Characterization of epithelial-neural communication

上皮神经通讯的表征

• 批准号: 9898306
• 负责人: Kathryn Marie Albers
• 金额: $ 60.13万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898306
• 关键词: Action Potentials; Activation Analysis; Affect; Afferent Neurons; Avil; Behavior; Behavioral; C Fiber; Cells; Characteristics; Coculture Techniques; Code; Communication; Complex; Cutaneous; Electrophysiology (science); Epidermis; Epithelial; Epithelial Cells; Epithelium; Exhibits; Fiber; Freund' s Adjuvant; Ganglia; Generations; Genetic Models; Genetic Transcription; Glutamates; Growth Factor; Halorhodopsins; Heterogeneity; Hyperalgesia; Hypersensitivity; Inflammation; Inflammatory; Keratin; Knowledge; Lasers; Light; Lighting; Measures; Mechanics; Mediating; Modeling; Mus; Nature; Nerve; Nerve Fibers; Nervous system structure; Neurons; Nociceptors; Outcome; Pain; Pathologic; Pattern; Peripheral; Peripheral Nerves; Pharmacology; Phenocopy; Phenotype; Physiological; Preparation; Process; Production; Property; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Role; Sensory; Skin; Spinal Cord; Spinal Ganglia; Stimulus; Temperature; Testing; Touch sensation; Transgenic Mice; afferent nerve; behavior measurement; behavioral response; cell type; cutaneous sensory neurons; experimental study; genetic approach; inflammatory pain; insight; keratinocyte; mouse model; optogenetics; public health relevance; relating to nervous system; response; sensory stimulus

 DESCRIPTION (provided by applicant): The transduction of cutaneous stimuli has been previously thought to be solely a function of sensory fibers. It is now recognized that production of growth factors and neuroactivators (e.g., NGF, ATP, ACh, glutamate) by epidermal keratinocytes can have a profound effect on this process. To unravel these complex interactions and advance our understanding of the mechanisms regulating neural-keratinocyte communication, we developed optogenetic mouse models in which light activated channelrhodopsin (ChR2) is targeted to cutaneous sensory neurons. Light stimulation of the skin of these mice was found to elicit a robust nocifensive behavioral response. Electrophysiological analysis of this activation using a skin-nerve-ganglia and spinal cord ex vivo preparation showed preferential activation of C-fiber nociceptors. Thus, blue-laser light penetrates the epidermis and activates ChR2 at levels that depolarize peripheral nerve terminals. Interestingly, light activation of some neurons did not elicit response properties identical to those obtained using direct mechanical or thermal stimulation of the skin. We hypothesized this lack of a full response reflected a missing stimulus from the skin. We therefore isolated mice in which ChR2 was targeted exclusively to K14 keratin expressing keratinocytes. Remarkably, light stimulation of keratinocytes expressing ChR2 evoked changes in behavioral and electrophysiologic response properties of cutaneous sensory neurons. We also found that different subtypes of cutaneous afferents are activated at different levels suggesting heterogeneity in skin-neural communication. Using these new genetic models we propose three specific aims to advance these findings: Aim 1 experiments will examine how light-induced release of neuroactivators (e.g., ATP) from ChR2- expressing keratinocytes activates subtypes of primary sensory afferents. Aim 2 will determine how light activation of ChR2 or halorhodopsin expressed by subtypes of sensory afferents or keratinocytes affects afferent response properties. We will also determine how this activation compares to mechanical and/or thermal stimulation of the skin. Aim 3 experiments will determine the contribution of changes in keratinocytes and sensory neurons to thermal and mechanical hyperalgesia in a model of inflammatory pain. These studies will determine if hyperalgesia is caused by changes in primary afferents, skin keratinocytes or both. The ability to control activation of either keratinocytes or sensory afferents will provide new insights into how the skin and sensory nervous system communicate under normal and inflamed conditions.

 描述（由申请人提供）： 皮肤刺激的转导先前被认为仅是感觉纤维的功能。现在人们认识到，表皮角质形成细胞产生的生长因子和神经激活剂（例如 NGF、ATP、ACh、谷氨酸）可以对此过程产生深远的影响。为了阐明这些复杂的相互作用并加深我们对调节神经角质形成细胞通讯机制的理解，我们开发了光遗传学小鼠模型，其中光激活视紫红质通道蛋白（ChR2）靶向皮肤感觉神经元。研究发现，对这些小鼠皮肤的光刺激会引发强烈的伤害行为反应。使用皮肤-神经-神经节和脊髓离体制剂对这种激活进行的电生理分析表明，C 纤维伤害感受器优先激活。因此，蓝色激光穿透表皮并激活 ChR2，使周围神经末梢去极化。有趣的是，一些神经元的光激活并没有引起与使用直接机械或热刺激皮肤所获得的反应特性相同的反应特性。我们假设这种缺乏充分反应反映了皮肤刺激的缺失。因此，我们分离了 ChR2 专门针对表达 K14 角蛋白的角质形成细胞的小鼠。值得注意的是，表达 ChR2 的角质形成细胞的光刺激引起皮肤感觉神经元的行为和电生理反应特性的变化。我们还发现不同亚型的皮肤传入神经在不同水平上被激活，这表明皮肤神经通讯的异质性。使用这些新的遗传模型，我们提出了三个具体目标来推进这些发现：目标 1 实验将检查光诱导表达 ChR2 的角质形成细胞释放神经激活剂（例如 ATP）如何激活初级感觉传入的亚型。目标 2 将确定感觉传入细胞或角质形成细胞亚型表达的 ChR2 或盐视紫红质的光激活如何影响传入响应特性。我们还将确定这种激活与皮肤的机械和/或热刺激相比如何。目标 3 实验将确定角质形成细胞和感觉神经元的变化对炎性疼痛模型中的热和机械痛觉过敏的影响。这些研究将确定痛觉过敏是否是由初级传入神经、皮肤角质形成细胞或两者的变化引起的。控制角质形成细胞或感觉传入的激活的能力将为皮肤如何发挥作用提供新的见解。 和感觉神经系统在正常和炎症条件下进行通信。