Retinoic acid signaling and incubation of cocaine craving

项目摘要 治疗药物滥用的一个主要问题是持续的易复发，即使在长期的 禁欲。在可卡因渴求复吸模型的孵育中，大鼠经历了延长的可卡因自我获得 行政管理。在戒断过程中，大鼠表现出线索诱导的可卡因逐渐增强(孵化)。 渴望。我们已经证明钙(Ca~(2+))-通透性AMPA受体(CP-AMPAR)是由 的GluA1亚基，在戒断后期积聚在伏核(NAC)，此后 是表达潜伏的线索诱导的渴望所必需的。因此，理解调节机制 CP-AMPAR的积累和维持可能产生新的治疗靶点，以减少渴求和 延长禁欲时间。我们实验室和其他实验室的研究表明，可卡因的戒断与 预测NAC中棘神经元(MSN)钙水平降低的多重适应。然而，几乎没有什么是 已知可能将撤药期间这种减少的活动与随后插入的 CP-AMPAR。一个有希望的候选涉及维甲酸(RA)信号级联。研究项目： 海马神经元已经证明，与基础突触传递相关的钙水平是 足以抑制RA合成。然而，在一段时间的不活动之后，RA的合成受到抑制，导致 增加GluA1的翻译和同源GluA1 CP-AMPAR的突触插入。CP-AMPAR 在孵化过程中NAC的积聚同样可能涉及活性的降低，这已经在 我们未发表的研究与GluA1的翻译增加有关。基于这些与RA的相似性- 诱导突触伸缩，我的中心假设是可卡因孵育导致NAC活动下降 神经元在基线水平，RA合成增加，CP-AMPAR的比例增加。目标1将确定是否存在钙离子 在可卡因渴求的孵化过程中，NAC核心的水平会发生变化。我将使用光纤光度法来 测量延长戒断的早期和晚期细胞内钙离子的变化，这是神经活动的一个相关性 获得可卡因(或生理盐水)自我给药。我将在基线和暴露于 与可卡因有关的线索。目标2将确定RA是否有助于CP-AMPAR在NAC中的蓄积 可卡因孵育大鼠的核心MSN。可卡因孵育脑片的全细胞膜片钳记录 或生理盐水大鼠将被用来确定对RA信号的药理学操作是否改变了AMPAR介导的 NAc中棘神经元的突触传递。AIM 3将确定RA是否介导突触伸缩 在培养的NAC MSN中。我将使用RA报告系统来确认RA在MSN中的表达是活性的- 依赖和药理学方法来确定CP-AMPAR的RA依赖的伸缩是否发生在 NAC神经元。在这些研究进行期间，我将参与一个多方面的培训计划，以制定 非替补席技能需要达到我在学术环境中成为PI的目标。

Project Summary A major problem in treating drug abuse is persistent vulnerability to relapse, even after long periods of abstinence. In the incubation of cocaine craving model of relapse, rats undergo extended access to cocaine self- administration. During withdrawal, rats exhibit a progressive intensification (incubation) of cue-induced cocaine craving. We have shown that calcium (Ca2+)-permeable AMPA receptors (CP-AMPAR), comprised exclusively of the GluA1 subunit, accumulate in the nucleus accumbens (NAc) during late withdrawal and thereafter are required for the expression of incubated cue-induced craving. Therefore, understanding mechanisms regulating CP-AMPAR accumulation and maintenance may yield novel therapeutic targets for reducing craving and prolonging abstinence. Work from our lab and others have shown that cocaine withdrawal is associated with multiple adaptations that predict reduced Ca2+ levels in NAc medium spiny neurons (MSN). However, little is known about mechanisms that may link this reduced activity during withdrawal to the subsequent insertion of CP-AMPARs. One promising candidate involves the retinoic acid (RA) signaling cascade. Studies in hippocampal neurons have demonstrated that Ca2+ levels associated with basal synaptic transmission are sufficient to suppress RA synthesis. However, following a period of inactivity, RA synthesis is disinhibited, leading to increased translation of GluA1 and synaptic insertion of homomeric GluA1 CP-AMPARs. CP-AMPAR accumulation in the NAc during incubation may likewise involve reduced activity and has been demonstrated in our unpublished studies to be associated with increased translation of GluA1. Based on these similarities to RA- induced synaptic scaling, my central hypothesis is that cocaine incubation leads to a decrease in activity in NAc neurons at baseline, increased RA synthesis, and scaling up of CP-AMPARs. Aim 1 will determine if Ca2+ levels are altered in the NAc core during incubation of cocaine craving. I will use fiber photometry to measure changes in intracellular Ca2+, a correlate of neural activity, in early and late withdrawal from extended access to cocaine (or saline) self-administration. I will measure Ca2+ at baseline and during exposure to a cocaine-associated cue. Aim 2 will determine whether RA contributes to CP-AMPAR accumulation in NAc core MSN of cocaine incubated rats. Whole-cell patch clamp recordings in brain slices from cocaine incubated or saline rats will be used to determine if pharmacological manipulation of RA signaling alters AMPAR-mediated synaptic transmission in NAc medium spiny neurons. Aim 3 will determine if RA mediates synaptic scaling in cultured NAc MSN. I will use a RA reporter system to confirm that RA expression in MSN is activity- dependent, and pharmacological methods to determine whether RA-dependent scaling of CP-AMPAR occurs in NAc neurons. While these studies are underway, I will participate in a multi-faceted training plan to develop the non-bench skills needed to reach my goal of becoming a PI in an academic setting.