Dynorphin Modulation of N-Methyl-D-Aspartate (NMDA) Receptor Function

描述（由申请人提供）：脊髓损伤是一种灾难性事件，它极大地改变了受害者的生活方式，并使个体终生活动能力降低、复发性医疗问题和慢性神经性疼痛。 损伤的最终严重程度不仅取决于脊髓束产生的初始损伤，还取决于一系列生化事件，这些事件在几天内进一步侵蚀脊髓的完整性。这些途径之一导致阿片肽强啡肽的浓度和释放急剧增加。强啡肽是一种神经毒性肽，通过激活兴奋性氨基酸受体的N-甲基-D-天冬氨酸（NMDA）亚类来损伤脊髓中的神经元。由强啡肽产生的损伤可以在非创伤性脊髓中产生从慢性异常性疼痛（疼痛到通常非疼痛的刺激）到完全瘫痪的症状。因此，强啡肽可能在创伤后脊髓损伤中起重要作用。这个项目的最终目标是了解强啡肽如何促进脊髓损伤。 强啡肽激活NMDA受体的机制目前尚不清楚。在该提议中，将检验的假设是强啡肽激活缺乏NR 1亚基（NRla）的外显子5编码区的低活性或静止的NMDA受体。全细胞膜片钳实验将在培养的脊髓神经元、脊髓切片和用NMDA受体亚基的已知组合转染的细胞系中进行。这些实验将证明：1）强啡肽增强NMDA受体的子集的活性，2）NRla亚基对于强啡肽增强NMDA受体功能是关键的，以及3）含有NMDA受体的低活性NRla负责强啡肽增强脊髓中的突触活性。这些实验将确定强啡肽的神经毒性机制，以及确定潜在的药理学目标，在脊髓损伤的关键创伤后时期进行干预。

DESCRIPTION (provided by applicant): Spinal cord injury is a catastrophic event that dramatically alters the lifestyle of the victim and subjects the individual to a lifetime of reduced mobility, recurrent medical problems and chronic neuropathic pain. The ultimate severity of the injury is not only determined by the initial damage produced by the tramna, but also by a cascade of biochemical events that further erodes the integrity of the spinal cord over the course of several days. One of these pathways leads to a dramatic increase in the concentration and release of the opioid peptide dynorphin. Dynorphin is a neurotoxic peptide that damages neurons in the spinal cord by activating the N-methyl-D-aspartate (NMDA) subclass of excitatory amino acid receptors. The damage produced by dynorphin can produce symptoms in non-traumatized spinal cord ranging from chronic allodynia (pain to normally non-painful stimuli) to full paralysis. Thus, it is likely that dynorphin contributes significantly to the post trauma spinal cord damage. The ultimate goal of this project is to understand how dynorphin contributes to spinal cord injury. The mechanism by which dynorphin activates NMDA receptors is currently not known. In this proposal the hypothesis that will be tested is that dynorphin activates low activity or quiescent NMDA receptors that lack the exon-5 coded region of the NR1 subunit (NRla). Whole cell patch clamp experiments will be conducted in cultured spinal cord neurons, spinal cord slices, and cell lines transfected with known combinations of NMDA receptor subunits. These experiments will demonstrate that 1) dynorphin enhances the activity of a subset of NMDA receptors, 2) the NRla subunit is critical for dynorphin enhancing NMDA receptor function, and 3) the low activity NRla containing NMDA receptors are responsible for dynorphin's enhancement of synaptic activity in the spinal cord. These experiments will identify the mechanism for dynorphin's neurotoxicity as well as identify potential pharmacological targets for intervention in the critical post trauma period of spinal cord injury.