Synaptic mechanisms maintaining persistent cocaine craving.

描述(由申请人提供)：迫切需要预防戒毒可卡因使用者因线索而复发的治疗方法。在持续复发易感性的大鼠模型中，延长可卡因自我给药导致线索诱导的可卡因渴求的依赖于戒断的“孵化”。停药1个月后，孵化依赖于伏核(NAC)突触强度的增加，这种增加是由高电导可渗透钙的AMPAR(CP-AMPAR)的持续升高和I组代谢性谷氨酸受体(MGluR)功能的相关变化所介导的。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。它是 众所周知，树突含有AMPAR亚单位和其他突触蛋白的mRNAs，这些mRNAs的树突状翻译(由NMDARs和mGluRs控制)对可塑性至关重要。然而，对于正常情况下突触传递是否调节NAC中的翻译，或者滥用药物是否会导致NAC或任何大脑区域中翻译的持续变化，目前尚不清楚。通过关注这些基本但尚未探索的问题，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)“孵化大鼠”NAC中的突触适应依赖于翻译的失调；2)这种失调是由于对蛋白质翻译的抑制调的丧失，通常由自发的NMDAR传递(MINI)提供，并由此导致钙内流；以及3)这种抑制调的丧失反映了含有GluN3的NMDAR水平的增加，这种NMDAR具有非常低的钙通透性。我们有三个目标。首先，我们将使用脑片中的代谢标记来确定孵育后蛋白质或关键蛋白质(GluA1或Arc)的整体基础翻译速率是否增加，以及NMDAR和mGluR1/5对翻译的调节是否发生改变。相关问题将在培养的NAC神经元中进行研究，这是一个可以明确测量树突翻译的系统。其次，阵列断层扫描和包埋后免疫金电子显微镜将被用来确定AMPAR亚单位、NMDAR亚单位和I组mGluRs在单个突触水平的表达和共定位。我们假设，在“孵化的大鼠”中，相对于GluA2富含GluA1的NAC突触，表明CP-AMPAs，也将富含GluN3。第三，全细胞膜片钳记录将确定孵育是否与NAC中含有GluN3的NMDAR对突触传递的贡献增加有关，以及刺激mGluR1是否也去除了突触中的CP-AMPAR，是否也去除了GluN3-NMDAR。总体而言，我们正在使用最先进的方法测试创新假设，其中一些方法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是一个可行的治疗靶点，以及最近有趣的证据表明，改变的蛋白质翻译是氯胺酮快速抗抑郁作用的基础，我们的发现将对旨在使各种大脑疾病的异常蛋白质翻译正常化的研究具有广泛的意义。

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.