Determination of morphological and molecular adaptations in ventral tegmental area dopamine neurons by chronic morphine

鸦片类药物滥用在美国是一种流行病，在美国，鸦片类药物的复发率很高，而且无意中过量服用 从2001年到2013年，死亡人数增加了两倍。尽管鸦片类药物滥用盛行，但人们对此知之甚少。 因长期使用而发生的神经适应。我们之前确定慢性阿片类药物暴露 导致腹侧被盖区(VTA)的多巴胺(DA)神经元的大小发生独特的变化，VTA是大脑的关键区域 脑缺血后，VTA DA胞体大小也明显减小。 海洛因成瘾者的尸检样本，提示与翻译相关。此外，我们还展示了 在啮齿动物模型中，胞体大小的变化与VTA DA神经元活动和奖赏处理相关， 暗示了一种功能上的联系。然而，对阿片类药物诱导的结构和功能的进一步理解 VTA细胞的异质性和分离技术限制了VTA DA神经元的神经适应 并观察VTA DA神经元的特定亚群。例如，无偏见的基因组方法对于 识别新的分子介质仅限于使整个VTA均质，这包括 多种神经元类型，不只是DA细胞。此外，通过光遗传学研究，这一点变得越来越清楚。 VTA DA神经元本身是不同的，因为VTA DA神经元的亚群是由 奖赏或厌恶刺激取决于其投射目标，例如伏隔核(NAC)或 前额叶皮质(PFC)因此，本提案旨在解决VTA蜂窝网络所带来的限制 通过使用尖端病毒和分子技术的异质性，解决了中心假设 慢性吗啡以投射特异性方式诱导VTA DA神经元结构可塑性 由VTA DA神经元的转录变化所介导。目标1将确定慢性吗啡是否会导致 结构可塑性(胞体大小和树突棘密度)的电路特异性变化 以Cre重组酶依赖的方式表达荧光蛋白的逆转腺相关载体 进入酪氨酸羟化酶(TH)-Cre小鼠的NAC和PFC。初步数据显示，在基础和 吗啡诱导NAC和PFC投射的VTA DA神经元之间的胞体大小。Aim 2将使用一种创新的 细胞类型特异性纯化技术，翻译核糖体亲和纯化(TRAP)以分离mRNA 并评估候选基因的基因表达变化，以及新的 通过RNA测序，可能介导吗啡诱导的结构和功能适应的基因。我们的 先前的RNA测序数据表明，慢性吗啡诱导了一种独特的基因表达模式 VTA与可卡因的比较；这些研究将确定这种变化是否特定地由VTA驱动 DA神经元。总而言之，这项工作有望推进对长期接触阿片类药物如何改变的理解。 VTA DA神经元的结构和功能及其在中皮质边缘的新机制 电路功能障碍。

Opiate abuse is an epidemic in the US, where opiates have a high relapse rate, and unintentional overdose deaths have tripled from 2001 to 2013. Despite the prevalence of opiate abuse, relatively little is known about the neuroadaptations that occur with chronic use. We previously determined that chronic opiate exposure induces a unique change in the size of dopamine (DA) neurons in the ventral tegmental area (VTA), a key brain region in the mesocorticolimbic reward circuit, and decreased VTA DA soma size was also observed in post- mortem human samples of heroin addicts, suggesting translational relevance. Moreover, we have demonstrated that changes in soma size are correlated with VTA DA neuronal activity and reward processing in rodent models, suggesting a functional link. However, further understanding of opiate-induced structural and functional neuroadaptations of VTA DA neurons has been limited by VTA cellular heterogeneity and techniques to isolate and examine specific subsets of VTA DA neurons. For example, unbiased genomic approaches necessary to identify novel molecular mediators have been limited to homogenization of the entire VTA, which includes multiple neuron types, not just DA cells. Further, it has become increasingly clear through optogenetic studies that VTA DA neurons themselves are diverse, as subsets of VTA DA neurons are differentially activated by rewarding or aversive stimuli depending on their projection target, e.g. the nucleus accumbens (NAc) or prefrontal cortex (PFC). Thus, this proposal seeks to address the limitations stemming from VTA cellular heterogeneity through the use of cutting-edge viral and molecular techniques, addressing the central hypothesis that chronic morphine induces structural plasticity in VTA DA neurons in a projection-specific manner and is mediated by transcriptional changes in VTA DA neurons. Aim 1 will determine whether chronic morphine induces circuit-specific changes in structural plasticity (soma size and dendritic spine density) through injection of retrograde adeno-associated vectors that express fluorescent proteins in a Cre-recombinase dependent manner into the NAc and PFC of tyrosine hydroxylase (TH)-Cre mice. Preliminary data suggest differences in basal and morphine-induced soma size between NAc- and PFC-projecting VTA DA neurons. Aim 2 will use an innovative cell type-specific purification technique, Translating Ribosome Affinity Purification (TRAP) to isolate mRNA specifically from VTA DA neurons and assess gene expression changes in candidate genes, as well as novel genes via RNA-sequencing, that may mediate morphine-induced structural and functional adaptations. Our previous RNA-sequencing data have shown that chronic morphine induces a unique gene expression pattern in the VTA compared to cocaine; these studies will determine whether such changes are driven specifically by VTA DA neurons. Together, this work is expected to advance the understanding of how chronic opiate exposure alters the structure and function of VTA DA neurons and to identify novel mechanisms underlying mesocorticolimbic circuit dysfunction.