Characterization of epithelial-neural communication

 描述(申请人提供)：皮肤刺激的转导以前一直被认为仅仅是感觉纤维的功能。目前已经认识到，表皮角质形成细胞产生的生长因子和神经激活剂(如NGF、ATP、ACh、谷氨酸)可以对这一过程产生深远的影响。为了解开这些复杂的相互作用并提高我们对神经-角质形成细胞通讯调控机制的理解，我们建立了光遗传小鼠模型，在该模型中，光激活的通道视紫红质(ChR2)靶向于皮肤感觉神经元。研究发现，光刺激这些小鼠的皮肤可以引起强烈的神经行为反应。使用皮肤-神经节和脊髓体外制剂对这种激活进行的电生理分析表明，C纤维伤害性感受器优先激活。因此，蓝色激光穿透表皮，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直接机械或热刺激皮肤相同的反应特性。我们假设，缺乏完整的反应反映了皮肤刺激的缺失。因此，我们分离了ChR2专门针对K14角蛋白表达角质形成细胞的小鼠。值得注意的是，光刺激表达ChR2的角质形成细胞引起了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在不同的水平上被激活，这表明皮肤-神经交流的异质性。利用这些新的遗传模型，我们提出了三个特定的目标来推进这些发现：目的1实验将研究光诱导表达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。目的2将确定光激活ChR2或由感觉传入或角质形成细胞亚型表达的卤视紫质如何影响传入反应特性。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。目的研究炎性痛模型中角质形成细胞和感觉神经元的变化在热痛敏和机械性痛敏中的作用。这些研究将确定痛觉过敏是由初级传入细胞、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细胞的激活的能力将提供对皮肤如何 和感觉神经系统在正常和发炎的情况下进行通信。

 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.