Synaptic mechanisms maintaining persistent cocaine craving.

描述（由申请人提供）：迫切需要治疗，以防止戒断可卡因使用者的线索诱导复发。在持续复发脆弱性的大鼠模型中，长期接触可卡因自我给药导致线索诱导的可卡因渴望的戒断依赖性“孵化”。在>1个月的戒断后，潜伏期取决于由高电导Ca 2+渗透性AMPAR（CP-AMPAR）的非常持续的升高和I组代谢型谷氨酸受体（mGluR）功能的相关变化介导的丘脑核（NAc）中突触强度的增加。我们的目标是确定这些长期适应的维持是否依赖于NAc中蛋白质翻译的失调。是 树突含有AMPAR亚基和其他突触蛋白的mRNA，这些mRNA的树突翻译（由NMDAR和mGluR控制）对于可塑性至关重要。然而，目前还不清楚突触传递是否在正常情况下调节NAc的翻译，或者滥用药物是否会在NAc或任何大脑区域的翻译中产生持续的改变。通过关注这些基本的尚未探索的问题，我们将推进我们对正常NAc功能以及可卡因成瘾的理解。我们的中心假设是：1）“孵育大鼠”的NAc中的突触适应依赖于失调的翻译; 2）这种失调是由于通常由自发NMDAR传递（minis）提供的对蛋白质翻译的抑制性张力的丧失以及由此产生的Ca 2+内流;以及3）这种抑制性张力的丧失反映了含GluN 3的NMDAR的水平增加，其具有非常低的Ca 2+渗透性。我们有三个目标。首先，我们将使用脑切片中的代谢标记来确定孵育后蛋白质降解的总体基础速率或关键蛋白质（GluA 1或Arc）的翻译是否增加，以及NMDAR和mGluR 1/5的翻译调节是否改变。相关的问题将在培养的NAc神经元中进行研究，在该系统中可以明确地测量树突翻译。第二，阵列断层扫描和植入后免疫金电子显微镜将被用来定义在单个突触水平的AMPAR亚基，NMDAR亚基，和I组mGluRs的表达和共定位。我们假设，NAc突触在“孵育大鼠”，是丰富的GluA 1相对于GluA 2，指示CP-AMPAR，也将丰富的GluN 3。第三，全细胞膜片钳记录将确定孵育是否与含GluN 3的NMDAR对NAc中突触传递的贡献增加有关，以及从突触中去除CP-AMPAR的mGluR 1刺激是否也去除了GluN 3-NMDAR。总的来说，我们正在使用最先进的方法测试创新的假设，其中一些从未应用于成瘾研究。此外，鉴于脆性X研究已经确定异常蛋白质翻译为可行的治疗靶点，并且最近有有趣的证据表明，改变的蛋白质翻译是氯胺酮快速抗抑郁作用的基础，我们的研究结果将对旨在使各种脑部疾病中的异常蛋白质翻译正常化的研究具有广泛意义。

DESCRIPTION (provided by applicant): Treatments to prevent cue-induced relapse in abstinent cocaine users are urgently needed. In a rat model of persistent relapse vulnerability, extended access cocaine self-administration leads to a withdrawal-dependent "incubation" of cue-induced cocaine craving. After >1 month of withdrawal, incubation depends on increased synaptic strength in the nucleus accumbens (NAc) mediated by a very persistent elevation of high conductance Ca2+-permeable AMPARs (CP-AMPARs) and by related changes in group I metabotropic glutamate receptor (mGluR) function. Our objective is to determine if maintenance of these long-lasting adaptations depends on dysregulation of protein translation in the NAc. It is well established that dendrites contain mRNAs for AMPAR subunits and other synaptic proteins, and dendritic translation of these mRNAs (controlled by NMDARs and mGluRs) is critical for plasticity. However, nothing is known about whether synaptic transmission regulates translation in the NAc under normal conditions or if drugs of abuse produce persistent alterations in translation in the NAc or in any brain region. By focusing on these fundamental yet unexplored questions, we will advance our understanding of normal NAc function as well as cocaine addiction. Our central hypothesis is: 1) synaptic adaptations in the NAc of "incubated rats" depend upon dysregulated translation; 2) this dysregulation is due to loss of inhibitory tone on protein translation normally provided by spontaneous NMDAR transmission (minis) and the resulting Ca2+ influx; and 3) loss of this inhibitory tone reflects increased levels of GluN3-containing NMDARs, which have very low Ca2+ permeability. We have 3 Aims. First, we will use metabolic labeling in brain slices to determine if the overall basal rate of protein translatio or the translation of key proteins (GluA1 or Arc) increases after incubation, and if NMDAR and mGluR1/5 regulation of translation is altered. Related questions will be studied in cultured NAc neurons, a system in which dendritic translation can be unequivocally measured. Second, array tomography and postembedding immunogold electron microscopy will be used to define expression and colocalization at the single synapse level of AMPAR subunits, NMDAR subunits, and group I mGluRs. We hypothesize that NAc synapses in "incubated rats" that are enriched for GluA1 relative to GluA2, indicative of CP-AMPARs, will also be enriched for GluN3. Third, whole-cell patch clamp recordings will determine if incubation is associated with an increased contribution of GluN3-containing NMDARs to synaptic transmission in the NAc, and whether mGluR1 stimulation, which removes CP-AMPARs from synapses, also removes GluN3-NMDARs. Overall, we are testing innovative hypotheses using state-of-the-art methods, several of which have never been applied to addiction research. Furthermore, given that fragile X research has identified aberrant protein translation as a viable therapeutic target, and given intriguing recent evidence that altered protein translation underlies the rapid antidepressant effect of ketamine, our findings will have broad significance for research aimed at normalizing aberrant protein translation in a variety of brain disorders.