Synaptic Transmission In Mammalian Cutaneous Touch Receptors

描述（由申请人提供）：触摸的重要性是不可否认的：它是我们生存的基本感觉，使我们能够在物理世界中导航。它不仅对简单的日常活动（如喂养和穿衣）至关重要，而且对更复杂的社会互动（如交配，结合和成功的育儿）也至关重要。尽管经过数十年的研究，触摸敏感性背后的分子和细胞机制在很大程度上是未知的。本研究的长期目标是阐明触摸接收机制，使用默克尔细胞-神经突复合体作为哺乳动物中机械感觉信号传导的模型。本申请的目的是测试Merkel细胞突触活性是否参与SAI反应的产生。 默克尔细胞与皮肤感觉传入一起形成轻柔触摸感受器，其产生缓慢适应I型（SAI）反应。SAI反应被认为介导依赖于辨别性触摸来识别和操纵物体的哺乳动物的高触觉敏锐度。默克尔细胞是一种机械感觉细胞，通过兴奋性神经传递将感觉信息传递给皮肤传入神经。另外，默克尔细胞可能是辅助细胞，通过神经传递调节机械敏感性SAI传入的感觉输出。超微结构和分子生物学研究表明，默克尔细胞与SAI传入突触。默克尔细胞与大量潜在的神经递质有关，包括谷氨酸、血清素、ATP、甲硫氨酸脑啡肽等。然而，功能研究已经产生了矛盾的结果，这些神经递质的参与。此外，经过世纪的研究，默克尔细胞具有功能性突触的直接证明仍然缺乏。 本申请的中心假设是默克尔细胞释放兴奋性神经递质以引发SAI传入中的动作电位。目的1将测试来自默克尔细胞的突触囊泡释放是否是触摸诱发的SAI反应所必需的。我将记录SAI传入的活动在小鼠的默克尔细胞表达破伤风神经毒素轻链（TeNT），突触囊泡释放的抑制剂。目标2将测试Merkel细胞突触活动是否足以引起SAI传入的动作电位，如果是这样，将确定潜在的兴奋性神经递质。由于默克尔细胞和SAI传入在皮肤中紧密并列，因此触摸刺激不能用于原位独立激活每种细胞类型。为了打破这一障碍，在目标2中，我使用选择性表达的光门控离子通道通道视紫红质-2（ChR 2）在默克尔细胞。这使我能够测试，如果选择性地激发默克尔细胞与光唤起动作电位在SAI传入。

DESCRIPTION (provided by applicant): The importance of touch is undeniable: it is an essential sense for our survival that allows us to navigate the physical world. It is vital not onl for simple daily activities such as feeding and dressing, but also for more complicated social interactions such as mating, bonding and successful child rearing. Despite decades of research, the molecular and cellular mechanisms underlying touch sensitivity are largely unknown. The long- term goal of this research is to elucidate mechanisms of touch reception using the Merkel cell-neurite complex as a model for mechanosensory signaling in mammals. The objective of this application is to test whether Merkel-cell synaptic activity is involved in generation of SAI responses. Merkel cells together with cutaneous sensory afferents form gentle-touch receptors, which generate slowly adapting type I (SAI) responses. SAI responses are thought to mediate high tactile acuity in mammals that rely on discriminative touch to recognize and manipulate objects. It has long been proposed that Merkel cells are mechanosensory cells that transduce sensory information to cutaneous afferents via excitatory neurotransmission. Alternatively, Merkel cells might be accessory cells that modulate sensory output of mechanosensitive SAI afferents via neurotransmission. Ultrastructural and molecular studies suggest that Merkel cells synapse with SAI afferents. A plethora of potential neurotransmitters have been associated with Merkel cells, including glutamate, serotonin, ATP, met-enkephalin and others. However, functional studies have yielded contradictory results regarding the involvement of these neurotransmitters. Moreover, a direct demonstration that Merkel cells have functional synapses is still lacking after more than a century of investigation. This application's central hypothesisis that Merkel cells release excitatory neurotransmitters to elicit action potentials in SAI afferents Aim 1 will test whether synaptic vesicle release from Merkel cells is necessary for touch-evoked SAI responses. I will record SAI afferent's activity in mice whose Merkel cells express tetanus neurotoxin light chain (TeNT), an inhibitor of synaptic vesicle release. Aim 2 will test whether Merkel-cell synaptic activity is sufficient to elicit action potentials in SAI afferents and, if so will identify underlying excitatory neurotransmitters. 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, in Aim 2 I use selective expression of the light-gated ion channel channelrhodopsin-2 (ChR2) in Merkel cells. This allows me to test if selective excitation of Merkel cells with light evokes action potentials in SAI afferents.