VTA dopamine connectivity and functional responses to drugs of abuse

摘要 药物使用和成瘾对公共卫生构成日益严重的挑战。的分布 多巴胺从腹侧被盖区（VTA）传入延髓核（NAc）和内侧核 前额叶皮质（mPFC）被假设影响药物滥用的倾向。此外该 中皮层投射持续成熟到成年早期使其更易受危险因素影响 例如在青春期接触滥用药物。Netrin 1/DCC介导的信号传导已经成为一种新的信号传导途径。 腹侧被盖区多巴胺回路形成和成熟的关键调节因子。然而，在降低小鼠DCC活性的同时， 显示增加中皮质投射，发现携带单等位基因DCC突变的人具有 中皮层连接性降低这一明显的差异反映了在知识上的巨大差距， DCC信号引导腹侧被盖区多巴胺轴突靶向，进而影响成瘾相关行为。 我们最近的研究已经确定了新的遗传手段，通过过度表达DCC抑制和提高DCC功能， 不同的受体变体。结合新的遗传方法，先进的全脑成像，以及实时 体内多巴胺记录，我们将测试中心假设，轴突靶向中皮质边缘 途径影响对药物滥用的功能反应。在这项试点研究中，我们将调查 DCC信号的双向变化如何改变中皮层连接，特别是在回路中 通过青春期的成熟。我们将确定解剖和功能变化， 这些变化与小鼠的加法相关行为有关。 目的1：确定DCC活动的变化如何影响皮质中轴突连接。 使用Cre-loxP系统，我们将通过过表达不同的DCC亚型来抑制或提高DCC信号传导 特别是腹侧被盖区多巴胺神经元和青少年发育期间。我们将追踪由此产生的腹侧被盖区轴突 使用高速高分辨率带式扫描共聚焦显微镜， 组织清理后的显微镜检查。这一目标的完成将使我们能够比较 DCC信号影响中皮层回路的成熟。这也将有助于区分不同的可能性， 导致小鼠和人类之间关于中皮层连接的差异。 目标2.确定DCC活性的变化如何影响行为过程中mPFC中的多巴胺释放 对滥用药物的反应。 使用与目标1相同的遗传方法来诱导DCC信号传导的双向变化，我们还将 确定mPFC中多巴胺释放的相应变化以及对动物行为的影响。 使用dLight1.3b荧光指示剂和纤维光度法，我们将跟踪多巴胺的实时动态。 条件性位置偏爱测试中的mPFC。这将有助于确定改变的功能结果。 中皮层回路成熟和描绘多巴胺回路中的解剖结构关系。

Abstract Substance use and addiction pose significant and increasing challenges to public health. The distribution of dopamine efferents from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and to the medial prefrontal cortex (mPFC) has been hypothesized to influence the predisposition to drug abuse. In addition, the continued maturation of mesocortical projection into early adulthood renders it more susceptible to risk factors such as exposure to drugs of abuse during adolescence. Netrin1/DCC-mediated signaling has emerged as a key regulator of VTA dopamine circuit formation and maturation. However, while reducing DCC activity in mice is shown to increase mesocortial projection, humans carrying monoallelic DCC mutations are found to have reduced mesocortial connectivity. This notable discrepancy reflects a significant gap in knowledge as to how DCC signaling directs VTA dopamine axon targeting and in turn influences addiction-related behaviors. Our recent study has identified novel genetic means to dampen and elevate DCC function by overexpressing distinct receptor variants. Combining the new genetic approach, advanced whole brain imaging, and real-time in vivo dopamine recording, we will test the central hypothesis that axon targeting in the mesocorticolimbic pathway influences the functional responses to drugs of abuse. Specifically in this pilot study, we will investigate how bidirectional changes in DCC signaling alters mesocortical connection, particularly during the circuit maturation through adolescence. We will determine both anatomical and functional changes and also correlate these changes with addition-related behaviors in mice. Aim 1: Determine how changes in DCC activity affects mesocortical axon connection. Using the Cre-loxP system, we will dampen or elevate DCC signaling by overexpressing distinct DCC isoforms specifically in VTA dopamine neurons and during adolescent development. We will follow the resulting VTA axon projection with a tdTomato reporter in the intact brain, using high-speed high-resolution ribbon scanning confocal microscopy after tissue clearing. Completion of this aim will allow us to compare how bidirectional changes in DCC signaling impact mesocortical circuit maturation. It will also help distinguish different possibilities that could contribute to the discrepancy between mice and humans regarding mesocortical connectivity. Aim 2. Determine how changes in DCC activity affects dopamine release in the mPFC during behavioral responses to drugs of abuse. Using the same genetic approach to induce bidirectional changes in DCC signaling as in Aim 1, we will also determine the corresponding alterations in dopamine release in the mPFC and the effects on animal behaviors. Using dLight1.3b fluorescent indicator and fiber photometry, we will follow real-time dopamine dynamics in the mPFC during conditioned place preference testing. This will help determine the functional outcomes of altered mesocortical circuit maturation and delineate the anatomical-structural relationship in the dopamine circuit.