Role for novel ventral tegmental area neuromedin S neurons in morphine responses

总结 虽然阿片类药物依赖和成瘾继续构成主要的健康和经济负担，但我们的 对潜在神经生物学的有限理解限制了更好的诊断和干预。失调 中皮质边缘奖励回路的作用被认为与药物成瘾的各个方面有关， 腹侧被盖区（VTA）多巴胺（DA）神经元活动和输出的改变， 有助于毒品使用的回报方面。然而，这些变化背后的分子机制 在VTA中，DA功能仍然相对未被探索。因此，我们的首要目标是确定分子 腹侧被盖区DA神经元介导药物奖赏长期变化的机制， 吸毒成瘾。为此，我们采用翻译核糖体亲和纯化（TRAP）技术， 在慢性吗啡暴露后小鼠腹侧被盖区DA神经元中特异性发生的表达变化。 虽然我们在输入区和腹侧被盖区发现吗啡引起的基因表达变化数量大致相同， 我们的RNA测序分析发现这些数据集之间几乎没有重叠，强调了 这种方法对于确定腹侧被盖区DA神经元中的机制的重要性，因为这些机制可能在整体上被掩盖， 组织分析。我们已经通过qPCR验证了基因表达的变化，有趣的是，我们发现表达 的数量的神经肽，而不是传统上描述的腹侧被盖区强烈诱导吗啡暴露。 神经介肽S（NMS）是特别感兴趣的，因为它在腹侧被盖区DA神经元中富集，并且其表达在腹侧被盖区DA神经元中。 在慢性吗啡暴露后显著增加。虽然NMS神经元的作用已经在 视交叉上核参与昼夜节律的调节，NMS在此结构之外的功能一直没有得到 特征，使其成为研究的高度新颖的基因。已知NMS与之结合的主要受体 神经介肽U受体2（NMUR 2）在腹侧被盖区DA神经元的靶区域内表达， 核（nucleus nucleus）。然而，是否所有VTA DA神经元都表达NMS，以及它们的潜力， 功能影响尚待确定。因此，在本申请中，我们寻求表征表达式， 共表达NMS的VTA DA神经元通过完成三个特定目标的功能影响：1）鉴定 表达NMS的腹侧被盖区DA能神经元及其投射部位，2）决定是否调制腹侧被盖区NMS 神经元活动改变吗啡引起的行为，以及3）确定VTA DA神经元中的NMS敲除是否改变 吗啡引起的行为我们的研究将利用细胞类型特异性CRISPR介导的缺失来操纵 这些新的VTA NMS表达神经元的输出，以评估它们在成年小鼠中的功能作用，沿着 转基因小鼠和DREADD来鉴定和改变VTA-NMS神经元活性。关键是，我们新的初步调查 数据表明腹侧被盖区-核管理系统神经元的激活会影响吗啡引起的行为，支持这一假设 和可行性预计这些研究将为今后调查特定VTA的作用奠定基础， DANMS电路，它们在行为过程中的活动，以及它们作为治疗干预靶点的潜力。

Summary Although opioid dependence and addiction continue to constitute a major health and economic burden, our limited understanding of the underlying neurobiology limits better diagnostics and interventions. Dysregulation of the mesocorticolimbic reward circuit is acknowledged to contribute to various aspects of drug addiction, with alteration in the activity and output of dopamine (DA) neurons in the ventral tegmental area (VTA) known to contribute to the rewarding aspects of drug use. However, the molecular mechanisms underlying these changes in VTA DA function remain relatively unexplored. Therefore, our overarching goal is to identify molecular mechanisms in VTA DA neurons mediating long-term changes in drug reward that contribute to the persistence of drug addiction. To this end, we used translating ribosome affinity purification (TRAP) to identify gene expression changes in mice that specifically occur in VTA DA neurons following chronic morphine exposure. While we found approximately the same number of gene expression changes by morphine in our input and VTA DA fractions, our RNA sequencing analysis found very little overlap between these datasets emphasizing the importance of this approach to identify mechanisms in VTA DA neurons as these are likely obscured in whole tissue analyses. We have validated gene expression changes via qPCR and intriguingly, we find that expression of number of neuropeptides not traditionally described in the VTA are robustly induced by morphine exposure. Neuromedin S (NMS) was of particular interest as it was enriched in VTA DA neurons, and its expression was robustly increased following chronic morphine exposure. While a role for NMS neurons has been described in the suprachiasmatic nucleus in circadian regulation, NMS function outside this structure has not been characterized, making it a highly novel gene of study. It is known that the primary receptor with which NMS interacts, neuromedin U receptor 2 (NMUR2), is expressed within the target regions of VTA DA neurons, such as the nucleus accumbens (NAc). However, whether all VTA DA neurons express NMS, and their potential functional impact has yet to be determined. Thus, in this application we seek to characterize the expression and functional impact of VTA DA neurons that co-express NMS via completion of three specific aims: 1) identify the VTA DA neurons that express NMS and their projection sites, 2) determine whether modulation of VTA NMS neuronal activity alters morphine-elicited behavior, and 3) determine if NMS knockout in VTA DA neurons alters morphine-elicited behavior. Our studies will utilize cell type-specific CRISPR-mediated deletion to manipulate the output of these novel VTA NMS-expressing neurons to assess their functional role in adult mice along with transgenic mice and DREADDs to identify and alter VTA-NMS neuron activity. Critically, our new preliminary data suggest that activation of VTA-NMS neurons affects morphine-elicited behavior, supporting the premise and feasibility. These studies are expected to set the stage for future work investigating the role of specific VTA- DANMS circuits, their activity during behavior, and their potential as targets for therapeutic intervention.