Role of Spinal GABA Receptors in Neuropathic Pain

描述(由申请人提供)：脊髓中GABA介导的抑制作用的丧失被认为有助于神经病理性疼痛的发展和维持。然而，支持性数据出人意料地疲软。此外，这些研究没有考虑到GABA受体的复杂分子生物学，以及在神经损伤后的疾病状态和重复活动条件下亚基组成和药理的变化。我们假设，神经损伤会导致脊髓中GABAA或GABAB受体的表达、分布和亚单位组成的即刻和长期变化。我们建议在明确的传入疼痛通路的突触前和突触后成分的背景下描述这些变化，以及一个表征良好的脊神经结扎模型，该模型展示了神经病理性疼痛行为的时间依赖变化。尤其重要的是，在神经损伤后，这些受体在未损伤的、受损的初级传入神经元和背角神经元上的表达和分子组成随时间的变化。用免疫印迹法检测GABAA和GABAB受体亚基在脊髓背角、同侧和对侧L4、L5背根节的表达和亚基组成的变化。用双色间接免疫荧光方法结合体视学测量，观察GABAA和GABAB受体亚单位在结扎和假手术大鼠L4和L5背根节初级传入神经元中不同亚群的分布。免疫组织化学方法还将结合结扎和假手术大鼠脊髓丘脑和脊髓臂神经细胞的逆行标记，以确定神经损伤后脊髓背角突触后GABAA和GABAB受体的分布和组成是否也发生了变化。分析的时间点从受伤后7天到140天不等。这些研究将为GABAA和GABAB受体在幼年动物初级传入神经元和背角神经元中的分布提供新的信息。他们将描述这些受体的分布和分子组成是如何在神经损伤后随时间变化的，并为抑制脊髓突触传递的分子和神经解剖学基础提供新的见解。

DESCRIPTION (provided by applicant): Loss of GABA-mediated inhibition in the spinal cord is proposed to contribute to the development and maintenance of neuropathic pain. However, the supporting data are surprisingly weak. Further, these studies have not considered the complex molecular biology of GABA receptors, and how subunit composition and pharmacology change in disease states and under conditions of repetitive activity as occurs after nerve injury. We hypothesize that nerve injury causes immediate, as well as long-term changes in the expression, distribution and subunit composition of GABAA or GABAB receptors in the spinal cord. We propose to characterize these changes in the context of defined presynaptic and postsynaptic elements of the afferent pain pathways and a well-characterized model of spinal nerve ligation that exhibits time-dependent changes in neuropathic pain behaviors. Of particular importance are the changes induced in the expression and molecular composition of these receptors on uninjured, and on injured primary afferent neurons and on dorsal horn neurons as a function of time after nerve injury. Changes in the expression and subunit composition of GABAA and GABAB receptor subunits in the spinal cord dorsal horn, and in ipsilateral and contralateral L4 and L5 DRG of ligated and sham rats will be determined by Western blot. Two-color indirect immunofluorescence methods will be used with stereological measurements to examine the distribution of GABAA and GABAB receptor subunits on different populations of immunohistochemically-identified primary afferent neurons in the L4 and L5 DRG of ligated and sham-operated rats. Immunohistochemical methods will also be used in conjunction with retrograde labeling of spinothalamic and spinoparabrachial neurons in ligated and sham rats to determine whether the distribution and composition of postsynaptic GABAA and GABAB receptors in the spinal cord dorsal horn is also altered after nerve injury. Time points for analysis will range 7 to 140 days after injury. These studies will provide new information about the distribution of GABAA and GABAB receptors on identified populations of primary afferent neurons and dorsal horn neurons in the naive animal. They will describe how the distribution and molecular composition of these receptors changes as a function of time after nerve injury and provide new insights into the molecular and neuroanatomical bases of inhibitorv svnaptic transmission in the spinal cord.