Optogenetic analysis of epidermal-neuronal interaction

描述（由申请人提供）：传递温度，触觉和疼痛刺激的各种感觉传入神经支配皮肤的表皮层。虽然刺激检测被认为是传入特异性的，但表皮角质形成细胞产生的生长因子（NGF）、神经调节剂（如ATP、ACh、CGRP）和离子通道（如TRP、Na+）的激活表明表皮也影响感觉信号。为了研究神经上皮间的通讯，我们首先建立了光遗传学小鼠模型，其中光激活通道视紫红质（ChR2）使用外周蛋白启动子驱动Cre重组酶靶向外周神经元。外周蛋白- chr2小鼠皮肤的光刺激引起强烈的伤害行为反应，使用皮肤神经神经节和脊髓离体制备的电生理分析显示c纤维伤害感受器优先激活。因此，蓝色激光穿透表皮，感觉神经元特异性基因启动子驱动ChR2在体内的表达，其水平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所获得的相同的反应特性。因此，在这个R21应用程序中，我们建议测试这样一个假设，即光刺激与皮肤直接机械或热刺激之间的差异是由于皮肤对机械或热操作的反应所释放的因素。为了验证这一假设，我们分离了光遗传小鼠菌株，其中ChR2使用K14角蛋白基因启动子靶向表皮角化细胞。使用离体皮肤-神经-神经节-脊髓制备对这些小鼠进行的初步分析表明，光刺激角化细胞会引起行为和电生理反应特性的变化。我们还发现，不同类型的皮肤传入神经被激活，并且使用光和机械或热刺激对皮肤的联合刺激会引起单个纤维的更大激活。在Aim 1中，我们将在解剖、行为和生化水平上进一步表征K14-ChR2小鼠。目的2实验将确定角化细胞的光激活如何影响皮肤感觉神经元功能定义亚群的反应特性，并确定这种激活如何与皮肤的机械和/或热刺激进行比较。我们将使用在R21先导研究中产生的数据来设计未来的研究，以调查神经调节剂释放机制，并确定角化细胞释放的神经调节剂是否激活特定的感觉传入亚型。

DESCRIPTION (provided by applicant): Diverse sets of sensory afferents that transmit temperature, touch and painful stimuli innervate the epidermal layer of the skin. Although stimulus detection was thought to be afferent specific, production of growth factors (NGF), neuromodulators (e.g., ATP, ACh, CGRP) and activation of ion channels (e.g., TRP, Na+) by epidermal keratinocytes suggests the epidermis also impacts sensory signaling. To examine nerve-epithelial communication we first developed optogenetic mouse models in which light activated channelrhodopsin (ChR2) is targeted to peripheral neurons using a peripherin promoter driving Cre recombinase. Light stimulation of the skin of peripherin-ChR2 mice elicits a robust nocifensive behavioral response and electrophysiological assays 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 sensory neuron specific gene promoters drive in vivo expression of ChR2 at levels that depolarize peripheral nerve terminals. Interestingly, light activation of some neurons did not elicit response properties identical to thoe obtained using direct mechanical or thermal stimulation of the skin. In this R21 application we therefore propose to test the hypothesis that the difference between light-stimulation and direct mechanical or thermal stimulation of skin is due to factors released by the skin in response to mechanical or thermal manipulation. To test this hypothesis we isolated optogenetic mouse strains in which ChR2 is targeted to epidermal keratinocytes using the K14 keratin gene promoter. Preliminary analysis of these mice using an ex vivo skin-nerve-ganglia-spinal cord preparation show that light stimulation of keratinocytes evokes changes in behavioral and electrophysiologic response properties. We also found that different subtypes of cutaneous afferents are activated and that combined stimulation of the skin using light and mechanical or thermal stimuli evokes greater activation of individual fibers. In Aim 1 we will further characterie K14-ChR2 mice at anatomical, behavioral and biochemical levels. Aim 2 experiments will determine how light activation of keratinocytes affects response properties of functionally defined subsets of cutaneous sensory neurons and determine how this activation compares to mechanical and/or thermal stimulation of the skin. We will use the data generated in this R21 pilot study to design future studies that will investigate neuromodulator release mechanisms and determine if neuromodulators released from keratinocytes activate specific sensory afferent subtypes.