Spinal modulation of non-peptidergic C-nociceptor input: A role for inhibitory calretinin interneurons

疼痛和瘙痒是主要的临床问题，不仅造成巨大的个人痛苦，而且由于许多患者无法工作，还具有重大的经济影响。据估计，慢性疼痛影响约20%的人口，但只有三分之一的患者对目前可用的治疗有反应。为了改善对这些疾病的治疗，我们需要更多地了解神经系统处理疼痛和瘙痒信息的复杂回路。这些信息最初由专门的神经细胞群传递到脊髓，包括一种称为非肽能伤害感受器的类型，并且已经表明，这些可以根据它们所包含的不同类型的蛋白质分为三个功能组。这些伤害感受器负责瘙痒的感觉，以及某些类型的疼痛，例如由皮肤的挤压或针刺引起的疼痛。它们也被认为有助于身体不同部位炎症引起的慢性疼痛，例如手术后的疼痛。我们最近在脊髓中发现了一种独特的神经细胞群，它们可能会阻止来自这些伤害感受器的信息传递。这些细胞含有一种称为钙视网膜蛋白的蛋白质，我们将其称为“抑制性钙视网膜蛋白细胞”（iCR）。我们的初步研究结果表明，iCRs可能被所有三种类型的非肽能伤害感受器激活。我们还发现，iCRs的位置非常理想，可以防止伤害感受器激活其他神经细胞，从而阻断疼痛和瘙痒信号。我们认为，在正常情况下，这些细胞在切断进入中枢神经系统的疼痛和瘙痒输入方面发挥着重要作用。在这个项目中，我们将使用几种不同的实验方法来研究iCRs在疼痛和瘙痒处理中的作用。我们的研究将涉及转基因小鼠，因为这些将使我们能够选择性地靶向iCRs。我们将首先使用显微镜，使我们能够在非常高的放大倍率下检查这些细胞之间的连接（突触）。特别是，我们将询问iCRs是否是唯一一种能够产生能够阻断非肽能伤害感受器活动的突触类型的神经细胞。然后，我们将使用一种方法，使我们能够研究iCRs的活动，并测试它们是否被所有三类伤害感受器激活。为了评估iCRs如何有效地阻断来自伤害感受器的信号传输，我们将记录特定脊髓神经细胞群（称为投射神经元）的活动。这些细胞形成了感觉信息从脊髓传递到大脑的主要途径，使我们能够感受到疼痛和瘙痒。我们将测试激活iCRs是否会阻止伤害感受器携带的感觉信息到达投射神经元，然后评估皮肤受到刺激时这对投射神经元活动的影响。我们将继续使用最近开发的技术，使我们能够选择性地改变小鼠iCRs的活性。我们将首先确定激活这些细胞是否可以减少炎症引起的疼痛和某些化学物质注射到皮肤中时看到的瘙痒。然后，我们将使用类似的方法对iCR进行检测，看看这是否会导致自发性疼痛或瘙痒行为。如果是这样的话，这将表明在正常情况下iCRs的活动会阻断这些感觉。这个项目将提供有关脊髓内神经回路的有价值的信息，这些神经回路控制传入的感觉信息，导致疼痛和瘙痒。重要的是，它还将揭示iCRs是否代表了可用于缓解慢性疼痛和瘙痒的新治疗方法的目标。

Pain and itch are major clinical problems that not only cause great personal suffering, but also have a substantial economic impact since many sufferers are unable to work. It is estimated that chronic pain affects ~20% of the population, but only 1 in 3 patients respond to currently available treatments. To improve treatment for these conditions, we need to find out more about the complex circuits through which the nervous system processes pain and itch information. This information is initially transmitted to the spinal cord by specialised populations of nerve cells, including a type known as non-peptidergic nociceptors, and it has been shown that these can be divided into three functional groups based on the different types of proteins that they contain. These nociceptors are responsible for the sensation of itch, as well as for certain types of pain such as that resulting from pinching of the skin or a pinprick. They are also thought to contribute to the chronic pain that results from inflammation in different parts of the body, for example the pain after surgical operations. We have recently identified a distinct population of nerve cells in the spinal cord that are likely to block the passage of information from these nociceptors. These cells contain a protein called calretinin, and we refer to them as "inhibitory calretinin cells" (iCRs). Our preliminary findings suggest that the iCRs may be activated by all three types of non-peptidergic nociceptors. We also find that the iCRs are ideally positioned to prevent the nociceptors from activating other nerve cells, thus blocking pain and itch signals. We propose that under normal circumstances, these cells play an important role in switching off pain and itch input at the point of entry into the central nervous system. In this project, we will use several different experimental approaches to investigate the role of iCRs in pain and itch processing. Our studies will involve genetically-modified mice, as these will allow us to target the iCRs selectively. We will initially use a microscope that allows us to examine the connections (synapses) between these cells at very high magnification. In particular, we will ask whether iCRs are the only type of nerve cell that make the types of synapse that can block activity in the non-peptidergic nociceptors. We will then use an approach that allows us to study the activity of the iCRs, and test whether they are activated by all three of the nociceptor classes. In order to assess how effectively iCRs block the transmission of signals from the nociceptors, we will record the activity of a specific population of spinal cord nerve cells, known as projection neurons. These cells form the major route through which sensory information is transmitted from the spinal cord to the brain, allowing us to feel pain and itch. We will test whether activating the iCRs prevents sensory information carried by the nociceptors from reaching the projection neurons, and then assess the impact that this has on the activity of the projection neurons when the skin is stimulated. We will go on to use a recently developed technique that allows us to selectively alter the activity of the iCRs in mice. We will first determine whether activating these cells reduces both the pain that results from inflammation and the itch that is seen when certain chemicals are injected into the skin. We will then use a similar approach to inactivate the iCRs and see whether this leads to spontaneous pain or itch behaviours. If so, this would indicate that activity of the iCRs blocks these sensations under normal circumstances. This project will provide valuable information about the nerve circuits within the spinal cord that control the incoming sensory information that results in pain and itch. Importantly, it will also reveal whether the iCRs represent a target for new treatments that could be used to alleviate chronic pain and itch.