Inflammation-induced changes in spinal GABA-A receptor signaling

描述（由申请人提供）：GABA-A受体（GAR）介导的脊髓传入输入的突触前抑制对于伤害性阈值的建立至关重要。在没有损伤的情况下，GAR拮抗剂的脊髓给药产生痛觉过敏和异常性疼痛，而GAR激动剂是镇痛剂。然而，我们最近证明，在持续性炎症的存在下，GAR信号传导发生了变化，使得脊髓加尔斯的激活实际上有助于炎症性痛觉过敏：脊髓注射GAR拮抗剂可逆转炎性痛觉过敏，而GAR激动剂则可加重炎性痛觉过敏。我们的初步数据表明，这种转变反映了GAR信号传导的突触前变化，包括不同GAR受体亚单位的出现和GAR受体亚单位的增加。阴离子平衡电位（Eanion）的去极化位移。E1阴离子的去极化移位可使GAR激活变为兴奋性。然而，我们最近的数据也表明，咪达唑仑，苯二氮卓类受体激动剂，在炎症的存在下保留镇痛功效，表明Eanion单独的去极化转变不足以解释炎症诱导的GAR信号传导的转变。脊髓GAR信号传导的明显转变对1）我们对持续性疼痛的潜在机制的理解，2）一系列全身麻醉剂（如异氟烷，丙泊酚和依托咪酯）的临床使用，以及3）开发用于治疗疼痛的新型治疗干预措施具有深远的意义。因此，我们提出了一系列实验，描述了4个具体的目标，旨在确定在存在和不存在炎症的情况下脊髓GAR信号转导的机制。在具体目标1中，我们将描述炎症诱导的GAR介导电流的生物物理特性和药理学变化以及皮肤感觉神经元中阴离子稳态的调节。在特定目标2中，我们将描述炎症诱导的皮肤感觉神经元中GAR亚基表达和分布的变化。在具体目标3中，我们将评估炎症诱导的GAR亚基表达和细胞内阴离子稳态变化的功能后果。在具体目标4中，我们将确定皮肤感觉神经元中的特定GAR亚基在炎症存在下介导GAR信号传导的原伤害感受作用的出现的程度。在这4个目标下描述的实验，涉及从行为药理学到病毒载体介导的基因表达操纵的一系列方法，旨在测试中心假设，即持续性炎症导致GAR信号传导的变化，反映了初级传入神经元中Eanion和GAR亚基表达变化的组合。公共卫生相关性：初级传入神经元中枢终末上的3-氨基丁酸-A（GABA-A）受体在抑制急性痛和维持损伤后的持续痛中起重要作用。GABA-A受体信号传导的这种转变的潜在机制仍有待确定，但它对1）我们对持续性疼痛的潜在机制的理解，2）一系列全身麻醉剂（如异氟烷，丙泊酚和依托咪酯）的临床使用，以及3）开发用于治疗疼痛的新型治疗干预措施具有深远的意义。因此，最终目标是最大限度地减少全身麻醉剂的有害后果，并确定新的治疗干预靶点，我们提出了一系列实验，旨在确定在炎症存在下脊髓GABA-A受体信号转导的变化机制。

DESCRIPTION (provided by applicant): GABA-A receptor (GAR) mediated presynaptic inhibition of afferent input to the spinal cord is critical for the establishment of nociceptive threshold. In the absence of injury, spinal administration of GAR antagonists produce hyperalgesia and allodynia whereas GAR agonists are analgesic. We recently demonstrated, however, that in the presence of persistent inflammation, there is a shift in GAR signaling such that activation of spinal GARs actually contributes to inflammatory hyperalgesia: spinal administration GAR antagonists reverse inflammatory hyperalgesia while GAR agonists exacerbate it. Our preliminary data suggest this shift reflects a presynaptic change in GAR signaling which includes the emergence of distinct GAR receptor subunits and a depolarizing shift in the anion equilibrium potential (Eanion). A depolarizing shift in Eanion may enable GAR activation to become excitatory. However, our recent data also indicate that midazolam, a benzodiazepine receptor agonist, retains analgesic efficacy in the presence of inflammation, indicating that a depolarizing shift in Eanion alone is insufficient to account for the inflammation-induced shift in GAR signaling. The apparent shift in spinal GAR signaling has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, we have proposed a series of experiments described under 4 specific aims, designed to identify mechanisms underlying spinal GAR signaling in the presence and absence of inflammation. In Specific Aim 1, we will characterize inflammation-induced changes in the biophysical properties and pharmacology of GAR mediated currents and the regulation of anion homeostasis in cutaneous sensory neurons. In Specific Aim 2, we will characterize inflammation-induced changes in the expression and distribution of GAR subunits in cutaneous sensory neurons. In Specific Aim 3, we will assess the functional consequences of inflammation-induced changes in GAR subunit expression and intracellular anion homeostasis. And in Specific Aim 4, we will determine the extent to which specific GAR subunits in cutaneous sensory neurons mediate the emergence of pronociceptive actions of GAR signaling in the presence of inflammation. The experiments described under these 4 aims, involving an array of approaches ranging from behavioral pharmacology to viral vector mediated manipulation of gene expression, are designed to test the central hypothesis that persistent inflammation results in changes in GAR signaling that reflect a combination of changes in Eanion and GAR subunit expression in primary afferent neurons. PUBLIC HEALTH RELEVANCE: 3-aminobutyric acid-A (GABA-A) receptors on the central terminals of primary afferent neurons appear to play critical role both in the inhibition of acute pain and the maintenance of persistent pain in the presence of injury. The mechanisms underlying this shift in GABA-A receptor signaling remain to be identified, but it has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, with the ultimate goals of both minimizing deleterious consequences of general anesthetics and identifying novel targets for therapeutic interventions, we have proposed a series of experiments designed to identify mechanisms underlying the shift in spinal GABA-A receptor signaling in the presence of inflammation.