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重大和日益严重的挑战。分布情况： 腹侧被盖区(VTA)向伏核(NAC)和内侧核的多巴胺传出 前额叶皮质(MPFC)被认为影响药物滥用的易感性。此外， 成年期早期中皮质投射的持续成熟使其更容易受到危险因素的影响 例如，在青春期接触滥用药物。Netrin1/DCC介导的信令已作为一种 VTA多巴胺回路形成和成熟的关键调节因子。然而，在降低小鼠的DCC活性的同时 研究表明，携带单等位基因DCC突变的人有更多的中皮质投射 大脑皮层连通性降低。这一显著的差异反映了关于如何 DCC信号引导VTA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发现了通过过度表达来抑制和提高DCC功能的新的遗传方法 独特的受体变异体。结合了新的遗传方法、先进的全脑成像和实时 活体多巴胺记录，我们将检验轴突靶向中皮质边缘的中枢假说 途径影响滥用药物的功能反应。具体地说，在这个试点研究中，我们将调查 DCC信号的双向变化如何改变中皮质连接，特别是在电路过程中 通过青春期的成熟。我们将确定解剖和功能的变化，并将 这些变化与小鼠的添加相关行为有关。 目的1：确定DCC活性的变化如何影响中皮质轴突连接。 使用Cre-loxP系统，我们将通过过度表达不同的DCC亚型来抑制或提升DCC信号 特别是在VTA多巴胺神经元和青少年发育期间。我们将跟踪生成的VTA轴突 用tdTomato记者在完整的大脑投影，使用高速高分辨率带状扫描共焦 组织清除后的显微镜检查。完成这一目标将使我们能够比较双向变化如何 DCC信号影响中脑皮层回路的成熟。它还将有助于区分不同的可能性，这些可能性可能 导致小鼠和人类之间在中脑皮质连接性方面的差异。 目的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信号的双向变化，我们还将 确定mPFC中多巴胺释放的相应变化及其对动物行为的影响。 使用dLight1.3b荧光指示器和纤维光度法，我们将跟踪实时的多巴胺动态 在条件性位置偏好测试中的mPFC。这将有助于确定改变的功能结果 中皮质环路的成熟和描绘多巴胺环路的解剖-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.