Investigating non-canonical mechanisms of endogenous opioids on motivation in dorsal midbrain

项目摘要 本R01的主要目标是确定内源性阿片类药物在脑内的计算和功能作用。 特定的中脑背侧核团对动机行为的影响。精神疾病在美国的优势 导致数千万美元的医疗成本。虽然许多神经精神疾病可能是 根据特定特征的存在或不存在而分离，精神疾病的一个共同主题是 情绪失调症情绪或动机行为的失调已知内源性阿片系统对 调节情感和动机神经回路。历史上，中脑背核(包括腹外侧核) 中脑导水管周围灰核和邻近的中缝背核)被证明是 阿片类药物作用。最近，中缝背外侧核亚区(LDRN)和伏核 被证明是阿片类药物介导的食欲动机的中脑边缘回路中的重要部位。然而，虽然 这个低密度脂蛋白伏核回路中的下游阿片类药物活性特别增强了食欲动机， 趋同研究表明，内源性阿片类物质可能在LDRN内起到动机抑制作用 它本身。例如，20世纪80年代的实验证明，向腹外侧部注射吗啡 PAG/LDRN可抑制大鼠的摄食。相应地，我们实验室使用CRISPR-Cas9介导的试点研究 阿片肽的抑制反而促进了食物的摄取。此外，出现局部LDRN阿片活性 抑制食欲和厌恶的动机行为(例如，LDRN中的局部阿片拮抗增加 防御性或逃逸行为)。这些广泛的、反动机的阿片类药物效应表明，失调可能 影响广泛的情绪化或激动型行为。因此，此R01应用程序的目标是确定 内源性阿片类药物如何通过多途径调节LDRN的食欲和厌恶动机行为 药理学、活体成像和光基因技术。首先，我们将识别解剖学上的 中脑背核内阿片样物质的特征(目标1)。接下来，我们将测试功能本地化 通过无线射流设备进行受体选择性药理拮抗 CRISPR-Cas9阿片肽的敲除。接下来，我们将使用双色1光子内窥镜成像来 研究局部阿片能神经元和非阿片能神经元如何相互作用来编码食欲和厌恶行为 (目标2)。后续实验将使用同时的单光子成像和细胞类型选择性光遗传 神经调节以增强或干扰LDRN的编码和动机行为的表达。最后，我们会 多路单光子成像、CRISPR-Cas9基因敲除和细胞类型特异性光遗传刺激 确定内源性阿片肽如何随意增强LDRN的编码和表达 行为(目标3)。总之，这些研究可能为神经精神障碍是如何经常发生的提供洞察力。 以情感和动机失调为特征，并提示特定的神经化学靶点 治疗性干预。

Project Summary The primary goal of this R01 is to determine the computational and functional role of endogenous opioids in specific dorsal midbrain nuclei on motivated behaviors. The preponderance of mental illness in the United States results in tens of millions of dollars in healthcare costs. While many neuropsychiatric conditions can be dissociated based on the presence or absence of specific features, a common theme across mental illnesses is the dysregulation of affective or motivated behaviors. The endogenous opioid system is known to powerfully modulate affective and motivational neural circuits. Historically, dorsal midbrain nuclei (including ventrolateral periaqueductal gray nucleus and the adjacent dorsal raphe nucleus) have been shown to be important sites for opioid action. More recently, the lateral dorsal raphe nucleus subregion (LDRN) and nucleus accumbens were shown to be important sites in an opioid-mediated mesolimbic circuit of appetitive motivation. However, while downstream opioid activity in this LDRNaccumbens circuit specifically enhances appetitive motivation, convergent studies indicate that endogenous opioids may play a motivationally suppressive role within LDRN itself. For example, experiments in the 1980s demonstrated morphine microinjections into ventrolateral PAG/LDRN could suppress food intake. Correspondingly, pilot studies in our lab using a CRISPR-Cas9 mediated knockdown of opioid peptides oppositely facilitated food intake. Furthermore, local LDRN opioid activity appears to suppress both appetitive and aversive motivated behaviors (e.g., local opioid antagonism in LDRN increases defensive or escape behaviors). These broad, anti-motivational opioid effects suggest that dysregulation could affect a wide range of affective or motivated behaviors. Therefore, the goal of this R01 application is to determine how endogenous opioids regulate appetitive and aversive motivated behaviors in LDRN by multiplexing genetic, pharmacological, in vivo imaging, and optogenetic technologies. First, we will identify the anatomical characteristics of opioids within dorsal midbrain nuclei (Aim 1). In tandem we will test the functional localization of opioids by performing receptor selective pharmacological antagonism via wireless fluidic devices and CRISPR-Cas9 knockdown of opioid peptides. Next, we will use dual-color 1-photon endoscopic imaging to examine how local opioidergic and non-opioidergic neurons interact to encode appetitive and aversive behaviors (Aim 2). Follow up experiments will use simultaneous 1-photon imaging with cell-type selective optogenetic neuromodulation to augment or disrupt LDRN encoding and expression of motivated behaviors. Finally, we will multiplex 1-photon imaging, CRISPR-Cas9 knockdown, and cell-type specific optogenetic stimulation to determine how endogenous opioid peptides casually augment LDRN encoding and expression of motivated behaviors (Aim 3). Together, these studies may provide insights into how neuropsychiatric disorders are often characterized by affective and motivational dysregulation, and suggest specific neurochemical targets for therapeutic intervention.