The role of NPY-containing inhibitory interneurons in spinal pain pathways

进入脊髓的神经纤维将各种感觉信息传递到一个叫做背角的区域。传入的感觉信息被传递给一类被称为投射神经元的神经细胞，这些神经细胞将信息传递给大脑，使其产生有意识的感知。然而，脊髓中的绝大多数神经细胞是中间神经元，它们负责在感觉信息到达投射神经元之前对其进行局部处理和调节。大约三分之一的中间神经元释放化学信使（神经递质），减少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控制着感觉信息的流动，包括被感知为疼痛的信息。人们认为，它们活动的减少有助于某些类型的慢性疼痛，例如神经损伤后可能发生的疼痛。尽管背角在疼痛机制中很重要，但我们对它的神经细胞和回路的组织，以及它们如何处理传入的感觉信息，仍然知之甚少。这主要是因为很难确定中间神经元的特定功能群。我们实验室的工作表明，基于特定化学标记的存在，可以识别几种不同类型的抑制性中间神经元。其中一组由产生神经肽Y （NPY）物质的细胞组成，这些细胞占背角表面抑制性中间神经元的15%左右，并分散在背角较深的部分。最近的研究选择性地沉默或激活了另外两个抑制性中间神经元群，并表明它们在减轻疼痛方面具有不同的作用。根据我们已经知道的NPY细胞之间的联系，我们预测它们会抑制几种不同类型的急性疼痛（但不包括瘙痒），它们会抑制由炎症或神经损伤引起的慢性疼痛。该项目的主要目的是验证这一假设，我们将通过使用一种基因改造的小鼠来实现这一目标，在这种小鼠中，NPY细胞可以被特异性靶向。我们将在这些老鼠的脊髓中注射病毒，这些病毒会使这些细胞沉默或激活，而对其他神经元没有影响。然后，我们将测试抑制NPY细胞导致疼痛阈值降低的预测，而激活它们导致炎症或神经损伤后疼痛减轻。我们将验证只有NPY细胞受到影响，因此我们可以将任何行为变化归因于对这些细胞的影响。脊髓中间神经元使用两种不同的抑制性神经递质，GABA和甘氨酸，我们将测试背角表面的NPY细胞仅使用GABA的预测，并且它们与该区域的许多其他类型的神经元建立突触连接。这将通过“光遗传学”来实现，其中光被用来激活特定的神经细胞群。此外，我们将测试背角深处的NPY细胞是否也是抑制性中间神经元，并找出它们使用的是哪种神经递质。我们还将使用病毒注射来可视化NPY细胞，而不改变它们的活性。通过这种方式，我们将更多地了解这些神经元的特定子集的功能，这些神经元强烈抑制对疼痛刺激作出反应的特定投射神经元组。我们将测试这一预测，即它们被触觉和疼痛刺激激活，因此有助于通过触摸抑制疼痛，并设定一个与刺激强度相适应的疼痛水平。这些实验将为了解脊髓中的抑制性神经细胞如何参与抑制疼痛提供重要的见解。这种类型的信息在寻找新的镇痛药物是必不可少的。

Nerve fibres entering the spinal cord carry various types of sensory information to a region called the dorsal horn. Incoming sensory information is transmitted to a class of nerve cells called projection neurons, which convey it to the brain for conscious perception. However, the vast majority of nerve cells in the spinal cord are interneurons, which are responsible for local processing and modulation of sensory information before it reaches the projection neurons. Around a third of the interneurons release chemical messengers (neurotransmitters) that reduce the activity of other nerve cells, and therefore have an inhibitory function. These inhibitory interneurons control the flow of sensory information, including that which is perceived as pain. It is thought that a reduction of their activity contributes to some types of chronic pain, for example the pain that can occur after nerve injury. Despite the importance of the dorsal horn in pain mechanisms, we still know relatively little about the organisation of its nerve cells and circuits, or about how they process the incoming sensory information. This is largely because of the difficulty in defining specific functional populations among the interneurons. Work from our laboratory has shown that several different types of inhibitory interneuron can be recognised, based on the presence of specific chemical markers. One group consists of cells that make a substance called neuropeptide Y (NPY), and these account for around 15% of inhibitory interneurons in the superficial part of the dorsal horn and are scattered through its deeper part. Recent studies have selectively silenced or activated two other populations of inhibitory interneurons, and shown that these have different roles in reducing pain. From what we already know about the connections of the NPY cells, we predict that they will inhibit several different types of acute pain (but not itch), and that they will suppress chronic pain caused by inflammation or nerve injury. The main aim of this project is to test this hypothesis, and we will do this by using a strain of genetically altered mouse in which the NPY cells can be specifically targeted. We will make injections into the spinal cords of these mice of viruses that will either silence or activate these cells, while having no effect on other neurons. We will then test the prediction that inhibiting the NPY cells causes a reduction of pain thresholds, while activating them results in less pain following inflammation or nerve injury. We will verify that only NPY cells have been affected, and that we can therefore attribute any behavioural changes to an effect on these cells.Two different inhibitory neurotransmitters, GABA and glycine, are used by spinal cord interneurons, and we will test the predictions that the NPY cells in the superficial part of the dorsal horn use only GABA, and that they make synaptic connections with many other types of neuron in this region. This will be achieved with "optogenetics", in which light is used to activate specific populations of nerve cells. In addition, we will test whether the NPY cells in the deep part of the dorsal horn are also inhibitory interneurons, and find out which neurotransmitter they use.We will also use viral injections to visualise the NPY cells without altering their activity. In this way, we will find out more about the functions of a specific subset of these neurons, which strongly inhibit a particular group of projection neurons that respond to painful stimuli. We will test the prediction that they are activated by both tactile and painful stimuli, and therefore contribute to suppression of pain by touch, as well as setting a level of pain that is appropriate to the strength of the stimulus. These experiments will provide important insight into how inhibitory nerve cells in the spinal cord are involved in suppressing pain. This type of information is essential in the search for new analgesic drugs.