Itch neurons studied in vivo

描述（由申请人提供）：慢性瘙痒，像慢性疼痛一样，是临床上公认的痛苦和生活质量下降的主要原因。虽然瘙痒伴随许多神经、皮肤和全身性疾病，但在潜在的周围神经机制的研究中，它受到的关注比疼痛少。有两种一般的方法来研究背根神经节神经元对化学刺激的生理特性，这些化学刺激会引起人类的瘙痒或动物的瘙痒相关行为。第一种方法是电生理记录这些神经元对瘙痒性化学物质施加于其皮肤感受野的反应所引起的动作电位。第二种方法是检查化学感觉转导的细胞分子机制，使用细胞体（体细胞），分离并放置在培养中，作为外周末梢的模型。在该模型的测试中，我们将结合这两种方法，并在体内比较完整、可视化、伤害性DRG神经元的体细胞和感受野的反应特性。我们将使用遗传分子荧光标记来识别麻醉小鼠中一种特定类型DRG神经元的胞体。当皮肤感受野受到瘙痒和痛觉刺激时，细胞外的动作电位将被记录下来。然后，激活感受野的瘙痒性化学物质将局部应用于体细胞，并测量染色体膜兴奋性或细胞内钙的变化。从具有相同荧光标记的急性分离体细胞中获得类似的测量结果进行比较。如果成功，这一结果将为研究瘙痒介导化学物质如何在周围神经元中被感知的潜在细胞机制提供新的方法，这是开发能够特异性阻断人类病理性瘙痒的药物的重要先驱。

DESCRIPTION (provided by applicant): Chronic itch, like chronic pain, is clinically recognized as a major cause of suffering and loss in the quality of life. Although itch accompanies many neurological, dermatological and systemic diseases, it has received less attention than pain in studies of underlying peripheral neural mechanisms. There have been two general approaches to studying the physiological properties of dorsal root ganglion neurons that respond to chemical stimuli that evoke itch in humans or itch-associated behavior in animals. The first is to electrophysiologically record action potentials evoked in these neurons in response to a pruritic chemical applied to their cutaneous receptive fields. The second approach is to examine the cellular molecular mechanisms of chemical sensory transduction using the cell body (soma), dissociated and placed in culture, as a model of the peripheral terminal endings. In a test of this model, we will combine these two approaches and compare the response properties of the soma and the receptive field in the intact, visualized, nociceptive DRG neuron, in vivo. We will use genetic-molecular fluorescent markers to identify the soma of a particular type of DRG neuron in the anesthetized mouse. Action potentials will be recorded extracellularly from the soma in response to pruritic and algesic stimuli delivered to its cutaneous receptive field. Then pruritic chemicals that activated the receptive field will be topically applied to the soma and alterations in somal membrane excitability or intracellular calcium will be measured. Similar measurements will be obtained, for comparison, from acutely dissociated somas with the same fluorescent markers. If successful, the outcome will point to new methods of studying the underlying cellular mechanisms of how itch mediating chemicals are sensed in peripheral neurons, an important precursor to the development of drugs that will act specifically to block pathological itch in humans. PUBLIC HEALTH RELEVANCE: The experiments will test the feasibility of a new physiological preparation that will allow cellular-molecular studies of chemically evoked itch to be carried out in functionally identified peripheral sensory neurons, in vivo. The approach is to use genetic-molecular markers to identify specific subtypes of sensory neurons and use electrophysiological recording and calcium imaging methods both in vivo and also in vitro to determine their functional properties. The outcome will point to new methods of studying the underlying cellular mechanisms of how itch mediating chemicals are sensed in peripheral neurons, an important precursor to the development of drugs that will act specifically to block pathological itch in humans.