The roles of functionally defined populations of lamina I projection neurons

神经性疼痛是一种令人痛苦的疾病，通常发生在疾病或神经损伤后，影响近10%的人口。治疗往往是不够的，这是一个主要原因是我们有限的了解神经回路在脊髓中参与这种情况。先前的研究已经确定了脊髓浅层的一组神经细胞，称为椎板I投射神经元，它们将与疼痛和皮肤温度相关的感觉信息直接传递到大脑。还表明这些细胞在神经性疼痛中起作用。然而，投射神经元在功能上是异质的，这使得很难理解它们相互作用的神经回路，并定义它们在不同类型的疼痛中的作用。在最近的一项研究中，我们发现，两个很大程度上独立的群体，其中占约85%的板层I投射神经元，可以确定在小鼠中基于两种不同的蛋白质的存在。这些蛋白质由称为Tacr1和Gpr83的基因编码。我们发现，Tacr1和Gpr83细胞对施加在皮肤上的疼痛或热刺激的反应不同，这意味着它们负责疼痛和温度感觉的不同方面。该研究构成了当前项目的起点。我们将首先通过检查对皮肤刺激的反应来识别特定的I层投射神经元群体，不仅针对表达这些受体中的每一种的细胞，而且针对表达两种受体或两种受体都不表达的较小群体。以前在其他物种中的研究表明，不同类型的投射神经元具有特定的形态学特征，我们将确定这是否适用于小鼠的投射神经元。然后，我们将测试这些不同类型的投射神经元参与脊髓内不同神经回路的预测。为了做到这一点，我们将首先研究它们来自另一类细胞的输入，称为兴奋性中间神经元。我们还将测试他们对一种名为阿片类药物（包括吗啡）的药物的反应，这种药物可以有效地抑制疼痛。阻断一组不同的脊髓细胞（称为抑制性中间神经元）的活动会导致夸张的感觉，这种机制被认为是导致神经性疼痛的原因。已经表明，当这种抑制被阻断时，一些板层I投射神经元对皮肤上的毛发的刷洗产生新的反应，这可能与神经损伤患者中常见的触摸诱发的疼痛相关。我们将测试哪类投射神经元表现出这种类型的反应。然后，我们将使用体内实验方法，使我们能够单独沉默Tacr1或Gpr83细胞，或同时沉默两个群体。我们将首先测试两种不同的方法，涉及细胞特异性毒性蛋白或光敏蛋白，这将阻止特定细胞的活动。我们将确定哪一种更有效，然后将其应用于神经损伤的小鼠模型，并测试沉默这些群体中的每一个是否会抑制神经性疼痛的不同方面，例如自发性疼痛，或者机械或热超敏反应。最后，我们将研究一个特定的功能群体的第一层投射神经元，专门响应冷却的皮肤，而不是痛苦的刺激。我们将测试这些细胞缺乏Tacr 1和Gpr83受体的预测，并研究它们参与的神经回路。该项目将提供有关脊髓如何处理感知为疼痛和温度的感觉信息的有价值的信息，以及神经病理性疼痛的细胞和回路。这对于开发治疗这种令人痛苦的疾病的新疗法至关重要。

Neuropathic pain is a distressing condition that commonly occurs following disease or injury to nerves, and affects nearly 10% of the population. Treatment is often inadequate, and a major reason for this is our limited understanding of the nerve circuits in the spinal cord that are involved in this condition. Previous studies have identified a group of nerve cells in the superficial part of the spinal cord, known as lamina I projection neurons, which carry sensory information related to pain and skin temperature directly to the brain. It has also been shown that these cells have a role in neuropathic pain. However, the projection neurons are functionally heterogeneous, and this has made it difficult to understand the nerve circuits with which they interact, and to define their roles in different types of pain. In a recent study, we showed that two largely separate populations, which account for ~85% of lamina I projection neurons, could be identified in the mouse based on the presence of two different proteins. These proteins are encoded by genes known as Tacr1 and Gpr83. We found that the Tacr1 and Gpr83 cells differed in their responses to painful or thermal stimuli applied to the skin, implying that they are responsible for different aspects of pain and temperature sensation. That study forms the starting point for the current project. We will initially identify specific populations of lamina I projection neurons by examining responses to skin stimulation not only for cells that express each of these receptors, but also for the smaller populations that express both receptors, or neither receptor. Previous studies in other species have suggested that different types of projection neuron have specific morphological features, and we will determine whether this is true for projection neurons in the mouse. We will then test the prediction that these different types of projection neuron are involved in different nerve circuits within the spinal cord. To do this, we will first investigate their inputs from another class of cell, known as excitatory interneurons. We will also test their responses to a class of drug known as opioids (which includes morphine) that can powerfully suppress pain. Blocking activity in a different group of spinal cord cells, known as inhibitory interneurons, leads to exaggerated sensations and this mechanism is thought to contribute to neuropathic pain. It has been shown that some lamina I projection neurons develop novel responses to brushing of hairs on the skin when this inhibition is blocked, and this is probably a correlate of the touch-evoked pain often seen in patients with nerve damage. We will test which classes of projection neuron show this type of response. We will then use an in vivo experimental approach that will allow us to silence the Tacr1 or Gpr83 cells individually, or to silence both populations simultaneously. We will initially test two different methods, involving either a cell-specific toxic protein, or a light-sensitive protein that will block activity in specific cells. We will determine which of these is more effective, and then apply it to a mouse model of nerve injury and test whether silencing each of these populations suppresses different aspects of neuropathic pain, such as spontaneous pain, or either mechanical or thermal hypersensitivity. Finally, we will examine a specific functional population of lamina I projection neurons that respond exclusively to cooling of the skin, rather than painful stimuli. We will test the prediction that these are the cells that lack both Tacr1 and Gpr83 receptors, and we will investigate the nerve circuits to which they contribute.The project will provide valuable information about how the spinal cord processes sensory information perceived as pain and temperature, as well as the cells and circuits that underlie neuropathic pain. This is vital for the development of new therapies for treating this distressing condition.