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

阿片类药物滥用在美国是一种流行病，阿片类药物的复发率很高， 从2001年到2013年，死亡人数增加了两倍。尽管阿片类药物滥用普遍存在，但人们对阿片类药物的使用知之甚少。 长期使用会产生的神经适应。我们之前确定长期接触阿片类药物 诱发腹侧被盖区（VTA）多巴胺（DA）神经元大小的独特变化， 在中皮质边缘奖赏回路中，也观察到VTA DA索马体大小减少。 海洛因成瘾者的尸体样本，这表明翻译相关性。此外，我们已经证明， 在啮齿动物模型中，索马大小的变化与腹侧被盖区DA神经元活动和奖赏处理相关， 这表明存在功能性联系然而，进一步了解阿片诱导的结构和功能 腹侧被盖区DA神经元的神经适应受到腹侧被盖区细胞异质性和分离技术的限制， 检查腹侧被盖区DA神经元的特定子集例如，公正的基因组方法， 鉴定新的分子介质仅限于整个VTA的均质化，其包括 多种类型的神经元，而不仅仅是DA细胞此外，通过光遗传学研究， 腹侧被盖区DA神经元本身是多样的，因为腹侧被盖区DA神经元的子集被不同的激活， 奖励或厌恶刺激，取决于它们的投射目标，例如丘脑核（NAc）或 前额叶皮层（PFC）。因此，该提议寻求解决VTA蜂窝网络所产生的限制。 通过使用尖端的病毒和分子技术，解决中心假设 慢性吗啡以投射特异性方式诱导腹侧被盖区DA神经元的结构可塑性， VTA DA神经元的转录变化介导的。目的1将确定慢性吗啡是否诱导 通过注射，在结构可塑性（索马大小和树突棘密度）的电路特异性变化 以Cre重组酶依赖性方式表达荧光蛋白的逆行腺相关载体 酪氨酸羟化酶（TH）-Cre小鼠的NAc和PFC。初步数据表明，基础和 吗啡诱导的索马体在投射NAc和PFC的VTA DA神经元之间的大小。Aim 2将采用创新的 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 特别是从腹侧被盖区DA神经元，并评估候选基因的基因表达变化，以及新的 基因通过RNA测序，这可能介导吗啡诱导的结构和功能适应。我们 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与可卡因相比;这些研究将确定这种变化是否是由VTA专门驱动的 DA神经元。总之，这项工作有望促进对慢性阿片类药物暴露如何改变 腹侧被盖区DA能神经元结构和功能，并确定新的机制， 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.