Do Rorb/calretinin interneurons (CR islet cells) gate spinal nociceptive inputs?

进入脊髓的神经纤维(初级传入纤维)携带各种类型的感觉信息。其中一些纤维(伤害性感受器)对通常被认为是疼痛的组织破坏性刺激做出反应。虽然伤害性感受器存在于全身的组织中，但有一种特殊的类别，称为C-mrgD传入细胞，专门供应皮肤。选择性地破坏小鼠的C-mRgD传入可以减少机械刺激后的疼痛行为，但不会导致热刺激或冷刺激。这表明，这些传入是机械性疼痛的正常感觉所必需的。初级传入纤维激活脊髓中的各种神经细胞。这些细胞中的大多数是中间神经元，它们会产生局部电路，在传入的感觉信息被传达到大脑进行意识感知之前，对其进行处理和修改。大约三分之一的中间神经元释放化学信使(神经递质)，这些化学信使降低其他神经细胞的活动，因此具有抑制功能。这些抑制性中间神经元使用两种基本机制：突触后抑制和突触前抑制。突触后抑制包括抑制附近神经细胞的活动，而突触前抑制通过降低传入感觉神经纤维激活其靶细胞的能力而直接作用于它们。这具有对特定类型的感觉信息提供高度选择性抑制的优点。已知突触前抑制作用于C-mRgD传入，这可能会抑制疼痛。直到最近，对与此相关的抑制性中间神经元一无所知，但我们现在已经确定了一组细胞，它们在突触前抑制C-MRgD传入。这些细胞之所以能被识别，是因为它们含有两种蛋白质：钙视黄素(CR)和RorB。这些蛋白质只在这个群体中共定位，由于它们的形态，我们称之为CR胰岛细胞。这些发现很重要，因为它们提供了一种研究突触前痛觉受器抑制在疼痛机制中的作用的方法。在这个项目中，我们将使用多学科的方法来测试一组关于CR胰岛细胞的假说。我们将使用转基因小鼠，在这些小鼠中，特定群体的神经细胞被标记有荧光标记和/或含有蛋白质，这些蛋白质允许通过应用光脉冲或通过给予高度选择性的药物来操纵它们的功能。我们将首先进行生理学研究，以验证这一假设，即CR胰岛细胞是C-MRgD传入突触前抑制的主要来源，并且它们可以阻断这些传入所引起的活动在脊髓神经细胞中的传递。我们知道C-mRgD传入并不是CR胰岛细胞的唯一靶点，因此我们将测试它们是否也抑制由这些传入激活的兴奋性神经细胞。如果是这样的话，这将允许CR胰岛细胞产生强大的抑制作用，将疼痛信息从伤害性初级传入传递到将该信息传递到大脑的投射细胞，并代表脊髓痛觉回路的主要输出。最后，我们将使用转基因小鼠在体内选择性地激活CR胰岛细胞。我们预测，这将缓解病理性疼痛状态下对机械刺激的过敏性，我们将在炎症和神经损伤诱发疼痛的标准模型中使用行为测试来确定是否如此。这些实验将极大地提高我们对脊髓中负责控制疼痛的神经回路的理解。这些信息在寻找治疗疼痛的新药方面很重要，也对未来研究慢性疼痛状态下脊髓的变化很重要。

Nerve fibres that enter the spinal cord (primary afferent fibres) carry various types of sensory information. Some of these fibres (nociceptors) respond to tissue-damaging stimuli that are normally perceived as pain. Although nociceptors are found in tissues throughout the body, one particular class, known as C-MrgD afferents, exclusively supplies the skin. Selective destruction of the C-MrgD afferents in mice leads to reduced pain behaviour following mechanical stimuli, but not hot or cold stimuli. This suggests that these afferents are required for the normal perception of mechanical pain. Primary afferent fibres activate a variety of nerve cells in the spinal cord. Most of these cells are interneurons, which give rise to local circuits that process and modify the incoming sensory information before it is conveyed to the brain for conscious perception. Around a third of these interneurons release chemical messengers (neurotransmitters) that reduce the activity of other nerve cells, and therefore have an inhibitory function. These inhibitory interneurons use two basic mechanisms: postsynaptic and presynaptic inhibition. Postsynaptic inhibition involves suppressing activity of nearby nerve cells, while presynaptic inhibition operates directly on the incoming sensory nerve fibres by reducing their ability to activate their target cells. This has the advantage of providing a highly selective inhibition of specific types of sensory information. Presynaptic inhibition is known to operate on C-MrgD afferents, and this will presumably suppress pain. Until recently nothing was known about the inhibitory interneurons responsible for this, but we have now identified a population of cells that presynaptically inhibit the C-MrgD afferents. These cells can be recognised because they contain two proteins: calretinin (CR) and Rorb. These proteins are only co-localised in this population, and because of their morphology we have called them CR islet cells. These findings are important because they provide a way of investigating the role of presynaptic inhibition of nociceptors in pain mechanisms.In this project, we will use a multi-disciplinary approach to test a set of hypotheses concerning the CR islet cells. We will use genetically altered mice in which specific populations of nerve cells are labelled with fluorescent markers and/or contain proteins that allow their functions to be manipulated, either by application of light pulses or through the administration of highly selective drugs. We will initially carry out physiological studies to test the hypothesis that the CR islet cells are the major source of presynaptic inhibition of the C-MrgD afferents and that they can block the transmission of activity evoked by these afferents in spinal cord nerve cells. We know that the C-MrgD afferents are not the only target for the CR islet cells, and we will therefore test whether they also inhibit excitatory nerve cells that are activated by these afferents. If so, this would allow the CR islet cells to generate a powerful inhibition of the transmission of pain information from nociceptive primary afferents to the projection cells that convey this information to the brain, and represent the main output for spinal cord pain circuits. Finally, we will use genetically altered mice to activate the CR islet cells selectively in vivo. We predict that this will alleviate the hypersensitivity to mechanical stimuli that occurs in pathological pain states, and we will use behavioural testing in standard models of inflammatory and nerve injury-evoked pain to determine whether this is the case.These experiments will greatly improve our understanding of the nerve circuits in the spinal cord that are responsible for controlling pain. This information is important in the search for new drugs to treat pain, and also for future studies to investigate changes in the spinal cord that underlie chronic pain states.