Contributions of Glial Glutamate Transport and NMDA Receptors in Nicotine Relapse

描述（由申请人提供）：尼古丁滥用和成瘾是一个很大的健康责任。事实上，全世界每年估计有600万人死于与吸烟有关的疾病，但目前有20%的成年人吸烟，而在那些试图戒烟的人中，超过90%的人复发。尼古丁是烟草中的主要活性生物碱，由动物自我给药，并在与药物奖赏相关的大脑区域（如延髓核）产生细胞适应性。由于吸烟行为的线索依赖性，暴露于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的作用， 和NMDA受体在尼古丁复吸易感性中的作用。我发现，尼古丁复吸中涉及到神经核的核心突触能机制，包括突触强度增加（以棘直径和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GLT 1减少，AMPA亚基GluA 1和NMDA亚基GluN 2B增加）。在拟议的奖励期间，我将探讨机制介导线索尼古丁复吸和可能的神经胶质细胞相互作用的基础复发的脆弱性。在K99的目标，我建议功能特点尼古丁介导的下调GLT 1，并确定其在恢复尼古丁寻求的作用。为此，我将学习谷氨酸摄取和全细胞膜片钳电生理学策略，以及运用我在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GLT 1对尼古丁寻求恢复的影响。我将在R 00期间采用这些技术，以进一步表征GluN 2B在线索恢复尼古丁寻求中的作用，并探索潜在的神经元-胶质细胞相互作用介导尼古丁复发。在R 00期间，我将从电生理学的角度来确定我在尼古丁熄灭的动物中发现的不受调节的GluN 2B受体是否是突触外的，是否是线索尼古丁寻求所必需的。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凝血因子降解程序，以及给予siRNA构建体来下调尼古丁熄灭动物中的GluN 2B，以确定正常化这种蛋白质是否会抑制尼古丁熄灭动物中的尼古丁寻求。这将 表明谷氨酸溢出和突触外NMDA受体激活在复发脆弱性中的关键作用。接下来，我将确定用siRNA恢复GluN 2B或用头孢曲松恢复GLT 1是否分别间接恢复GLT 1或GluN 2B，这表明线索诱导的尼古丁复发中的神经元-神经胶质相互作用。最后，我将研究恢复GluN 2B或GLT 1是否会阻止我以前在尼古丁恢复过程中发现的快速，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诱导的神经生物学改变的新治疗方案。

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.