Retinoic acid, homeostatic plasticity and cocaine craving

项目摘要 我们将利用‘渴望的孵化’模型来研究治疗可卡因成瘾的一个主要挑战，即 即使在长期禁欲之后，对线索诱发的复发的脆弱性仍然存在。在这个模型中，大鼠 在退出扩展通道后，线索诱导的渴求逐渐增强(孵化) 可卡因自我管理。人类也会产生渴望的潜伏期。在此之前，我们展示了钙离子- 通透性AMPAR(CP-AMPAR；同源GluA1)聚集在中等棘神经元的突触中 停药30天后，对伏核(NAC)核(NAC核)进行核团核团(MSN)的定位。此后，需要激活它们 用来表达培养出来的可卡因欲望。因此，了解CP-AMPAR的可塑性可能会提供关于 渴望的强度是如何被调节的。我们实验室和其他实验室的研究表明，可卡因的戒断 与多种适应相关，这些适应预测NAC MSN中活动或钙信号的减少。然而，几乎没有 已知这可能与CP-AMPAR插入有关。有希望的候选人是一种动态平衡 涉及维甲酸(RA)信号的可塑性。对海马神经元的研究表明，钙水平 与正在进行的突触传递相关的蛋白足以抑制RA的合成。然而，在一段时间后， 在不活跃的情况下，RA合成是不受抑制的。这增加了GluA1的翻译，实现了体内平衡的增加 通过插入同源GluA1 CP-AMPAR增强突触强度。CP-AMPAR在NAC岩芯中的积累 在渴望的孵化过程中，同样可能涉及到钙离子的减少(见上文)，我们最近证明了它是 与增加的GluA1翻译有关。基于这些与RA介导的动态平衡可塑性的相似性 在海马体和其他数据中，我们假设可卡因的戒断与钙的降低有关 MSN中的信号，导致RA合成解除抑制，增加GluA1翻译和增加突触 同源GluA1 CP-AMPAR水平。目标1将测试兴奋性传递与类风湿关节炎之间的关系 在培养的NAC MSN(与皮质细胞共培养以恢复谷氨酸输入)中的合成和GluA1翻译。 目标2将使用光纤光度法来验证NAC核心MSN中的基线钙在 可卡因戒断，尽管在线索诱导的寻找测试中可能会短暂增加，这种增加可能 随着渴望的孵化发生而增加。在D1或A2a中表达Cre重组酶的转基因大鼠 受体阳性的MSN(A2a是D2 MSN的标志)将使这两个MSN群体得以研究。在《目标3》中， D1NAC或A2aNAC核心MSN的全细胞膜片钳记录将确定急性操作 RA信号改变CP-AMPAR水平以及是否涉及蛋白质翻译。然后，我们将确定敲门是否 可卡因戒断过程中NAC核心中一种关键的RA合成酶的下降可减少CP-AMPAR的积聚 以及长期戒断后潜伏的渴求的维持。相反，我们会击倒这种酶 降解生理盐水大鼠的RA，以确定RA的长期增加是否足以增加CP-AMPAR。 总体而言，我们的研究将使用最先进的工具来测试一个概念上新的关于渴望孵化的假说。

Project Summary We will use the `incubation of craving' model to study a major challenge in treating cocaine addiction, that is, the persistence of vulnerability to cue-induced relapse even after long periods of abstinence. In this model, rats exhibit progressive intensification (incubation) of cue-induced craving after withdrawal from extended-access cocaine self-administration. Incubation of craving also occurs in humans. Previously, we showed that Ca2+- permeable AMPARs (CP-AMPARs; homomeric GluA1) accumulate in synapses on medium spiny neurons (MSN) of the nucleus accumbens (NAc) core after ~30 days of withdrawal. Thereafter, their activation is required for expression of incubated cocaine craving. Thus, understanding CP-AMPAR plasticity may provide clues about how the intensity of craving is regulated. Work from our lab and others has shown that cocaine withdrawal is associated with multiple adaptations that predict reduced activity or Ca2+ signaling in NAc MSN. However, little is known about how this might be linked to CP-AMPAR insertion. A promising candidate is a form of homeostatic plasticity involving retinoic acid (RA) signaling. Work in hippocampal neurons has shown that Ca2+ levels associated with ongoing synaptic transmission are sufficient to suppress RA synthesis. However, after a period of inactivity, RA synthesis is disinhibited. This increases GluA1 translation, enabling a homeostatic increase in synaptic strength via insertion of homomeric GluA1 CP-AMPARs. CP-AMPAR accumulation in the NAc core during incubation of craving may likewise involve reduced Ca2+ (see above) and we recently showed that it is associated with increased GluA1 translation. Based on these similarities to RA-mediated homeostatic plasticity in hippocampus and on other data, we hypothesize that cocaine withdrawal is associated with decreased Ca2+ signaling in MSN, leading to disinhibition of RA synthesis, increased GluA1 translation and increased synaptic levels of homomeric GluA1 CP-AMPARs. Aim 1 will test the relationship between excitatory transmission, RA synthesis, and GluA1 translation in cultured NAc MSN (co-cultured with cortical cells to restore glutamate inputs). Aim 2 will use fiber photometry to test the hypothesis that baseline Ca2+ in NAc core MSN is reduced during cocaine withdrawal, although it may transiently increase during cue-induced seeking tests and this increase may be augmented as incubation of craving occurs. Transgenic rats expressing Cre recombinase in D1 or A2a receptor-positive MSN (A2a is a marker for D2 MSN) will enable both MSN populations to be studied. In Aim 3, whole-cell patch clamp recordings in identified D1 or A2a NAc core MSN will determine if acute manipulation of RA signaling alters CP-AMPAR levels and if protein translation is involved. Then, we will determine if knocking down a critical RA synthetic enzyme in NAc core during cocaine withdrawal attenuates CP-AMPAR accumulation and maintenance of incubated craving after long withdrawal. Conversely, we will knock down the enzyme that degrades RA in saline rats to determine if a long-term increase in RA is sufficient to increase CP-AMPARs. Overall, our studies will use state-of-the-art tools to test a conceptually novel hypothesis for incubation of craving.