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，阿片类药物的复吸率很高，并且无意中过量服用 从 2001 年到 2013 年，死亡人数增加了两倍。尽管阿片类药物滥用现象普遍存在，但人们对阿片类药物滥用的了解相对较少。 长期使用时发生的神经适应。我们之前确定，长期阿片类药物暴露 引起腹侧被盖区（VTA）多巴胺（DA）神经元大小的独特变化，腹侧被盖区是大脑的关键区域 中皮质边缘奖励回路中的区域，并且在治疗后也观察到 VTA DA 体细胞大小减少。 海洛因成瘾者的尸检人体样本，表明转化相关性。此外，我们还证明了 在啮齿动物模型中，体细胞大小的变化与 VTA DA 神经元活动和奖励处理相关， 建议功能性链接。然而，进一步了解阿片引起的结构和功能 VTA DA 神经元的神经适应受到 VTA 细胞异质性和分离技术的限制 并检查 VTA DA 神经元的特定子集。例如，必须采用公正的基因组方法 识别新的分子介质仅限于整个 VTA 的均质化，其中包括 多种神经元类型，而不仅仅是 DA 细胞。此外，通过光遗传学研究已经变得越来越清楚 VTA DA 神经元本身是多样化的，因为 VTA DA 神经元的子集被不同程度地激活 奖励或厌恶的刺激取决于他们的预测目标，例如伏隔核 (NAc) 或 前额皮质（PFC）。因此，该提案旨在解决 VTA 蜂窝网络带来的限制 通过使用尖端病毒和分子技术的异质性，解决中心假设 慢性吗啡以投射特异性方式诱导 VTA DA 神经元的结构可塑性，并且 由 VTA DA 神经元的转录变化介导。目标 1 将确定慢性吗啡是否会诱发 通过注入电路特定的结构可塑性变化（体细胞大小和树突棘密度） 以 Cre 重组酶依赖性方式表达荧光蛋白的逆行腺相关载体 进入酪氨酸羟化酶 (TH)-Cre 小鼠的 NAc 和 PFC。初步数据表明基础和 吗啡诱导的 NAc 和 PFC 投射的 VTA DA 神经元之间的体细胞大小。目标 2 将使用创新的 细胞类型特异性纯化技术，翻译核糖体亲和纯化 (TRAP) 来分离 mRNA 特别是来自 VTA 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，可能介导吗啡诱导的结构和功能适应。我们的 先前的 RNA 测序数据表明，长期吗啡会诱导一种独特的基因表达模式 VTA 与可卡因相比；这些研究将确定这些变化是否是由 VTA 专门驱动的 DA 神经元。总之，这项工作预计将增进对慢性阿片类药物暴露如何改变的理解 VTA DA 神经元的结构和功能，并确定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.