Contributions of Glial Glutamate Transport and NMDA Receptors in Nicotine Relapse

项目摘要/摘要 尼古丁滥用和上瘾是一项巨大的健康负担。事实上，与吸烟有关的疾病 据估计，全世界每年有600万人死亡，但目前有20%的成年人吸烟，而且 那些试图戒烟的人，90%会复发。尼古丁是烟草中的主要活性生物碱，是自行给药的。 并在与药物奖励相关的大脑区域产生细胞适应，例如 伏隔核。由于吸烟行为的线索依赖性，接触与尼古丁相关的线索是 复发的风险因素。在这里，我研究了神经胶质谷氨酸转运和NMDA受体在尼古丁中的作用。 故态复萌。我发现伏核的核心谷氨酸能机制参与了 尼古丁复发，包括突触强度增加(测量为脊柱直径和AMPA增加 电流)和伴随的蛋白质变化(包括胶质谷氨酸转运体GLT1的减少， AMPA亚基GluA1和NMDA亚基GluN2B增加)。在建议的获奖期内，我 将探索介导线索尼古丁恢复和可能的神经元-神经胶质细胞相互作用的机制 潜在的复发脆弱性。在K99目标中，我建议从功能上描述尼古丁介导的 下调GLT1，并确定其在恢复尼古丁寻求中的作用。为了做到这一点，我将学习 谷氨酸摄取和全细胞膜片钳电生理策略，以及使用我的Western印迹 以及我在F32 NRSA期间学到的颅内显微注射技能。我也会用反义活体 吗啡和头孢曲松研究上调或下调GLT1对恢复的影响 尼古丁寻求者。我将在R00期间使用这些技术来进一步描述 GluN2B在线索恢复的尼古丁寻找中的作用，并探索一种潜在的神经元-神经胶质细胞相互作用介导尼古丁 旧病复发。在R00期间，我将电生理学地确定上调的GluN2B受体是否 在尼古丁熄灭的动物中发现的是突触外的，是寻找尼古丁线索所必需的。这就做 通过一套创新的技术来实现这一点，包括使用WALL的共生生物脱脂过程 细胞膜片钳和应用siRNA构建下调尼古丁熄灭后的GluN2B 以确定使这种蛋白正常化是否会抑制尼古丁熄灭动物的尼古丁寻找线索。 这将表明谷氨酸溢出和突触外NMDA受体激活在复发中起关键作用 脆弱性。接下来，我将确定使用siRNA恢复GluN2B或使用头孢曲松恢复GLT1是否间接恢复 GLT1或GluN2B，分别表明在线索诱导的尼古丁复发中神经元-胶质细胞相互作用。最后，我会 检查恢复GluN2B或GLT1是否会阻止我之前在 提示尼古丁恢复。这些实验有可能揭示新的神经生物学。 尼古丁成瘾的机制，并可能有助于开发新的治疗方案 逆转尼古丁引起的神经生物学改变。

Project Summary/Abstract 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 electrophysiologically determine if the upregulated GluN2B receptors I found in nicotine-extinguished animals are extrasynaptic and necessary for cued nicotine seeking. I will accomplish this with an innovative set of techniques including a coagonist degredation procedure using whole cell patch clamp, and administration of siRNA constructs to downregulate 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 extrasynaptic 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.