增加来衡量）和伴随的蛋白质变化（包括神经胶质谷氨酸转运蛋白 GLT1 的减少和 AMPA 的增加）亚基 GluA1 和 NMDA 亚基 GluN2B）。在拟议的奖励期间，我将探索介导提示尼古丁恢复的机制以及潜在的复发脆弱性背后的神经元-神经胶质细胞相互作用。在 K99 目标中，我建议从功能上表征尼古丁介导的 GLT1 下调，并确定其在恢复尼古丁寻求中的作用。为此，我将学习谷氨酸摄取和全细胞膜片钳电生理学策略，并运用我在 F32 NRSA 期间获得的蛋白质印迹和颅内显微注射技能。我还将使用反义体内吗啉和头孢曲松来检查上调或下调 GLT1 对恢复尼古丁寻求的影响。我将在 R00 期间使用这些技术来进一步表征 GluN2B 在提示恢复尼古丁寻找中的作用，并探索介导尼古丁复发的潜在神经元-神经胶质细胞相互作用。在 R00 期间，我将从电生理学角度确定我在尼古丁熄灭的动物中发现的不受调节的 GluN2B 受体是否是额外的突触，并且对于提示尼古丁寻找是必需的。我将通过一套创新的技术来实现这一目标，包括使用全细胞膜片钳的促效剂降解程序，以及施用 siRNA 构建体来下调尼古丁熄灭的动物中的 GluN2B，以确定标准化该蛋白质是否会抑制尼古丁熄灭的动物中提示的尼古丁寻找。这将表明谷氨酸溢出和突触外 NMDA 受体激活在复发易感性中发挥着关键作用。接下来，我将确定用 siRNA 恢复 GluN2B 或用头孢曲松恢复 GLT1 是否会分别间接恢复 GLT1 或 GluN2B，这表明提示诱导的尼古丁复吸中存在神经元-神经胶质细胞相互作用。最后，我将检查恢复 GluN2B 或 GLT1 是否会阻止我之前在提示尼古丁恢复期间发现的快速、短暂的突触可塑性。这些实验有可能揭示尼古丁成瘾的新神经生物学机制，并可能有助于开发旨在逆转尼古丁引起的神经生物学改变的新治疗方案。