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

摘要 尽管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了解有限，限制了更好的诊断和干预。调控失调 中皮质边缘奖赏环路的活动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变化 对毒品使用的有益方面作出贡献。然而，这些变化背后的分子机制 在VTA中，DA的功能还相对未被研究。因此，我们的首要目标是识别分子 VTA DA神经元介导药物奖赏长期变化的机制有助于持续 吸毒成瘾。为此，我们使用翻译核糖体亲和纯化(TRAP)技术来鉴定基因 慢性吗啡暴露后小鼠VTA DA神经元中特异性表达的变化。 虽然我们在我们的输入和VTA中发现了大约相同数量的吗啡引起的基因表达变化 DA组分，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 这种方法对确定VTA DA神经元的机制的重要性，因为这些机制可能是整体模糊的 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 许多传统上没有在VTA中描述的神经肽都是由吗啡暴露引起的。 神经递质S(NeuroMedin NMS，NMS)因其丰富的VTA DA神经元而引起人们的特别关注，其表达 在慢性吗啡暴露后显著增加。虽然NMS神经元的作用已在 视交叉上核在昼夜节律调节中，NMS的功能在这个结构之外一直没有得到 特点，使其成为一个高度新奇的研究基因。已知NMS与其主要受体 神经介素U受体2(NMUR2)在VTA DA神经元的靶区表达，如 作为伏隔核(NAC)。然而，是否所有的VTA DA神经元都表达NMS，以及它们的潜力 功能影响尚未确定。因此，在本申请中，我们寻求表征表达式和 通过完成三个特定目标共同表达NMS的VTA DA神经元的功能影响：1)确定 VTA DA神经元表达NMS及其投射部位，2)决定VTA NMS是否调制 神经元活动改变吗啡诱导的行为，以及3)确定VTA DA神经元中的NMS基因敲除是否改变 吗啡诱导的行为。我们的研究将利用特定细胞类型的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 转基因小鼠和DREADD以鉴定和改变VTA-NMS神经元活性。关键的是，我们新的初选 数据表明，VTA-NMS神经元的激活影响了吗啡诱导的行为，支持了这一前提 和可行性。这些研究有望为未来研究特定VTA的作用奠定基础。 DANMS回路，它们在行为过程中的活动，以及它们作为治疗干预目标的潜力。

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.