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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：接触感是无数基本行为的组成部分，例如喂养，社会交流和避免身体伤害。在哺乳动物中，具有不同生理特性和形态末端器官的触摸受体编码不同的触觉质量。但是，这种多样性的基础机制尚不清楚。这项研究的长期目标是确定哺乳动物的默克尔细胞神经突配合物如何将触摸刺激转换为神经信号，从而在我们的动态环境中为大脑提供有关对象的信息。该应用的目的是确定表皮默克尔细胞在皮肤触摸接收中是否起兴奋的作用。与髓鞘的皮肤传入复杂，它们形成了柔和的受体，可缓慢调整I型（SAI）反应。该项目与人类健康高度相关，因为1）认为皮肤中的SAI反应是人类和其他依靠歧视性触摸来识别和操纵物体的人类和其他哺乳动物的高触觉敏锐度； 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后的135年仍然未知。基于形态，长期以来，默克尔细胞已被认为是机械感知细胞，类似于内耳毛细胞。或者，默克尔细胞可能是构成机械敏感SAI传入的感觉输出的辅助细胞。该探索性项目将使用创新技术直接区分这两个模型。该应用程序的中心假设是，默克尔细胞释放兴奋性神经递质以唤起SAI传入中的动作电位。为了检验这一假设，必须选择性地激发默克尔细胞，而无需激活SAI传入中可能存在的内在机械密度机制。由于默克尔细胞和SAI传入在皮肤中紧密并置，因此触摸刺激不能用于独立激活每种细胞类型的原位。为了打破这种障碍，该方法将采用光遗传学，小鼠模型和离体皮肤录音的组合。由于小鼠默克尔细胞位于透明表皮中，因此光门控离子通道通道Ropopsin-2（ChR2）为激发完整皮肤中的默克尔细胞提供了理想的工具。目的是1）在体内默克尔细胞中CHR2的选择性和稳健表达，以及2）确定令人兴奋的默克尔细胞在没有触摸刺激的情况下是否会驱动SAI传入中的动作潜在发射。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，已经生成了一种新颖的Rosa26Chr2敲入小鼠模型，该模型在验证后可以由科学界广泛使用。