An Optogenetic Strategy to Determine if Merkel Cells Are Excitatory in the Skin

确定皮肤中默克尔细胞是否兴奋的光遗传学策略

• 批准号: 8331584
• 负责人: Ellen A Lumpkin
• 金额: $ 18万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8331584
• 关键词: Abbreviations; Action Potentials; Address; Antigen-Presenting Cells; Behavior; Biological Assay; Biological Process; Brain; Calcium; Calcium Channel; Cell Shape; Cell membrane; Cells; Code; Communities; Complex; Cutaneous; Environment; Epidermis; Excitatory Amino Acid Antagonists; Fluorescence; Fluorescence-Activated Cell Sorting; Gated Ion Channel; Glutamate Transporter; Glutamates; Goals; Hair Cells; Health; Human; In Situ; Inner Hair Cells; Ion Channel; Knock-in Mouse; Knockout Mice; Knowledge; Labyrinth; Life; Light; Mammals; Mechanics; Mediating; Merkel Cells; Modeling; Morphology; Mouse Strains; Mus; Nerve; Neurites; Neurons; Neurotransmitters; Organ; Output; Pain; Physiological; Play; Property; Proteins; Protocols documentation; Receptor Cell; Research; Role; Sensory; Sensory Receptors; Shapes; Signal Transduction; Skin; Stimulus; Synaptic Vesicles; Tactile; Tamoxifen; Testing; Texture; Touch sensation; Vibrissae; base; cell type; embryonic stem cell; feeding; gene induction; grasp; in vivo; innovation; innovative technologies; keratinocyte; light gated; mouse model; neurotransmission; novel; optogenetics; presynaptic; protein expression; receptor; relating to nervous system; response; social; somatosensory; tool; voltage

DESCRIPTION (provided by applicant): The sense of touch is an integral component of countless essential behaviors such as feeding, social exchange and avoiding bodily harm. In mammals, different tactile qualities are encoded by touch receptors with distinct physiological properties and morphological end organs; however, the cellular mechanisms underlying this diversity is unknown. The long-term goal of this research is to determine how mammalian Merkel cell-neurite complexes transduce touch stimuli into neural signals that inform the brain about objects in our dynamic environment. The objective of this application is to determine whether epidermal Merkel cells play an excitatory role in cutaneous touch reception. In complex with myelinated cutaneous afferents, they form gentle-touch receptors that mediate slowly adapting type I (SAI) responses. This project is highly relevant to human health because 1) SAI responses in the skin are thought to underlie high tactile acuity in humans and other mammals that rely on discriminative touch for recognizing and manipulating objects; and 2) Merkel cells are one of only four fundamental cell types in the mammalian epidermis and yet their biological function in the skin remains unknown 135 years after their discovery. Based on morphology, Merkel cells have long been proposed to be mechanosensory cells, analogous to inner-ear hair cells. Alternatively, Merkel cells might be accessory cells that shape sensory output of mechanosensitive SAI afferents. This exploratory project will use innovative technologies to directly distinguish between these two models. This application's central hypothesis is that Merkel cells release excitatory neurotransmitters to evoke action potentials in SAI afferents. To test this hypothesis, Merkel cells must be selectively excited without activating intrinsic mechanosensory mechanisms that might be present in SAI afferents. Since Merkel cells and SAI afferents are tightly juxtaposed in the skin, touch stimuli cannot be used to independently activate each cell type in situ. To break this barrier, the approach will employ a combination of optogenetics, mouse models and ex vivo skin-nerve recordings. Because mouse Merkel cells reside in the transparent epidermis, the light-gated ion channel channelrhodopsin-2 (ChR2) provides an ideal tool to excite Merkel cells in the intact skin. Aims are to 1) achieve selective and robust expression of ChR2 in Merkel cells in vivo, and 2) determine whether exciting Merkel cells in the absence of touch stimuli drives action potential firing in SAI afferents. The project is innovative because it represents first use of a light-activated protein to dissect the function of any skin cell type or to address mechanisms of touch reception. Furthermore, a novel Rosa26ChR2 knock-in mouse model has been generated that, when validated, can be broadly used by the scientific community.

描述(申请人提供)：触觉是无数基本行为的组成部分，如进食、社会交流和避免身体伤害。在哺乳动物中，不同的触觉质量是由具有不同生理特性和形态末端器官的触摸感受器编码的；然而，这种多样性背后的细胞机制尚不清楚。这项研究的长期目标是确定哺乳动物默克尔细胞-轴突复合体如何将触摸刺激转换为神经信号，从而向大脑通知我们动态环境中的物体。这项应用的目的是确定表皮默克尔细胞在皮肤触摸接受中是否起到兴奋作用。在与有髓皮肤传入的复合体中，它们形成温和的触觉受体，介导缓慢适应I型(SAI)反应。该项目与人类健康高度相关，因为1)皮肤中的SAI反应被认为是人类和其他哺乳动物高触觉敏锐度的基础，这些哺乳动物依靠辨别触觉来识别和操纵物体；2)默克尔细胞是哺乳动物表皮中仅有的四种基本细胞类型之一，但它们在皮肤中的生物学功能在发现135年后仍然未知。基于形态，长期以来，默克尔细胞被认为是机械感觉细胞，类似于内耳毛细胞。或者，默克尔细胞可能是辅助细胞，形成机械敏感的SAI传入的感觉输出。这个探索性项目将使用创新技术直接区分这两种模式。这一应用的中心假设是，默克尔细胞释放兴奋性神经递质，以在SAI传入中诱发动作电位。为了验证这一假设，必须选择性地兴奋Merkel细胞，而不激活SAI传入中可能存在的内在机械感觉机制。由于默克尔细胞和SAI传入细胞紧密并列在皮肤中，触摸刺激不能用来在原位独立激活每种细胞类型。为了打破这一障碍，该方法将结合光遗传学、小鼠模型和体外皮肤神经记录。由于小鼠的Merkel细胞驻留在透明的表皮中，光门控离子通道-视紫红质-2(ChR2)提供了一个理想的工具来刺激完整皮肤中的Merkel细胞。目的是1)在体内实现ChR2在Merkel细胞中的选择性和强势表达，以及2)确定在没有触摸刺激的情况下兴奋的Merkel细胞是否会驱动SAI传入的动作电位放电。该项目具有创新性，因为它代表了首次使用光激活蛋白来剖析任何皮肤细胞类型的功能或解决触摸接收机制。此外，已经产生了一种新的Rosa26ChR2敲入小鼠模型，经过验证后，可以被科学界广泛使用。