Populations of inhibitory interneurons in the dorsal horn of the spinal cord

进入脊髓的神经纤维携带各种类型的感觉信息。其中一些纤维（伤害感受器）选择性地对组织损伤刺激做出反应。由伤害感受器传递的信号被传递到脊髓中负责撤回反射的局部神经回路，并且还被传递到将信息传递到大脑的一组神经细胞（投射神经元），在那里它被感知为疼痛。脊髓中的绝大多数神经细胞只参与局部回路，这些被定义为中间神经元。这些细胞中的许多释放化学信使，降低其他神经细胞的活性，因此具有抑制功能。脊髓内有复杂的神经回路，将传入的感觉纤维与中间神经元和投射神经元连接起来。这些回路在调节感觉信息的流动和调节疼痛的强度方面发挥着重要作用。例如，已经表明，阻断脊髓中抑制性中间神经元的功能会导致过度疼痛，并且影响这些细胞的病症会导致慢性疼痛状态。然而，尽管它们非常重要，我们仍然对脊髓内处理疼痛信号的神经回路的组织知之甚少。其主要原因是脊髓中间神经元的外观非常多样，因此很难将它们分类为不同的功能群体。在我们能够做到这一点之前，我们不可能解开不同类型神经细胞之间的复杂联系，因此也不可能理解它们在疼痛处理中的作用。我们已经发现，脊髓中的几组抑制性中间神经元可以根据它们所含的不同物质来识别。该项目的主要目的是测试这些组与其他神经细胞的连接是否不同，从而测试它们的功能是否不同。我们将对基因改变的小鼠进行实验，其中天然荧光蛋白存在于这些不同的抑制性中间神经元组中。这将使我们能够记录这些细胞的活动，并用另一种荧光染料标记它们，以便研究每个细胞的结构和连接。我们有初步的证据表明，属于其中一组的细胞不会被疼痛刺激激活，因此我们将测试这一组细胞是否从伤害感受器获得更少的连接。我们将使用一种称为“聚类分析”的强大技术来观察从这些组中汇集的大量细胞样本。聚类分析比较了每个细胞获得的广泛测量，并使用这些来提供样本中不同群体数量的客观评估。该技术已被用于定义几个大脑区域中不同功能的中间神经元群体，但尚未应用于脊髓中的疼痛通路。我们还发现，属于这些化学定义的组中的两个的细胞为两种不同类型的疼痛激活投射神经元提供了强大的抑制输入。因此，我们将测试这些细胞是否被伤害感受器激活，以及它们是否对应于通过聚类分析鉴定的群体。该项目将提供有关参与调节疼痛的不同类型的抑制性中间神经元的重要信息。通过揭示它们与传入感觉纤维和投射神经元的联系，它将大大增加我们对脊髓神经回路如何组织的知识。识别直接抑制投射神经元的中间神经元可能会为开发治疗疼痛的药物提供新的靶点。

Nerve fibres that enter the spinal cord carry various types of sensory information. Some of these fibres (nociceptors) respond selectively to tissue damaging stimuli. The signals conveyed by nociceptors are transmitted to local nerve circuits in the spinal cord that are responsible for withdrawal reflexes, and also to a group of nerve cells (projection neurons) that carry the information to the brain, where it is perceived as pain. The great majority of nerve cells in the spinal cord are only involved in local circuits, and these are defined as interneurons. Many of these cells release chemical messengers that reduce the activity of other nerve cells, and therefore have an inhibitory function. There are complex nerve circuits within the spinal cord that connect incoming sensory fibres with interneurons and projection neurons. These circuits play an important part in modulating the flow of sensory information and regulating the intensity of pain. For example it has been shown that blocking the function of inhibitory interneurons in the spinal cord causes excessive pain, and disorders affecting these cells can lead to chronic pain states. However, despite their great importance, we still know little about the organisation of the nerve circuits that process pain signals within the spinal cord. A major reason for this has been that spinal cord interneurons are very diverse in their appearance, and it has therefore been difficult to classify them into distinct functional populations. Until we can do this, it will not be possible to unravel the complex connections between the different types of nerve cell, and therefore to understand their roles in pain processing. We have found that several groups of inhibitory interneurons in the spinal cord can be recognised on the basis of different substances that they contain. The main aim of this project is to test whether these groups differ in their connections with other nerve cells, and therefore in their function. We will perform experiments on genetically altered mice in which a naturally fluorescent protein is present in these different groups of inhibitory interneurons. This will allow us to record the activity of these cells and to label them with another fluorescent dye, so that the structure and connections of each cell can be investigated. We have preliminary evidence that cells belonging to one of these groups are not activated by painful stimuli, and we will therefore test whether cells in this group receive fewer connections from nociceptors. We will use a powerful technique known as 'cluster analysis' to look at a large sample of cells pooled from each of these groups. Cluster analysis compares a wide range of measures obtained for each cell, and uses these to provide an objective assessment of the number of different populations within the sample. This technique has been used to define different functional populations of interneurons in several brain regions, but has not yet been applied to pain pathways in the spinal cord. We have also found that cells belonging to two of these chemically-defined groups provide a powerful inhibitory input to two different types of pain-activated projection neuron. We will therefore test whether these cells are activated by nociceptors, and whether they correspond to populations identified by the cluster analysis. The project will provide important information about the different types of inhibitory interneuron that are involved in regulating pain. By revealing their connections with incoming sensory fibres and projection neurons, it will add a great deal to our knowledge of how the nerve circuits in the spinal cord are organised. Identifying the interneurons that directly inhibit projection neurons may reveal new targets for the development of drugs designed to treat pain.