Using dual intersectional genetics to understand and modulate itch

描述(由申请人提供)：慢性瘙痒(瘙痒)是一种广泛存在的疾病，严重降低生活质量。然而，几乎没有有效的治疗慢性瘙痒的方法，部分原因是对瘙痒的神经基础仍然知之甚少。因此，我们研究的长期目标是在特定的神经回路水平上更好地了解瘙痒是如何在神经系统中编码的，以期开发出更有效的瘙痒治疗方法。我们之前发现，转录因子Bhlhb5是脊髓中正常瘙痒感觉所需的抑制性中间神经元(这里称为B5-I神经元)的子集生存所必需的；缺乏这些脊髓中间神经元的小鼠会遭受持续性病理性瘙痒。这些发现表明，B5-I神经元具有抑制瘙痒的功能；然而，这些证据只是相互关联的。在这项应用中，我们建议使用交叉遗传策略来操纵B5-I神经元的活动，以建立B5-I神经元的活动与小鼠抓挠行为之间的因果关系。在这里，我们建议通过三个特定的目标来验证这一假设：1)表征Bhlhb5-flpO敲击小鼠，这是我们双重交叉策略的关键工具，并使用该小鼠与Ptf1a-cre系一起从基因上定义B5-I神经元。2)研究B5-I神经元对皮肤自然刺激的功能反应特性，并证实药物遗传学方法特异性地操纵其活动的能力。3)用胞外发生和药物发生的方法描述B5-I神经元在体内对瘙痒介导的抓挠行为的功能。这些实验的结果将使我们能够在体外和体内可视化、表征和功能操纵B5-I神经元，从而开始解码瘙痒背后的神经回路。此外，所收集的洞察力可能对患有慢性瘙痒的人具有重大临床意义。

DESCRIPTION (provided by applicant): Chronic itch (pruritis) is a widespread condition that severely diminishes quality of life. However, there are few effective treatments for chronic itch, in part because the neural basis for itch remains poorly understood. Thus, the long-term goal of our research is to gain a better understanding of how itch is encoded in the nervous system at the level of specific neural circuits with the view of developing more effective therapies for pruritis. We previously discovered that the transcription factor Bhlhb5 is required for the surviva of a subset of inhibitory interneurons in the spinal cord (which are here termed B5-I neurons) that are required for normal itch sensation; mice lacking these spinal interneurons suffer from persistent pathological itch. These findings imply that B5-I neurons function to inhibit itch; however, the evidence is merely correlative. In this application we propose to use intersectional genetic strategies to manipulate the activity of B5-I neurons in order to establish cause-and-effect relationships between the activity of B5-I neurons and scratching behavior in mice. Here we propose to test this hypothesis through 3 specific aims: 1) Characterize the Bhlhb5-flpO knockin mouse, a key tool for our dual intersection strategy, and use this mouse together with the Ptf1a-cre line to genetically define B5-I neurons. 2) Investigate the functional response properties of B5-I neurons to natural stimulation of the skin and to confirm the ability of pharmacogenetic approaches to specifically manipulate their activity. 3) Use exocytogenetic and pharmacogenetic approaches to delineate function of B5-I neurons in vivo for itch-mediated scratching behavior. Results from these experiments will begin decoding the neural circuits that underlie itch by enabling us to visualize, characterize, and functionally manipulate B5-I neurons in vitro and in vivo. Moreover, the insight gleaned may have major clinical implications for people that suffer from chronic itch.