Retinoic acid, homeostatic plasticity and cocaine craving

项目摘要 我们将使用"渴望潜伏期"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，仍然容易受到线索诱导的复发。在这个模型中，大鼠 在从扩展访问中退出后，表现出线索诱导的渴望的进行性强化（潜伏期） 可卡因自我管理人类也会产生渴望。之前，我们发现Ca2 +- 渗透性AMPAR（CP-AMPAR;同型GluA1）在中型棘神经元的突触中积累 (MSN)停药约30天后，观察到核小体（NAc）核心的变化。此后，需要激活它们 来表达对可卡因的渴望因此，了解CP-AMPAR可塑性可能会提供线索， 渴望的强度是如何调节的我们实验室和其他实验室的工作表明， 与预测NAc MSN中活性降低或Ca2+信号传导的多种适应相关。不过小 已知这可能与CP-AMPAR插入有关。一个有希望的候选人是一种形式的自我平衡 涉及视黄酸（RA）信号传导的可塑性。海马神经元的研究表明， 与正在进行的突触传递相关的突触素足以抑制RA的合成。但经过一段时间 不活动时，RA合成被解除抑制。这增加了GluA1的翻译，使体内稳态增加， 通过插入同源GluA1 CP-AMPAR增强突触强度。CP-AMPAR在NAc核心中的累积 在孵化的渴望可能同样涉及减少钙（见上文），我们最近表明， 与GluA1翻译增加有关。基于这些与RA介导的稳态可塑性的相似性 在海马和其他数据中，我们假设可卡因戒断与Ca2+减少有关 MSN中的信号传导，导致RA合成的去抑制，增加GluA1翻译和增加突触 同源GluA1 CP-AMPAR水平。目的1将测试兴奋性传递、RA 合成和GluA1翻译（与皮质细胞共培养以恢复谷氨酸输入）。 目标2将使用光纤光度法来检验NAc核心MSN中的基线Ca2+在 可卡因戒断，虽然它可能会短暂增加线索诱导寻求测试，这种增加可能 随着渴望的孵化而增强。在D1或A2a中表达Cre重组酶的转基因大鼠 受体阳性MSN（A2a是D2MSN的标志物）将使两个MSN群体都能够被研究。在目标3中， 在已鉴定的D1或A2a NAc核心MSN中的全细胞膜片钳记录将确定是否急性操纵 RA信号改变CP-AMPAR水平，如果蛋白质翻译参与。然后，我们将确定是否敲门 可卡因戒断过程中NAc核心中一种关键的RA合成酶的下降减弱了CP-AMPAR的蓄积 以及长期戒断后对潜伏的渴望的维持。相反地，我们会敲除 在盐水大鼠中降解RA，以确定RA的长期增加是否足以增加CP-AMPAR。 总的来说，我们的研究将使用最先进的工具来测试一个概念上新颖的渴望孵化假设。

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.